Molecular Biology Reports

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Article
Transposable elements, also known as “jumping genes,” have the ability to hop within the host genome. Nonetheless, this capacity is kept in check by the host cell defense systems to avoid unbridled TE mobilization. Diferent types of stressors can activate TEs in Drosophila, suggesting that TEs may play an adaptive role in the stress response, especially in generating genetic variability for adaptive evolution. TE activation by stressors may also lead to the notion, usually found in the literature, that any form of stress could activate all or the majority of TEs. In this review, we defne what stress is. We then present and discuss RNA sequencing results from several studies demonstrating that stress does not trigger TE transcription broadly in Drosophila. An explanation for the LTR order of TEs being the most overexpressed is also proposed.
 
An increase in the concentration of yohimbine showing a dose-dependent decline in the cell viability of KB-ChR-8–5 cells
Alteration in the cell morphology of KB-ChR-8–5 cells in response to yohimbine treatment
Yohimbine treatment inducing apoptosis in KB-ChR-8–5 cells
Effects of yohimbine on the mitochondrial membrane potential of KB-ChR-8–5 cells
Effect of yohimbine on the intracellular ROS generation of KB-ChR-8–5 cells
Article
  • Nasimudeen R. JabirNasimudeen R. Jabir
  • Mohd Shahnawaz KhanMohd Shahnawaz Khan
  • Nouf Omar AlafaleqNouf Omar Alafaleq
  • [...]
  • Bakrudeen Ali AhmedBakrudeen Ali Ahmed
Background The demand for environmentally friendly and cost-effective plant-based products for the development of cancer therapeutics has been increasing. Yohimbine (α2-adrenergic receptor antagonist) is a stimulant and aphrodisiac used to improve erectile dysfunction. In this study, we aimed to evaluate the anticancer potential of yohimbine in drug-resistant oral cancer KB-ChR-8–5 cells using different biomolecular techniques. Methods We estimated the anticancer efficacy of yohimbine using different assays, such as MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cell cytotoxicity, cell morphology, cell apoptosis, reactive oxygen species (ROS) formation, and modulation in the mitochondrial membrane potential (MMP). Results Yohimbine showed a dose-dependent increase in cytotoxicity with a 50% inhibitory concentration (IC50) of 44 µM against KB-ChR-8–5 cancer cell lines. Yohimbine treatment at 40 µM and 50 µM resulted in a considerable change in cell morphology, including shrinkage, detachment, membrane blebbing, and deformed shape. Moreover, at the dose of IC50 and above, a significant induction was observed in the generation of ROS and depolarization of MMP. The possible mechanisms of action of yohimbine underlying the dose-dependent increase in cytotoxicity may be due to the induction of apoptosis, ROS generation, and modulation of MMP. Conclusion Overall, yohimbine showed a significant anticancer potential against drug-resistant oral cancer KB-ChR-8–5 cells. Our study suggests that besides being an aphrodisiac, yohimbine can be used as a drug repurposing agent. However, more research is required in different in vitro and in vivo models to confirm the feasibility of yohimbine in clinics.
 
A GST pull-down assay followed by western blot analysis confirmed the interaction between GST/MRPS23 and CDK11A p110 and p58 isoform. Lane 1, HEK293 whole cell lysate (input); lane 2, GST protein with input (negative control); lane 3 & 4, Bait protein GST/MRPS23 (~ 46 kDa) fusion protein (confirmed by anti-GST antibody) with input in duplicates showing the presence of CDK11A in pull-down fraction. B IP with mouse monoclonal anti-MRPS23 antibody using HEK293 whole cell lysate followed by western blot analysis using rabbit-anti CDK11A showing the p110 and p58 isoform in the IP fraction. Lane 1, HEK293 whole cell lysate (input); lane 2, mouse IgG isotype antibody bound with protein A/G beads and input (negative control); lane 3, anti-MRPS23 antibody-bound protein A/G bead, showing the presence of CDK11A in IP fraction. C IP with rabbit anti-CDK11A antibody using HEK293 whole cell lysate followed by WB analysis showing the presence of MRPS23 in the pull-down fraction. Lane 1,rabbit IgG isotype antibody bound with protein A/G beads and input (negative control); lane 2, HEK293 whole cell lysate (input); lane 3, rabbit anti-CDK11A antibody-bound protein A/G beads, showing the presence of CDK11A p58 and p110 isoform along with MRPS23 in pull-down fraction. D IP with anti-FLAG antibody bound agarose beads using FLAG/CDK11-p58 overexpressed HEK293 whole cell lysate followed by WB analysis showing the presence of MRPS23 in the pull-down fraction. Lane 1, FLAG/CDK11-p58 overexpressed HEK293 whole cell lysate; lane 2, control vector FLAG alone overexpressed HEK293 whole cell lysate; lane 3, anti-FLAG antibody bound with protein A/G beads and control vector overexpressed HEK293 whole cell lysate (negative control); lane 4: anti-FLAG antibody bound with protein A/G beads and FLAG/CDK11-p58 overexpressed HEK293 whole cell lysate which shows the presence of FLAG/CDK11-p58 and MRPS23 in pull-down fraction. E The subcellular fractionation of HEK293 cells followed by western blot analysis of MRPS23 and CDK11A protein expression showed expression of MRPS23 and CDK11A in both cytoplasmic and nuclear fractions respectively. The extracts were analyzed using an anti-GAPDH antibody, a cytoplasmic marker. F IP with mouse monoclonal anti-MRPS23 antibody using a nuclear fraction of HEK293 cells followed by WB using rabbit anti-CDK11A antibody showing the presence of CDK11A in the pull-down fraction. Lane 1, HEK293 nuclear extract (input); lane 2, mouse IgG isotype antibody bound with protein A/G beads and input (negative control); lane 3, anti-MRPS23 antibody-bound protein A/G beads showing the presence of CDK11Ap58 in pull-down fraction
A IP with pan-phosphoserine antibody followed by WB analysis showed the presence of MRPS23 in the pull-down fraction. Lane 1, HEK293 whole cell lysate (input); lane 2, mouse IgG isotype antibody bound with protein A/G beads and input (negative control); lane 3, mouse monoclonal pan-phosphoserine antibody-bound protein A/G beads, showing the presence of MRPS23 in pull-down fraction. B Phosphoprotein fraction (eluate) and flow-through fraction flow-through were extracted from three breast tumor tissues and corresponding paired normal tissues and WB analysis was done. Western blot analysis showed the presence of MRPS23 in phosphoprotein enriched fraction in breast tumor tissue and paired normal tissues. Phospho Akt was used as a marker to assess phosphoprotein enrichment in flowthrough and elute fraction. C Graphical representation of the wildtype (WT) and deletion mutant plasmid constructs of MRPS23. FLAG-tagged MRPS23 wilt type (WT) and FLAG-tagged deletion construct (δTM) were overexpressed in MCF7 cells and confirmed by western blot using anti-FLAG antibody. D MRPS23 (δTM) mutant overexpressing MCF7 cells showed increased cell viability when compared with MRPS23 (WT) transfected cells, indicating a role of the N-terminal sequence of MRPS23 in cell viability. The experiment was performed independently thrice in four replicates. E Cell cycle analysis using the propidium iodide staining method showed the increased entry of cells into the S phase and a higher percentage of proliferating cells in MRPS23 (δTM) mutant overexpressing cells. F Western blot analysis of expression of Cyclin B1, E-cadherin, and vimentin in MRPS23 (WT) and MRPS23 (δTM) mutant overexpressed MCF7 cell lysates. G The cytoplasmic and nuclear extract was isolated from MRPS23 (WT) and δTM mutant overexpressing MCF cells respectively. The subcellular fractionation followed by western blot analysis showed that MRPS23 (WT) and MRPS23 (δTM) mutant protein expressionm are present in both cytoplasmic and nuclear fractions, while MRPS23 (δTM) mutant showed significantly increased levels in cytoplasmic extract when compared to MRPS23 (WT) overexpressing cells. The extracts were analyzed using an anti-HMGB1 antibody, nuclear fraction marker, and α-tubulin, cytoplasmic marker
A Graphical representation of the MRPS23 (S11A) and MRPS23 (S11G) plasmid constructs of MRPS23. FLAG-tagged MRPS23 (S11A) mutant and FLAG-tagged MRPS23 (S11G) mutant was overexpressed in MCF7 cells and confirmed by western blot using anti-FLAG antibody. B Increased cell viability can be observed in MRPS23 (S11G) transfected cells when compared with MRPS23 (S11A) transfected cells. The experiment was performed independently thrice in four replicates. C Cell cycle analysis using the propidium iodide staining method showed the increased entry of cells into the S phase and a higher percentage of proliferating cells in MRPS23 (S11G) mutant overexpressing cells (51%) when compared to MRPS23 (S11A) mutant overexpressing cells (40%). D Western blot analysis of expression of Cyclin B1, E-cadherin, and vimentin in MRPS23 S11A and S11G overexpressed MCF7 cell lysates respectively. The protein levels showed overexpression of Cyclin B1 and Vimentin in MRPS23 (S11G) mutant overexpressing cells when compared with MRPS23 (S11A) overexpressing cells. E Western blot analysis of expression of PI3K-PKB/Akt Pathway proteins (AKT, p-AKT (Ser 473, p-AKT (Thr 308), p-GSK-3β (Ser9), p-c-Raf (Ser259), pPDK (Ser241), p-PTEN (Ser380)), in MRPS23 (S11A) and (S11G) overexpressed MCF7 cell lysates respectively. The whole cell lysates used were prepared 72 h post transfection. The intensity of protein expression showed significant level of p-AKT (Ser473)/pan AKT, p-AKT (Ser308)/pan AKT p-PDK (Ser241), in the MRPS23 (S11G) mutant overexpressing cells when compared to the MRPS23 (S11A) mutant overexpressing cells. P-PTEN (Ser380) and p-c-Raf (Ser 259) were downregulated in MRPS23 (S11G) mutant overexpressing cells when compared to MRPS23 (S11A) mutant overexpressing cells. F Western blot analysis of expression levels of the pro-survival BCL-2 family proteins was analyzed which showed that BCL2, BCL-xL,MCL-1, p-BCL-2 (Ser70)), and were overexpressed in MRPS23 (S11G) mutant overexpressing cells when compared to MRPS23 (S11A) mutant overexpressing cells. The experiment was performed independently twice. G Western blot analysis of cytoplasmic and nuclear extracts were isolated from MRPS23 (S11A) and MRPS23 (S11G) mutant overexpressing MCF cells. The quality extracts were analyzed using anti-HMGB1 antibody, nuclear fraction marker, and α-tubulin, cytoplasmic marker. (In all the Fig.s, the values are represented as mean ± SD). (p-value 0.0001: *****, 0.001: ****, 0.005: ***, 0.01: **, 0.05: *)
A ELISA confirming the specificity of p-MRPS23 (Ser 11) antibody (antiserum) which was raised in Wistar rats and isolated by negative selection using unphospho-MRPS23 synthetic peptide (SRLETVGSIFSRTRD). B WB analysis of phospho protein-extract isolated from HEK293 confirming Serine 11 phosphorylated form of MRPS23 in eluate fraction (phospho protein enriched fraction) when probed using p-MRPS23 (Ser11) antibody. Note: The total MRPS23 and Ponceau stained bot is presented in Supplementary Fig. 2 for reference. C Subcellular fractionation followed by WB analysis showed the presence of p-MRPS23 (Ser11) in nuclear extract. D Overexpression of p-MRPS23 (Ser11) was observed in MCF7 cells overexpressed with FLAG/CDK11A p58 and FLAG/ Cyclin D3 when compared control vector-transfected MCF7 lysates. E WB analysis of protein expression levels of p-MRPS23 (Ser11) protein and pan-MRPS23 in breast cancer cell lines MCF7, SKBR3, MDAMB468, and MDAMB231 and control MCF10A cells. F WB analysis of protein expression levels of CDK11A p110 and p58 isoform among breast cancer cell lines MCF7, SKBR3, MDAMB468, and MDAMB231 and control MCF10A cells
A Non-radioactive kinase assay was performed using Cyclin B1/CDK1 kinase and MRPS23 synthetic peptide as substrate and the fluorescence was read at excitation 540 nm and emission 590 nm which showed increase fluorescence in a reaction containing MRPS23 peptide substrate when compared with control (no substrate). The assay was performed independently twice in three replicates. B Non-radioactive kinase assay was performed using Cyclin B1/CDK1 kinase and purified GST-MRPS23 protein (1 µg) as a substrate which showed significant fluorescence owing to phosphorylation when compared with control (GST protein (1 µg) alone as substrate). The assay was performed independently twice in three replicates. C The gene co-expression of MRPS23 and CDK1 analyzed by GEPIA for normal breast tissue showed a moderate positive correlation (r = 0.31). D The gene co-expression of MRPS23 and CDK1 analyzed by GEPIA for breast cancer datasets showed that there is a moderate positive correlation (r = 0.27). E The MCF7 cells were treated with 2.5 µM of CDK1i and 50 nM of FP separately and the whole-cell lysates were obtained in RIPA buffer. Immunoblotting showed that treatment with the inhibitors inhibited the phosphorylation of Ser 11 of MRPS23, which is evident by probing with p-MRPS23 (Ser 11) antibody. F Western blot analysis of control siRNA and MRPS23 siRNA transfected cell lysates showed two-fold downregulation in MRPS23 siRNA transfected cells 72 h post-transfection. The knockdown was performed independently thrice and confirmed. G Control siRNA and MRPS23 siRNA was transfected onto MCF7 cells and 72 h post-transfection, the cells were treated with CDK1i (2.5 µM) for 24 h followed by cell viability analysis for 24, 48, 72, and 96 h. The experiment was performed independently thrice in four replicates. H Similarly, Control siRNA and MRPS23 siRNA was transfected into MCF7 cells and 72 h post-transfection, the cells were treated with FP (50 nM) for 24 h followed by cell viability analysis for 24, 48, 72, and 96 h. The experiment was performed independently thrice in four replicates. I and J The cell viability was analyzed in FLAG/MRPS23 (WT) and pCDNA/FLAG (CV) overexpressed MCF7 cells post-treatment with CDK1i and FP for 24 h. All the values are represented as mean ± SD (p-value 0.0001: *****, 0.001: ****, 0.005: ***, 0.01: **, 0.05: *)
Article
Background Post-translational modification of some mitoribosomal proteins has been found to regulate their functions. MRPS23 has been reported to be overexpressed in various cancers and has been predicted to be involved in increased cell proliferation. Furthermore, MRPS23 is a driver of luminal subtype breast cancer. However, its exact role and function in cancer remains unknown. Methods and results Our previous study identified protein–protein interactions involving MRPS23 and CDK11A. In this study, we confirmed the interaction of MRPS23 with the p110 and p58 isoforms of CDK11A. Phosphoprotein enrichment studies and in vitro kinase assay using CDK11A/cyclin D3 followed by MALDI-ToF/ToF analysis confirmed the phosphorylation of MRPS23 at N-terminal serine 11 residue. Breast cancer cells expressing the MRPS23 (S11G) mutant showed increased cell proliferation, increased expression of PI3-AKT pathway proteins [p-AKT (Ser47), p-AKT (Thr308), p-PDK (Ser241) and p-GSK-3β (Ser9)] and increased antiapoptotic pathway protein expression [Bcl-2, Bcl-xL, p-Bcl2 (Ser70) and MCL-1] when compared with the MRPS23 (S11A) mutant-overexpressing cells. This finding indicated the role of MRPS23 phosphorylation in the proliferation and survival of breast cancer cells. The correlation of inconsistent MRPS23 phosphoserine 11 protein expression with CDK11A in the breast cancer cells suggested phosphorylation by other kinases. In vitro kinase assay showed that CDK1 kinase also phosphorylated MRPS23 and that inhibition using CDK1 inhibitors lowered phospho-MRPS23 (Ser11) levels. Additionally, modulating the expression of MRPS23 altered the sensitivity of the cells to CDK1 inhibitors. Conclusion In conclusion, phosphorylation of MRPS23 by mitotic kinases might potentially be involved in the proliferation of breast cancer cells. Furthermore, MRPS23 can be targeted for sensitizing the breast cancer cells to CDK1 inhibitors.
 
The expression level ofDNAH5andlinc02220in study group. A: Significant downregulation of DNAH5 in TAZ and AZ groups compared to NZ group. B: Meaningful upregulation of linc02220 in TAZ and AZ groups in comparison to NZ group (p < 0.0001). NZ: Normospermia, TAZ: terato-asthenozoospermia, AZ: asthenozoospermia
Correlation ofDNAH5expression tolinc02220expression in study groups. NZ: Normospermia, TAZ: terato-asthenozoospermia, AZ: asthenozoospermia
Article
Background Numerous pieces of evidence show that many environmental and genetic factors can cause male infertility. Much research in recent years has investigated the function of long non-coding RNAs (lncRNAs) in fertility. The main objective of the current study was to investigate the expression of Dynein Axonemal Heavy Chain 5 (DNAH5) as a gene that plays an essential role in sperm motility in individuals with asthenozoospermia and terato-asthenozoospermia. Alterations in linc02220 expression (located close to the DNAH5 gene), its action potential in DNAH5 regulating, and the correlation between their expression and normal sperm morphology and motility were also examined. Method and material This study examined the semen of 31 asthenozoospermia individuals (AZ), 33 terato-asthenozoospermia (TAZ) individuals, and 33 normospermia (NZ) individuals with normal sperm as a control group. The expression levels of DNAH5 and linc02220 in the sperm samples were analyzed by real-time PCR. Results Gene expression analysis revealed a significant association between DNAH5 expression and sperm motility and morphology (p < 0.0001). The DNAH5 expression levels in the TAZ and AZ groups were also significantly reduced; however, linc02220 was significantly upregulated in both TAZ and AZ groups compared to the NZ group (p < 0.0001). DNAH5 expression in the TAZ and AZ groups was negatively correlated with linc02220 expression, thus, DNAH5 downregulation was associated with linc02220 overexpression (p < 0.05). Conclusions The gene linc02220 could be a potential regulatory target for DNAH5, and both could affect sperm’s normal motility and morphology.
 
Assay of aprepitant effect on A2780 cells. The IC50 was observed at about 25.9 µM after 24 h. Results were presented by mean ± standard deviation (SD) of three distinct experiments
The effect of SP/NK1R system and aprepitant on MMP2 (A) and MMP9 (B) gene expression in A2780 cells. The results demonstrated that the level of mRNA expression of MMP2 and MMP9 is significantly reduced in cells exposed to aprepitant (10 µM) alone or in combination with SP (100 and 400 nM) compared to the control group (#. Compared to the treated group, *. Compared to control group, ##P < 0.01, ###P < 0.001, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001)
The effect of SP/NK1R system and aprepitant on VEGF (A) and its receptor (VEGF-R) (B) gene expression in A2780 cells. The results exhibited that the level of mRNA expression of VEGF and VEGF-R is significantly reduced in cells treated with aprepitant (10 µM) alone or in combination with SP (100 and 400 nM) compared to the control group. (#. Compared to the treated group, *. Compared to control group, # p < 0.05, ##P < 0.01, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001)
Results of cell migration study using scratch method after treatment with SP and aprepitant. Microscopic images show that A2780 cell migration to the cell-free region in the presence of SP is significantly increased compared to the control group, while aprepitant suppresses this effect (a). The graph shows the percentage of cell-free area at different time points during the in vitro scratching procedure (b). (#. Compared to the treated group, *. Compared to control group, # p < 0.05, *P < 0.05, **P < 0.01, ***P < 0.001)
Article
Background Substance P (SP) has a crucial role in cancer initiation and progression via binding to its specific receptor (NK1R). Various evidence confirmed the overexpression of NK1R and SP in the tissue of multiple cancers, including ovarian cancer. Despite numerous studies, the mechanism of the SP/NK1R system on migration and angiogenesis of ovarian cancer cells has not yet been deciphered. In this study, considering the critical factors in cell migration (MMP-2, MMP-9) and angiogenesis (VEGF, VEGFR), we investigated the possible mechanism of this system in inducing migration and angiogenesis of ovarian cancer cells. Methods and Results First, the resazurin assay was conducted to evaluate the cytotoxic effect of aprepitant (NK1R antagonist) on the viability of A2780 ovarian cancer cells. After that, the impact of this system and aprepitant on the mRNA expression of the factors mentioned above were studied using RT-PCR. Besides, the scratch assay was performed to confirm the effect of the SP/NK-1R system and aprepitant on cell migration. Our results implied that this system induced cell migration and angiogenesis by increasing the mRNA expression of MMP-2, MMP-9, VEGF, and VEGFR. The obtained results from the scratch assay also confirmed the positive effect of this system on cell migration. Meanwhile, the blocking of NK1R by aprepitant suppresses the SP effects on cell migration and angiogenesis. Conclusions Overall, the SP/NK1R system plays a vital role in ovarian cancer progression, and the inhibition of NK1Rusing aprepitant could inhibit the spread of ovarian cancer cells through metastasis and angiogenesis.
 
CMV PCR products. Well 1 was positive control, wells 2, 4, 5, 6 were positive CMV, wells 3 and 7 were negative CMV, well 8 was negative control. Add product size based on size marker
Electrophoresis of Tetra-amplification-refractory mutation system (Tetra-ARMS) polymerase chain reaction products for IL-6-174 genotyping. Lane M, 100-bp ladder; lanes 1, 3, 4, and 8 positive for GG genotypes; lanes 2, 5, and 6 positive for CC genotypes; lane 7 positive for GC genotypes
Electrophoresis of RFLP-PCR for IL-6-634 genotyping. Lane M, 100-bp ladder; lane 1 positive for CC genotypes; lane 2 positive for GC genotypes; lane 3 positive for GG genotypes
Article
Background Recurrent pregnancy loss (RPL) is described as two or more spontaneous abortions. To date, scientists in various fields of knowledge, such as genetics, endocrinology, anatomy, immunology, and microbiology, have identified some important factors that affect abortions; nonetheless, the precise basic etiology is not determined in up to 50% of RPL cases. Human cytomegalovirus (CMV) infection and host genetic background, like IL-6 SNP polymorphisms, play important roles in RPL etiology. Objective This study aimed to evaluate the relationships among single nucleotide polymorphisms (-634C/G and -174 G/C) in the IL-6 gene with CMV infection and the risk of RPL for early detection and treatment. Materials and methods This case–control study was carried on 80 Iranian females with RPL and 80 healthy females as controls. DNA was extracted from samples and CMV and IL6 SNPs were detected using Tetra ARMS-PCR. Statistics were analyzed by Epi Info TM and SPSS software by X2 test for the roles of CMV detection and two polymorphisms in RPL. Results The results indicated an increased rate of CMV infection in the RPL group (44%) compared to the control group (25.45%). The prevalence of IL-6-634C/G genotype among RPL patients with CMV infection was 80%, while the frequency of this genotype among RPL patients without CMV infection was 50%. Furthermore, no substantial relation was found between IL-6-174 G/C genotypes and RPL (p = 0.005). Conclusion This study not only indicated a significant role for CMV in RPL, but also showed an association between CMV and allele G in IL6-634 among Iranian women. In addition, the findings suggested the use of CMV and IL-6-634 GG genotypes as diagnostic and prognostic biomarkers for RPL in the Iranian population.
 
The anti-proliferative effects of VA on (A) A-375 melanoma and (B) epidermal melanocyte cells were determined by RTCA xCELLigence system. The cells were treated with VA at a concentration range between 100 and 1000 µM for 96 h. The data demonstrates the mean ± SD from two independent experiments (* p < 0.05)
The morphological evaluation of A-375 melanoma cell after treatment with VA (A) The effects of IC50 concentration of VA on (a) A-375 melanoma, (b) melanocyte cells and cell control images. Photos were taken under a light microscope at 10X. (B) The fold change of caspase-3 activity after VA treatment in A-375 melanoma cell (C: Control, VA: Vulpinic acid). (* p < 0.05)
The apoptotic effects of VA on A-375 melanoma cells were determined by flow cytometry (A) Apoptosis analysis evaluated by flow cytometry, A–: viable cells, A+-: early apoptotic cells, A++: late apoptotic cells, A-+: necrotic cells, (B) Cell cycle histograms of the cells after treatment with VA (C) The graphical representation of (a) apoptosis and (b) cell cycle analysis (C: Control, VA: Vulpinic acid) (*p < 0.05, ** p < 00.01)
Real time PCR analysis of the transcript expression levels of the (A) Caspase gene family, (B) TRAF and Kinase gene family, (C) other apoptosis-related gene family members, (D) BCL-2 gene family, (E) TNF gene family and (F) cell proliferation and cell cycle associated genes in VA treated A-375 melanoma cells compared with control (* p < 0.05, ** p < 0.01)
Article
Backgrounds Malignant melanoma is an aggressive skin tumor with a rapidly increasing incidence and there is not yet a successful treatment strategy. Vulpinic acid (VA) is derived from secondary metabolites from lichen species. In the current study, we, for the first time, investigated the anti-cancer effects of VA and the underlying mechanism VA induced programmed cell death in melanoma. Methods The anti-cancer effects of VA on melanoma cells were evaluated by the xCELLigence system, flow cytometry, caspase-3 activity and RT-PCR analysis. Results Our results showed that VA had a strong anti-proliferative effect on A-375 melanoma cells without damaging human epidermal melanocyte cells. Additionally, VA promoted apoptotic cell death through G2/M arrest and the activation of both intrinsic and extrinsic apoptosis pathways according to the analysis of 88 genes associated with apoptosis by qRT-PCR. Conclusions Our findings suggest that VA could become an alternative topical and transdermal treatment strategy in the treatment of maligned melanoma cancer. However, further investigations are needed to assess the underlying molecular mechanism of VA mediated apoptotic cell death in the treatment of melanoma.
 
Venn diagrams showing the a functional annotation [cellular components (CC), biological processes (BP) and molecular functions (MF)] and b Number of syntenic unigenes associated across different treatments in okra
Principal component analysis based bi-plot under a control, b drought and c heat stress
Population structure of okra genotypes used in the current study
UPGMA cluster showing diversity among okra genotypes
Linkage disequilibrium plot showing linkage between unigene markers used in current study
Article
Background Considerable production losses are caused by heat and drought stress in okra. Germplasm evaluation at genetic level is essential for the selection of promising genotypes. Lack of genomic information of okra limits the use of genetic markers. However, syntenic markers of some related family could be used for molecular characterization of major economic traits. Methods and results Herein, 56 okra genotypes were evaluated for drought and heat tolerance. Sixty-one expressed sequence tags (ESTs) identified for heat and drought tolerance in cotton were searched from literature surveys and databases. The identified ESTs were BLAST searched into okra unigene database. Primers of selected okra unigenes were synthesized and amplified in all genotypes using standard polymerase chain reaction (PCR) protocol. Marker trait association (MTA) of the syntenic unigenes were identified between genotypic and phenotypic data on the basis of linkage disequilibrium Functional syntenic analysis revealed that out of these 61 cotton ESTs 55 had functional homology with okra unigenes. These 55 unigenes were used as markers for further analysis (amplification). Okra genotypes showed significance variations for all the physo-morphological parameters under heat and drought stress. Genotypes Perbhani Karanti, IQRA-III, Selection Super Green, Anmol and Line Bourd performed better under drought stress whereas genotypes Perbhani Karanti, IQRA-III, Green Gold, OK-1501 and Selection Super Green showed heat tolerance. Fifty markers showed amplification in okra. Fifty-six okra genotypes were clustered into three distinct populations. LD analysis has shown most significant linkage between markers Unigene43786 and Unigene3662. MTAs using MLM and GLM models revealed that 23 markers have significant associations (p < 0.05) with different traits under control and stressed conditions. Relative water content is associated with four markers (Unigene10673, Unigene99547, Unigene152901, and Unigene129684) under drought conditions. Whereas, Electrolyte leakage was associated with 3 markers (Unigene109922, Unigene28667 and Unigene146907) under heat stress. Conclusion These identified unigenes may be helpful in the development of drought and heat tolerant genotypes in okra.
 
Article
Background Early and intermediate serological screening cannot detect sex chromosome abnormalities. Currently, noninvasive prenatal testing (NIPT) is the only procedure available for screening such disorders; however, its use is controversial. Methods and Results A total of 47,855 pregnant women underwent NIPT at our referral center from January 2014 to December 2020. Of the 314 patients with a positive NIPT indicating sex chromosome abnormalities, 260 were screened via karyotype analysis and single nucleotide polymorphism (SNP) array after amniotic fluid extraction; 96 cases were confirmed. Karyotype analysis and SNP array were consistent in the diagnosis of 88 out of the 96 fetuses. The positive predictive value (PPV) for sex chromosome abnormalities was found to be 36.9%. The PPV in patients aged 30–34 years was significantly higher than that in patients aged < 30 years. No statistically significant difference was observed on the PPV among patients with or without previous adverse pregnancy outcomes. Moreover, 83 women carrying fetuses were diagnosed with a sex chromosome abnormality terminated their pregnancy. Conclusions Improvements in detection and analytical technologies are needed to increase the accuracy of sex chromosome abnormalities detection. Pregnant women with a positive NIPT for these abnormalities may require invasive diagnostic procedures such as karyotype analysis and SNP array for better genetic counseling.
 
Schematic of the CRISPR/dCas9 system. The CRISPR/dCas9 system is composed of dCas9-VP64, sgRNA and p65-MS2, the sgRNA guides the dCas9 nuclease to specific target genes, dCas9 is mainly fused with VP64 and p65 for gene regulatory activation, and the transcriptional activators VP64 and p65 have strong transcriptional activation function on target genes
In vivo delivery and potential therapeutic strategies of CRISPR/Cas9 system in hepatic fibrosis therapy. (A) In vivo delivery of CRISPR/dCas9 system mainly include viral vectors and non-viral vectors. (B) HSCs may be converted into hepatocytes via CRISPR/dCas9 system
Article
Hepatic fibrosis is a pathological reaction of tissue damage and repair caused by various pathogenic factors acting on liver. At present, there is no effective anti-fibrotic specific therapy. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (dCas9) system is a new generation of gene editing technology. The CRISPR/dCas9 system provides a platform for studying site-specific transcriptional regulation, which has high efficiency in gene transcriptional activation for achieving robust. This system holds promise for hepatic fibrosis therapy via acting on liver fibrosis effector cells. However, there are some challenges associated with this novel technology, such as large structural variants at on-target, off-target sites, and targeted delivery efficiency. In this review, we present the potential implications and describe the challenges of CRISPR/dCas9 system that might be encountered in hepatic fibrosis therapy.
 
Examination of AR expression in different breast cancer cell lines (a) Western blot assays of AR, HER2, PR and ER in the indicated cell lines. Cells were cultured in media with 10% FBS. Whole cell lysates were analyzed by western blot. GAPDH was used as a loading control. (b) DHT treatment increased AR protein in the indicated cell lines. The 22Rv1 prostate cancer cell line served as a positive control. Cells were cultured in phenol red-free medium containing 10% charcoal-stripped FBS (CSS) for 48 h and then treated with vehicle or 5 nM of DHT for 24 h. Whole cell lysates were subjected to Western blot assay using AR antibody. (c) The average ratios of the AR band intensity to the GAPDH band intensity, which were calculated from the data of three independent assays. *, p < 0.05 by unpaired Student’s t-test
HC-1119 treatment inhibited the increase in AR protein elevated by DHT treatment in AR-positive TNBC cells Hs578T (a) and SUM159PT (b) cells were cultured in a medium with 10% CSS for 48 h, and then treated with vehicle, 5 nM of DHT, 0.5 μm (for Hs578T cells) or 10 μm (for SUM159PT cells) of HC-1119, or both DHT and HC-1119 as indicated for 24 h. Whole cell lysates were prepared from the treated cells and analyzed by Western blot using AR antibody. GAPDH served as a loading control. Experiments were repeated at least three times and the representative bands of Western blot assays are presented. *, p < 0.05 by One-Way ANOVA
HC-1119 treatment suppresses DHT-induced nuclear localization of AR in AR-positive TNBC cells Hs578T cells were cultured in a medium with 10% CSS for 48 h, and then seeded onto glass coverslips in a 24-well plate, and treated for one hour with vehicle (Ctrl), DHT (5nM), HC-1119 (0.5 μm), or both DHT and HC-1119 as indicated. Treated cells were processed for immunecytofluorescence staining using AR antibody. Images were recorded with a confocal microscope
HC-1119 inhibits DHT-stimulated migration and invasion of AR-positive TNBC cells (a, b) Effects of HC-1119 on the migration and invasion of AR-positive TNBC cells. Hs578T (a) and SUM159PT (b) cells in the upper chambers of the transwell plate for migration and invasion assays were treated with vehicle (Ctrl), DHT, HC-1119 or both DHT and HC-1119 as indicated for 24 h. Migrated or invaded cells attached to the lower surface of the membrane were imaged under microscope with 4X and 20X magnifications. The quantitative cell numbers of migrated and invaded cells were normalized to cell numbers of vehicle treated groups. *, **, and ***, p < 0.05, p < 0.01, and p < 0.001, which were compared with Ctrl groups by One-Way ANOVA. ##. and ###, p < 0.01, and p < 0.001, which were compared with DHT groups by One-Way ANOVA. (c, d) Hs578T (c) and SUM159T (d) cells were treated with vehicle (Ctrl), DHT, HC-1119, or both DHT and HC-1119 for 24 h as indicated. Whole cell lysates of the treated cells were analyzed for the indicated proteins by Western blot. Experiments were repeated at least three times and the representative images were presented. Each band intensity on Western blot was quantitatively measured by densitometry, and the average band intensity of each protein was normalized to the band intensity of GAPDH and presented in the bar graphs
HC-1119 treatment reduces DHT-promoted metastasis of AR-positive TNBC cells in mice (a) Visible metastatic nodules in the liver. Two-million SUM159PT cells were injected into one of the fourth mammary gland fat pads of the 6-week-old BALB/c nude mice (n = 8). One week later, mice were treated with vehicle, DHT, HC-1119 or both DHT and HC-1119 as indicated for 31 days. The representative liver images with metastatic nodules (indicated by arrows) are presented. The number of mice with liver metastasis in each group is also presented in the table. (b) Representative H&E-stained liver sections with metastasis lesion. The boxed area in the left panel is enlarged in the right panel. MFIL, metastatic foci in liver. HCs, hepatic cords. (c) Immunohistochemical staining for AR (brown color). Tissue sections were prepared from xenograft tumors in the mammary gland fat pads of mice treated with vehicle, DHT, HC-1119 or both DHT and HC-1119 as indicated. Immunohistochemical staining for AR was carried out by using an AR antibody. The boxed areas are shown in higher magnification as indicated
Article
Triple-negative breast cancers (TNBCs) are aggressive, and they develop metastasis at earlier stages, relapse more frequently, and exhibits poorer prognosis than other subtypes of breast cancer. Due to the lack of estrogen receptor for endocrine therapy and HER2 for targeted therapy, new targeted therapies for TNBCs are urgently needed. Enzalutamide is a second-generation androgen receptor (AR) inhibitor, and HC-1119 is a new synthetic deuterated enzalutamide. Owing to the isotope effect, HC-1119 has many advantages over enzalutamide, including slow metabolism, high plasma concentration and low brain exposure. However, the efficacy of HC-1119 in inhibition of AR function in triple-negative breast cancer (TNBC) has not been studied. In this study, we found high-level AR expression in both Hs578T and SUM159PT TNBC cell lines. Activation of AR by dihydrotestosterone (DHT) in both cell lines increased AR protein, induced AR-nuclear localization, enhanced cell migration and invasion in culture, and promoted liver metastasis in mice. Importantly, cotreatment with HC-1119 of these cells efficiently abolished all of these effects of DHT on both Hs578T and SUM159PT cells. These results indicate that HC-1119 is a very effective new second-generation AR antagonist that can inhibit the migration, invasion and metastasis of the AR-positive TNBC cells.
 
Patient enrollment flow-chart
Genotyping distribution of MMP-3 (-1612 5 A/6A) (a), MMP-9 (-1562 C/T) (b) according to occurring cardiovascular outcomes
Kaplan Meier Survival analysis for major cardiovascular outcomes in relation to MMP-3 (-1612 5 A/6A) polymorphism
Article
Background Matrix metalloproteinases (MMPs) are widely expressed in atherosclerosis lesions. The disequilibrium of MMPs driving to an overexpression or a lack of its level can be influenced by genetic variations. MMP-3 and MMP-9 may be affected by specific polymorphisms like − 1612 5 A/6A and the − 1562 C/T respectively. We aim in the present study to investigate prospectively the association between the − 1612 5 A/6A MMP-3 and − 1562 C/T MMP-9 polymorphisms and clinical outcomes in patients with coronary artery disease (CAD). This study is elaborated to reveal whether one of these polymorphisms is a probable predictor of cardiovascular complications in this CAD cohort. Methods and Results A total of 168 patients with CAD were prospectively followed up over a period of 5 years. Genotypes for the MMP-3 (-1612 5 A/6A) and MMP-9 (-1562 C/T) polymorphisms were performed using PCR-RFLP. Their levels were measured by ELISA in Sandwich test during the follow-up period, 39 cardiovascular outcomes occurred with 21 repeat targets for revascularization, 3 patients with Myocardial infarction, 8 for heart failure, 5 for Stroke and 2 for cardiovascular mortality. The MMP-3 5 A/6A polymorphism was related to the disease on the contrary of the MMP-9 -1562 C/T. Patients carrying the 5 A allele had a higher level of MMP-3 level and those who carried the 6 A allele had lower level (p = 0.04). After applied multivariable Cox-hazard models we revealed that the 6 A allele is independently associated to the disease complication. Kaplan–Meier survival test revealed that individuals having the 6 A allele had a lower survival rate than those with the 5 A allele (p = 0.04). Conclusion Our study suggests the disruption of the MMP-3 level may be due to the existence of the polymorphism − 1612 residing in its promoter region. MMP-3 can be considered as a marker of diagnosis and prediction in cardiovascular events.
 
Receiver operating characteristics (ROC) curve for serum vitamin D in differentiating children with ASD from healthy controls
Article
Objective This study aimed to examine the correlation between polymorphisms in vitamin D receptor (VDR) gene and serum vitamin D, and to determine their role in predicting childhood Autism Spectrum Disorder (ASD). Methods Children with ASD and age- and gender- matched healthy controls were recruited from the Chinese Han population. Their serum 25(OH) vitamin D was measured using competitive chemiluminescent immunoassays. The TaqMan probe approach was applied to analyze the common VDR SNPs rs731236 (Taq1), rs11568820 (Cdx2), rs1544410 (BsmI), and rs228570 (FokI). Both linear and logistic regressions were applied in data analysis. Results A total of 269 children with ASD and 320 healthy controls were recruited. Children with ASD had significantly lower levels of serum vitamin D and a significantly higher rate of vitamin D deficiency (< 20 ng/ml) compared to healthy controls (67.7% vs 34.1%). All these examined VDR SNPs were not correlated with serum vitamin D concentrations or vitamin D deficiency. Logistic regression analysis revealed that rs731236 and serum vitamin D were associated with childhood ASD. The area under the receiver operating characteristic (ROC) curve was 0.7285 for serum vitamin D. Children with both T/C genotype of rs731236 and vitamin D deficiency had a higher risk of being diagnosed with ASD. Conclusion All examined common VDR SNPs are not correlated with serum vitamin D concentrations or vitamin D deficiency. The combination of T/C phenotype of rs731236 and vitamin D deficiency are associated with a higher risk of childhood ASD. Vitamin D is a promising target in the prevention and treatment of this disease.
 
Article
Background Hyperhomocysteinemia (HHcy) is a common complication in Chinese hypertensive patients and associated with methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism, folate, and vitamin B12 (Vit B12) status. This study evaluated the associations of MTHFR C677T polymorphism, folate, and Vit B12 with H-type hypertension. Methods and results 887 eligible patients with essential hypertension were included. Patients were divided into two groups according to the Hcy level, the H-type hypertension group and the normal hypertension group. Related risk factors such as MTHFR polymorphism, folate and Vit B12 status were analyzed in the two groups. Age, gender, SBP, DBP, MTHFR C677T genotype, folate and Vit B12 differed significantly between H-type hypertension and normal hypertension groups (P < 0.05). MTHFR 677TT variant, gender, folate, and Vit B12 were independent risk factors for the occurrence of H-type hypertension. The risk for TT carriers was 8 times higher than that of CC and CT carriers [OR (95% CI) 8.248 (5.274–12.899)]. Male patients had almost fivefold higher odds than female patients [OR (95% CI) 4.923 (2.741–8.842)]. Folate level of patients with H-type hypertension decreased with the C to T substitution of MTFHR C677T gene (P < 0.05), while Vit B12 level was not associated with the gene (P > 0.05). Conclusions MTHFR 677TT variant, gender, folate, and Vit B12 were risk factors for the occurrence of H-type hypertension. Folate but not Vit B12 was associated with MTFHR C677T polymorphism in patients with H-type hypertension. Accordingly, the above factors may be considered in the prevention and treatment of hypertension.
 
Generation of VASN−/− mice using CRISPR-Cas9 genome editing. a Schematic diagram showing the genomic structure of mouse VASN with two exons. The site of genome editing is marked. The KO site is in the second exon of the VASN gene. b Electrophoresis and DNA sequence analysis were performed to identify the genotypes of the mice. c qRT-PCR analysis showed that the VASN mRNA levels in the lungs of VASN−/− mice lower than VASN+/+ mice. The 18S rRNA gene was used as an endogenous control. The data are shown as the mean ± SD, *p < 0.05 and **p < 0.01 (n = 6 per group). d Western blot analysis showed that the VASN protein levels in the lungs of VASN−/− mice lower than VASN+/+ mice
VASN knockout resulted in lung injury in mice. a Wet weight of lung tissue in VASN−/− mice and VASN+/+ mice. b–e (10X) and b′–e′ (40X): H&E staining of the lung area in VASN+/+ and VASN−/− mice at 21 days. b and b′: Thrombosis and inflammatory infiltration increased. c and c′: Haemorrhage occurred. d and d′: The septum widened, and alveoli dilated. e and e′: Normal lung. f–o: Ultrastructure of pulmonary cells of VASN+/+ and VASN−/− mice at 21 days. f: In VASN+/+ mice, alveolar cells were basically normal (1 µm). g–l: Type II alveolar cells from VASN−/− mice. g: Cell shrinkage, nuclear shrinkage, nuclear condensation, cell membrane damage and fragmentation (1 µm). h and i: Mitochondrial oedema, heterochromatin edge aggregation and concentration, basement membrane thickening, increases in lysosomes, increases in lipid droplets, reductions in cytoplasm, increases in endoplasmic reticulum, and apoptosis (500 nm). j Increases in lysosomes and apoptosis (500 nm). k Vacuolization, increases in lysosomes, and apoptosis (500 nm). l Apoptosis (500 nm). m Type I alveolar cells (500 nm) of VASN−/− mice; irregular cells with serrated edges (500 nm). n and o Vascular endothelial cells from VASN.−/− mice; cell variants with discontinuous membranes forming a rough vascular wall are shown (500 nm)
VASN knockout induces lung injury by upregulating FABP4 expression. a Volcano map of 159 upregulated and 53 downregulated genes in the lungs of 21-day-old VASN−/− mice compared to VASN+/+ mice. b Quantitative verification of the relative expression of enriched genes. c qRT-PCR analysis showed that FABP4 gene mRNA levels in the lungs of VASN−/− mice were higher than VASN± and VASN+/+ mice. The 18S rRNA gene was used as an endogenous control. The data are shown as the mean ± SD. *p < 0.05 and **p < 0.01(n = 6 per group). d Western blot analysis showed that FABP4 protein levels in the lungs of VASN−/− mice were higher than VASN± and VASN+/+ mice. e: Immunohistochemical analysis showed that the FABP4 protein levels in the lungs of VASN−/− mice were higher than VASN± and VASN+/+ mice
VASN knockout induces lung injury by mediating inflammation via FABP4. a qRT-PCR analysis showed that the TNF-α, IL-1β, and IL-6 mRNA levels in the lungs of VASN−/− mice were higher than VASN+/+ mice. The 18S rRNA gene was used as an endogenous control. The data are shown as the mean ± SD, *p < 0.05 and **p < 0.01 (n = 6 per group). b Western blot analysis showed that TNF-α, IL-1β, and IL-6 protein levels in the lungs of VASN−/− mice were higher than VASN+/+ mice. c Immunohistochemical analysis showed that TNF-α, IL-1β, and IL-6 protein levels in the lungs of VASN−/− mice were higher than VASN+/+ mice. (10X and 40X)
A schematic diagram showing the molecular signalling pathway by which VASN regulates lung injury
Article
Background Lung injury caused by pulmonary inflammation is one of the main manifestations of respiratory diseases. Vasorin (VASN) is a cell-surface glycoprotein encoded by the VASN gene and is expressed in the lungs of developing mouse foetuses. Previous research has revealed that VASN is associated with many diseases. However, its exact function in the lungs and the underlying mechanism remain poorly understood. Methods and results To investigate the molecular mechanisms involved in lung disease caused by VASN deficiency, a VASN gene knockout (VASN−/−) model was established. The pathological changes in the lungs of VASN−/− mice were similar to those in a lung injury experimental mouse model. We further analysed the transcriptomes of the lungs of VASN−/− mice and wild-type mice. Genes in twenty-four signalling pathways were enriched in the lungs of VASN−/− mice, among which PPAR signalling pathway genes (3 genes, FABP4, Plin1, AdipoQ, were upregulated, while apoA5 was downregulated) were found to be closely related to lung injury. The most significantly changed lung injury-related gene, FABP4, was selected for further verification. The mRNA and protein levels of FABP4 were significantly increased in the lungs of VASN−/− mice, as were the mRNA and protein levels of the inflammatory factors IL-6, TNF-α and IL-1β. Conclusions We believe that these data provide molecular evidence for the regulatory role of VASN in inflammation in the context of lung injury.
 
Exosome-free FBS accelerates heat stress-induced apoptosis in BMECs. A BMECs activity detected using the CCK-8 kit under heat stress. B, C mRNA levels of Bax and HSP70 in BMECs detected using RT-qPCR. D–F Protein expression levels of Bax and HSP70 in BMECs examined using western blot
Exosome-free FBS facilitates heat stress-induced oxidative stress in BMECs. A, B Reactive oxygen species (ROS) level was determined using DCFH-DA kit cultured after treatment of cells with exosome-free serum under heat stress. C The SOD2 content was determined using a manganese superoxide dismutase assay kit. Data are presented as the mean ± SEM (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001
Exosome-free serum increases heat stress-induced mitochondrial damage in BMECs. A, B Mitochondrial membrane potential examined using JC-1 kit. C–E The protein expression levels of Drp1, p-Drp1, and Fis1 determined using western blot. Data are presented as the mean ± SEM (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001
Exosome-free serum suppresses milk protein synthesis in BMECs. A-C The mRNA levels of SREBP1, ELF5, and CSN2 detected using RT-qPCR. D-G Western Blot used to detect the protein expression levels of SREBP1, ELF5 and CSN2 in BMECs. Data are presented as the mean ± SEM (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001
Exosome-free serum inhibits milk fat synthesis in BMECs. A Triglyceride (TG) content in BMECs determined using the triglyceride detection kit. B, C mRNA levels of mTOR and ADD1 detected using RT-qPCR. D–G Western blot used to detect the protein expression levels of mTOR, PPARγ, p-mTOR and ADD1 in BMECs. Data are presented as the mean ± SEM (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001
Article
Background Exosomes are involved in intercellular communication, affecting many physiological and pathological process. The present study evaluated the effects of serum exosomes on the function of bovine mammary epithelial cells (BMECs) and milk synthesis under heat stress. Methods and results We cultured the BMECs in fetal bovine serum (FBS) or exosome-free FBS medium and examined, their viability using CCK-8 kit. The results showed that culturing the cells in an exosome-free medium decreased viability and increased the levels of reactive oxygen species. The BMECs cultured in the exosome-free medium had reduced mitochondrial membrane potential, decreased manganese superoxide dismutase activity, and disrupted mitochondrial dynamics. They exhibited apoptosis due to upregulated Drp1, Fis1, Bax and HSP70. Lastly, we observed downregulation of milk fat and lactoprotein-related genes: mTOR, PPARγ, p-mTOR and ADD1 and SREBP1, ELF5, and CSN2, respectively, after culturing the cells in an exosome-free medium. These negative effects of the exosome-free medium on the BMECs could be further reinforced under heat stress. Conclusion Our results demonstrated that exosomes from serum are critical for maintaining the normal function of BMECs.
 
Schematic representation of modulatory effect of berberine on plasma Lipid metabolic pathways. Details of the processes and abbreviations are explained in the text
Article
Berberine is a bioactive isoquinoline alkaloid compound extracted from various medicinal plants, such as Barberry. Berberine shows various pharmacological properties that are mainly attributed to its anti-inflammatory and antioxidant effects. A growing body of evidence has shown that berberine influences cholesterol metabolism, and consequently, may ameliorate dyslipidemias and atherosclerosis. Plasma high-density lipoprotein cholesterol (HDL-C) is known to have an independent negative association with incident cardiovascular disease (CVD). However, several outcomes trials and genetic studies have failed to meet expecting the beneficial effects of elevating plasma HDL-C concentrations. Hence, investigations are currently focused on enhancing the functionality of HDL particles, independent of their plasma concentrations. HDL particles show various qualities because of a heterogeneous composition. Consistent with complex metabolism and composition, various biological functions are found for HDL, such as anti-inflammatory, antioxidant, anti-apoptotic, and anti-thrombotic activities. Protective effects of berberine may impact the functionality of HDL; therefore, the present literature review was intended to determine whether berberine can amplify HDL function. It was concluded that berberine may regulate markers of HDL activity, such as apo-AI, cholesterol efflux, LCAT, PON1, and S1P activities and levels. Consequently, berberine may recuperate conditions with dysfunctional HDL and, therefore, have the potential to emerge as a therapeutic agent. However, further human trials of berberine are warranted to evaluate its impact on HDL function and cholesterol metabolism.
 
Illustration of an overview of As uptake, transport and detoxification in rice
The figure is a generalized representation of known/presumed proteins, and processes involved in transport and detoxification of inorganic As. Arsenate (AsV) enters in roots via phosphate transporters (PHT) and arsenite (AsIII) enters via nodulin 26-like intrinsic proteins (NIP). Once entered, AsV is reduced to AsIII by the action of AsV reductase (HACs) by using glutathione (GSH) as a reductant. AsIII then forms complexes with GSH, phytochelatins (PCs) metallothionein (MTs) prior to sequestration in vacuoles. These complexes get transported via ATP binding cassette transporters (OsABCC1 in rice) and are sequestered by vacuoles. The OsABCC1 transporters are expressed in leaves also. The detoxification mechanism consequently predominates cellular As in the form of AsIII. AsIII is then effluxes by Lsi2 (an AsIII /H + antiporter) and NRAMP (Natural resistance-associated macrophage protein) into xylem, thus responsible for loading AsIII in xylem. The role of phloem in As movement from shoots to roots remains to be confirmed. Also, the transporters that are responsible for grain and phloem loading of AsIII in rice are still needed to be revealed
GSSG represents oxidized form of glutathione. M represents maternal tissue and F is filial tissue
Simplified model illustrating the role of WRKY TFs under As stress in rice
The figure provides a simplified model illustrating the role of WRKY TFs under As stress in rice by initiating signaling mechanisms that in a way respond to As and possibly minimize the As accumulation in root, shoot or grain, providing tolerance or having low As content that would be beneficial for the human health as well as agricultural yield. OsWRKY28 is involved in AsV accumulation. The mutation in OsWRKY28, results in slowing the rate of arsenate (AsV) accumulation in shoots of rice plant. OsWRKY28 regulates AsV concentration possibly by affecting Jasmonic acid (JA) signaling or other phytohormones. Red arrow indicates decreased accumulation
Article
Arsenic (As) is a global carcinogenic contaminant, and is one of the significant environmental constraints that limits the development and yield of crop plants. It is always tagged along with rice as rice takes up As and tends to accumulate it in grains. This amassment makes a way for As to get into the food chain that leads to unforeseen human health risks. Being viewed as parallel with toxicity, As in rice is an important global risk that calls for an urgent solution. WRKY Transcription Factors (TFs) seems to be promising in this area. The classical and substantial progress in the molecular mechanism of WRKY TFs, strengthened the understanding of innovative solutions for dealing with As in rice. Here, we review the potential of WRKY TFs under As stressed rice as a genetic solution and also provide insights into As and rice. Further, we develop an understanding of WRKY TF gene family and its regulation in rice. To date, studies on the role of WRKY TFs under As stressed rice are lacking. This area needs to be explored more so that this gene family can be utilized as an effective genetic tool that can break the As cycle to develop low or As free rice cultivar.
 
Methylation levels of patients and healthy controls
Article
Background Coronary artery disease (CAD) is a complex disease that is influenced by environmental and genetic factors. Lipid levels are regarded as a major risk factor for CAD, and epigenetic mechanisms might be involved in the regulation of CAD development. This study was designed to investigate the association between the DNA methylation status of 8 lipid metabolism-related genes and the risk of CAD in the Chinese Han population. Methods A total of 260 individuals were sampled in this study, including 120 CAD cases and 140 normal healthy controls. DNA methylation status was tested via targeted bisulfite sequencing. Results The results indicated a significant association between hypomethylation of the APOC3, CETP and APOC1 gene promoters and the risk of CAD. Individuals with higher methylation levels of the APOA5 and LIPC gene promoters had increased risks for CAD. In addition, ANGPTL4 methylation level was significantly associated with CAD in males but not females. There were no significant differences in the methylation levels of the APOB and PCSK9 gene promoters between CAD patients and controls. Conclusions The methylation status of the APOC3, APOA5, LIPC, CETP and APOC1 gene promoters may be associated with the development of CAD.
 
Article
Background: RNA-binding protein with serine-rich domain 1 (RNPS1) is a member of a splicing-dependent mega Dalton protein complex or exon junction complex (EJC). During splicing, RNPS1 acts as a protector of global transcriptome integrity by suppressing the usage of cryptic splice sites. Additionally, RNPS1 functions in almost all stages of mRNA metabolism, including constitutive splicing, alternative splicing, translation and nonsense-mediated mRNA decay (NMD). The aim of the present study was to generate a highly specific polyclonal antibody against human RNPS1. Methods and results: A plasmid, pHis-TEV-RNPS1, has been constructed to overexpress recombinant RNPS1 (22-305 amino acids) by cloning the nucleotide sequence downstream of an N-terminal His-tag in the parent plasmid pHis-TEV. The recombinant plasmid was then transformed into Rosetta and expression was induced using IPTG. The His-tagged RNPS1 protein was purified using Ni-NTA affinity chromatography. The rabbit antiserum was then obtained by immunizing rabbits with the purified recombinant RNPS1 protein. The antiserum was further purified by antigen-immunoaffinity chromatography. The sensitivity and the specificity of the polyclonal antibody were assessed by enzyme-linked immunosorbent assay (ELISA) and knockdown assay. ELISA demonstrated that the antibody has a high binding affinity for RNPS1 and the usable titre is 1:2000. Conclusion: The antibody detected RNPS1 in human, mouse cell lines and rat tissue in Western blot. Importantly, the antibody efficiently detected the decrease in RNPS1 expression in siRNA induced knockdown assay, indicating the specificity of the antibody. The polyclonal antibody against RNPS1 will be a useful tool for performing further functional studies on RNPS1.
 
The population structure of rice diversity panel of 100 rice genotypes using 204 SSR marker data. (a) Peak value observed by ?Structure Harvester v 0.6.93? indicates the maximum value of K=4. (b)Population structure of rice diversity panel based on Bayesian method analyzed with 204 polymorphic SSRs detecting 4 groups. The four different colors represent the four different groups or subpopulations showing the similar genetic background
The UPGAMA DARWIN tree displaying the distribution of rice genotypes in four groups, and presenting the genetic similarities and dissimilarities within and between groups
Linkage disequilibrium (LD) patterns among 100 accessions genotyped with 204 SSR markers. The squared correlation coefficients (r2) for each pair of markers are presented in the upper triangle and their corresponding tests in the lower triangle: As shown in the LD patterns display diagram, P<0.01 for blue, P<0.001 for green, P<0.0001 for red. R2 indicates the percentage of total variation explained. The values of r2 range from 0.00-1.00 represented by different colors
Article
Background Rice (Oryza sativa L.) is one of the staple foods worldwide. To feed the growing population, the improvement of rice cultivars is important. To make the improvement in the rice breeding program, it is imperative to understand the similarities and differences of the existing rice accessions to find out the genetic diversity. Previous studies demonstrated the existence of abundant elite genes in rice landraces. A genome-wide association study (GWAS) was performed for yield and yield related traits to find the genetic diversity. Design Experimental study. Methods and results A total of 204 SSRs markers were used among 17 SSRs found to be located on each chromosome in the rice genome. The diversity was analyzed using different genetic characters i.e., the total number of alleles (TNA), polymorphic information content (PIC), and gene diversity by Power markers, and the values for each genetic character per marker ranged from 2 to 9, 0.332 to 0.887 and 0.423 to 0.900 respectively across the whole genome. The results of population structure identified four main groups. MTA identified several markers associated with many agronomically important traits. These results will be very useful for the selection of potential parents, recombinants, and MTAs that govern the improvements and developments of new high yielding rice varieties. Conclusions Analysis of diversity in germplasm is important for the improvement of cultivars in the breeding program. In the present study, the diversity was analyzed with different methods and found that enormous diversity was present in the studied rice germplasm. The structure analysis found the presence of 4 genetic groups in the existing germplasm. A total of 129 marker-trait associations (MTAs) have been found in this study.
 
Macroscopic appearance of colonic tissues
Histopathological appearances of the colons between the groups (A-E). (A) normal histology in Control group, (B) almost normal appearance in AA + TH treated group, (C) severe hyperemia and slight hemorrhages in AA group, (D) reduced histopathological lesions in AA + SS treated group, (E) normal histology in TH group, HE, Bars = 50 μm
NF-kB expressions in the group (F-J). (F) Slight expression in Control group, (G) almost normal expression in AA + TH treated group, (H) marked increases in expression in AA group, (I) slight decrease in AA + SS treated group, (J) very slight expression in TH group, Streptavidin Biotin Peroxidase method, Bars = 50 μm
SAA expressions in the group (K-O). (K) Slight expression in Control group, (L) almost normal expression in AA + TH treated group, (M) marked increases in expression in AA group, (N) slight decrease in AA + SS treated group, (O) very slight expression in TH group, Streptavidin Biotin Peroxidase method, Bars = 50 μm
Histopathological and immunohistochemical results
AA: Acetic acid; TH: Theranekron; SS: Sulfasalazine; Nf-kB: Nuclear factor kappa beta; SAA: Serum amyloidA
CRP expressions in the group (A-E). (A) slight expression in Control group, (B) almost normal expression in AA + TH treated group, (C) severe increases in AA group, (D) reduced expression in AA + SS treated group, (E) very slight expression in TH group, Streptavidin Biotin Peroxidase method, Bars = 50 μm
GRO expressions in the group (F-J). (F) Slight expression in Control group, (G) almost normal expression in AA + TH treated group, (H) marked increases in expression in AA group, (I) slight decrease in AA + SS treated group, (J) very slight expression in TH group, Streptavidin Biotin Peroxidase method, Bars = 50 μm
OPN expressions in the group (K-O). (K) Slight expression in Control group, (L) almost normal expression in AA + TH treated group, (M) marked increases in expression in AA group, (N) slight decrease in AA + SS treated group, (O) very slight expression in TH group, Streptavidin Biotin Peroxidase method, Bars = 50 μm
Immunohistochemical results
AA: Acetic acid; TH: Theranekron; SS: Sulfasalazine;; CRP: C reactive protein; GRO: Growth-related oncogene OPN: Osteopontin
Article
Background Inflammatory bowel disease (IBD) is characterized with chronic inflammation of gastrointestinal track. In the pathogenesis of IBD, inflammation is the main mechanism. Induction of inflammation triggers the oxidative stress that subsequently leading to apoptosis. Considering the all pathological mechanisms, many therapeutic agents have been used for IBD but because of serious side effects there is still a need for new therapeutic drugs. In this study, we aim to evaluate the possible protective effects of Theranekron (TH) on acetic acid (AA)- induced colonic damage and to describe the probable effect mechanisms of TH. Materials and results Fourty female adult Wistar albino rats were divided into 5 groups. Following 24 h fasting, colitis was induced by rectal instillation of AA. In TH group, a single dose of subcutaneous 0.2 ml TH was used. In treatment groups, 0.2 ml TH single dose or 100 mg/kg sulfasalazine (SS) for 7 days were used after colitis induction. Normal salin was used for all applications in control group. Histopathologically hemorrhage, edema and inflammatory reactions were seen in AA group. TH and SS decreased the severity of lesions. Nuclear factor kappa B, Serum amyloid A, C-reactive protein, Growth-related oncogene, and Osteopontin expressions were markedly increased in AA group and TH markedly reduced these expressions. In Western analysis, decreased NF-kB and caspase-3 levels were observed with TH. Oxidative markers did not changed significantly. Conclusions TH has a prominent anti-inflammatory effect on AA-induced colonic inflammation via NF-kB signaling whereas antiapoptic effects seem to be independent from this pathway.
 
Article
Background Most of the bioactive peptides exhibit antioxidant effect and do elicit inhibitory effect on proliferation of cancer cells. This study investigates the in-vitro antioxidant and anti-cancer properties of NV14 peptide, derived from serine O-acetyltransferase (SAT) of spirulina, Arthrospira platensis. Methods The anti-cancer effect of the peptide was evaluated using human adenocarcinoma epithelial cells (MCF-7), while the anti-oxidant potential, as in reduction in ROS concentration, has been established using the H2O2-exposed, Madin-Darby canine kidney (MDCK) cells. The outcome of the in vitro analyses has been evaluated by in silico molecular docking analyses. Results The peptide, dose-dependently, reduced oxidative stress as well as cell proliferation. Besides, based on the binding scores between NV14 peptide and the important proteins associated with apoptosis and antioxidant defense, it is evident that the peptide has antioxidant and anti-cancer effect, in vitro. Conclusions Together, this study demonstrates that NV14 has a potent antioxidant and anti-cancer capability; however, further direction needs to be focused on clinical or pharmacodynamics aspects.
 
The image depicts the in silico analysis of alterations in tenascin-C (TNC) expression in serum exosomes and during endothelial cell differentiation.A) The image illustrates the upregulation of TNC in the serum of patients with coronary artery disease (CAD) by comparison with individuals without CAD. B) The image demonstrates the receiver-operator characteristic (ROC) curve analysis for TNC expression in the bioinformatics data of the study population (area under the ROC curve, 0.805; P = 0.078). C) The expression pattern of TNC in the course of endothelial cell transitions is illustrated herein. The image shows human embryonic stem cells (Day0), mesoderm committed cells (Day2), early vascular progenitor cells (Day4), late vascular progenitor cells (Day8), endothelial cells, and human umbilical vein endothelial cells (HUVECs) as endothelial control cells
The image presents TNC expression levels in the serum samples of 40 patients with coronary artery disease (CAD) and 20 individuals without CAD. A) The expression level of Tenascin-C (TNC) significantly rose in the CAD samples compared with the non-CAD controls (P = 0.010). B) The image illustrates the receiver-operator characteristic (ROC) curves analysis for TNC expression in the CAD and non-CAD samples. TNC expression significantly discriminated CAD from non-CAD samples (area under the ROC curve, 0.0.744; P = 0.011)
Article
Background Coronary artery disease (CAD), is the leading cause of mortality and morbidity worldwide. Tenascin-C (TNC) with high expression levels in inflammatory and cardiovascular diseases, leads to the rupture of atherosclerotic plaques. The origin of plaque destabilization can be associated to endothelial dysfunction. Given the high prevalence of CAD, finding valuable biomarkers for its early detection is of great interest. Using serum samples from patients with CAD and individuals without CAD, we assessed the efficacy of TNC expression levels in serum exosomes and during endothelial cell differentiation as a noninvasive biomarker of CAD. Methods TNC expression was analyzed using the RNA-sequencing data sets of 6 CAD and 6 normal samples of blood exosomes and endothelial differentiation transitions. Additionally, TNC expression was investigated in the serum samples of patients with CAD and individuals without CAD via qRT-PCR. ROC curve analysis was employed to test the suitability of TNC expression alterations as a CAD biomarker. Results TNC exhibited higher expression in the exosomes of the CAD samples than in those of the non-CAD samples. During endothelial differentiation, TNC expression was upregulated and then consistently downregulated in mature endothelial cells. Moreover, TNC was significantly upregulated in the serum of the CAD group (P = 0.02), with an AUC of 0.744 for the expression level (95% confidence interval, 0.582 to 0.907; P = 0.011). Hence its expression level can be discriminated CAD from non-CAD samples. Discussion Our study is the first to confirm that altered TNC expression is associated with pathological CAD conditions in Iranian patients. The expression of TNC is involved in endothelial differentiation and CAD development. Accordingly, TNC can serve as a valuable noninvasive biomarker with potential application in CAD diagnosis.
 
Article
Background: The genus Trichoglossus belongs to the family Psittacidae and includes fourteen species distributed worldwide. According to the International Union for Conservation of Nature and Natural Resources (IUCN) Red List of Threatened Species, most Trichoglossus species have shown a decreasing population trend recently. In particular, Trichoglossus forsteni is listed as "Endangered" in the IUCN Red List of Threatened Species. Moreover, Trichoglossus haematodus and Trichoglossus moluccanus are one of the most traded and illegally traded parrots. However, only a few genetic studies have been conducted regarding the conservation of this genus. Methods and results: In the present study, complete mitochondrial genomes of three species (T. forsteni, T. haematodus, and T. moluccanus) were sequenced and compared with Trichoglossus rubritorquis, species whose mitochondrial genome is already reported. Results indicate that the complete mitochondrial genomes of the three species were similar in length (17,906 bp for T. haematodus to 17,909 bp for T. forsteni). Furthermore, the organization and order of these three mitochondrial genomes were identical, including thirteen protein-coding genes (PCGs), two ribosomal RNA genes, 22 transfer RNA genes, and two control regions (CRs) categorized into three domains containing nine conserved motifs. In addition, the genus Trichoglossus formed a well-supported monophyletic lineage. Conclusions: The results of this study may be useful for future genetic studies toward the conservation of the genus Trichoglossus.
 
The figure depicts various mechanisms exhibited by icariin via the PI3K-AKT pathway
The figure depicts various mechanisms exhibited by icariin via the Nrf-2 pathway
Article
Icariin is a primary active component of the traditional Chinese medicinal plant Epimedium grandiflorum. A range of pharmacological effects of icariin has been researched by modern science to explain its traditional medicinal uses. Attributing to the wide range of pharmacological properties like anti-osteoporosis, anti-inflammation, anti-oxidative stress, anti-depression, and anti-tumor property possessed by icariin, it is now being considered a potential therapeutic agent for a wide variety of disorders ranging from neoplasm, neurodegenerative disorders, osteoporosis, and cardiovascular diseases. Various signaling pathways including NFκB/NALP3, IGF-1, MiR-223-3p/ NALP3, TLR4/ NFκB, and WNT1/β-catenin are involved in the different biological actions exerted by icariin. Apart from these pathways, PI3K-AKT (Phosphoinositide 3 kinase-Protein kinase B) and Nrf-2 (nuclear erythroid 2-related factor 2) signaling pathways are two important pathways that form the fundamental basis for the pharmaceutical efficacy of icariin. This review gives an overview of previous in vitro and in vivo studies that investigated the potential role of icariin via PI3K-AKT and Nrf-2 signaling pathways to provide greater insights into its potential clinical use in a variety of disorders.
 
Determination of in vitro effect of HCM on cell proliferation and mRNA expression. (A) The process of attaining HCM which includes 24-h hypoxic preconditioning. (B) Cell viability assay showing that the HCM-treated group exhibited the highest cell viability in the in vitro toxic hepatitis model. (C) Realtime PCR demonstrating the effects of HCM and NCM on the mRNA expression of apoptosis-related and autophagy-related markers. Control conditioned medium, NCM, and HCM were reacted with the control mouse AML12 hepatocytes and thioacetamide (TAA)-treated AML12 hepatocytes, respectively. Twenty-four hours after treatment, the mRNA expression of a pro-apoptotic marker (Bim), an anti-apoptotic marker (Mcl-1), and autophagy-related markers (LC3B and p62) was compared. Values are presented as mean ± standard deviation of three independent experiments. * P < 0.05. Abbreviations: Bim, Bcl-2-like protein 11; Ct, control; NCM, ASC-secretome obtained under normoxia; HCM, ASC-secretome obtained under hypoxic preconditioning; LC3B, microtubule-associated proteins 1 A/1B light chain 3B; Mcl-1, myeloid cell leukemia 1; TAA, thioacetamide
Determination of in vitro effect of HCM on apoptosis and autophagy. (A) Western blot analysis demonstrating the in vitro effect of HCM on the protein expression of apoptosis-[Left] and autophagy-related [Right] markers. The relative densities of individual markers were quantified using Image J and then normalized to the density of β-actin in each group. (B) [Left] GFP-LC3 puncta formation assay showing the HCM-treated group exhibiting significantly higher accumulation of GFP-LC3 puncta formation than that of controls. [Right] Percentages of GFP-LC3 puncta formation were measured using Image J and expressed as relative values to those in normal livers. (C) [Left] Monodansylcadaverine (MDC) staining showing that the HCM-treated group had a significantly higher number of MDC-positive autophagic vacuoles than that of controls. [Right] Percentages of immunoreactive areas were measured using Image J and expressed as relative values to those in normal livers. Values are presented as mean ± standard deviation of three independent experiments. * P < 0.05. Abbreviations: Bak, Bcl2-antagonist/killer 1; Bim, Bcl-2-like protein 11; c-Cas3, cleaved caspase-3; Ct, control; Mcl-1, myeloid cell leukemia 1; NCM, ASC-secretome obtained under normoxia; HCM, ASC-secretome obtained under hypoxic preconditioning; TAA, thioacetamide
Autophagy blockage by bafilomycin A1 for the determination of the role of autophagy. (A) [Left] GFP-LC3 puncta formation assay demonstrating successful autophagic inhibition by bafilomycin A1. [Right] Percentages of GFP-LC3 puncta formation were measured using Image J and expressed as relative values to those in normal livers. (B) [Left] Monodansylcadaverine (MDC) staining demonstrating successful autophagic inhibition by bafilomycin A1. [Right] Percentages of immunoreactive areas were measured using Image J and expressed as relative values to those in normal livers. (C) Western blot analysis with/without autophagy inhibition by bafilomycin A1. Autophagy blockage by bafilomycin-A1 significantly abrogated the anti-apoptotic effect of HCM. The relative densities of individual markers were quantified using Image J and then normalized to the density of β-actin in each group. Values are presented as mean ± standard deviation of three independent experiments. * P < 0.05. Abbreviations: BafA1, bafilomycin A1; Bim, Bcl-2-like protein 11; c-Cas3, cleaved caspase-3; Ct, control; Mcl-1, myeloid cell leukemia 1; NCM, ASC-secretome obtained under normoxia; HCM, ASC-secretome obtained under hypoxic preconditioning; TAA, thioacetamide
Autophagy blockage by ATG5 siRNA for the determination of the role of autophagy. (A) [Left] Monodansylcadaverine (MDC) staining demonstrating successful autophagic inhibition by ATG5 siRNA. [Right] Percentages of immunoreactive areas were measured using Image J and expressed as relative values to those in normal livers. (B) Western blot analysis with/without autophagy inhibition by ATG5 siRNA. Autophagy blockage by ATG5 siRNA significantly abrogated the anti-apoptotic effect of HCM. The relative densities of individual markers were quantified using Image J and then normalized to the density of β-actin in each group. Values are presented as mean ± standard deviation of three independent experiments. * P < 0.05. Abbreviations: ATG5, autophagy-related protein 5; Bim, Bcl-2-like protein 11; Ct, control; NCM, ASC-secretome obtained under normoxia; HCM, ASC-secretome obtained under hypoxic preconditioning; TAA, thioacetamide
Determination of in vivo effect of HCM on apoptosis and autophagy. (A) Design of animal model which includes four groups: sham, saline, NCM, and HCM groups. (B) Western blot analysis demonstrating the in vivo effect of HCM on the protein expression of apoptosis-[Left] and autophagy-related [Right] markers. The relative densities of individual markers were quantified using Image J and then normalized to the density of β-actin in each group. (C) [Left] GFP-LC3 puncta formation assay showing the HCM-treated group exhibiting significantly higher accumulation of GFP-LC3 puncta formation than controls. [Right] Percentages of GFP-LC3 puncta formation were measured using Image J and expressed as relative values to those in normal livers. (D) [Left] P62 staining showing that the HCM-treated group had a significantly higher number of p62-positive autophagic vacuoles than controls. [Right] Percentages of immunoreactive areas were measured using Image J and expressed as relative values to those in normal livers. Values are presented as mean ± standard deviation of three independent experiments. * P < 0.05. Abbreviations: Bim, Bcl-2-like protein 11; c-Cas3, cleaved caspase-3; Ct, control; Mcl-1, myeloid cell leukemia 1; NCM, ASC-secretome obtained under normoxia; HCM, ASC-secretome obtained under hypoxic preconditioning; PH, partial hepatectomy
Article
Background Hypoxic preconditioning (HP) is a stem cell preconditioning modality designed to augment the therapeutic effects of mesenchymal stem cells (MSCs). Although autophagy is expected to play a role in HP, very little is known regarding the relationship between HP and autophagy. Methods and results The adipose-derived stem cell (ASC)-secretome obtained under normoxia (NCM) and ASC-secretome obtained under HP (HCM) were obtained by culturing ASCs for 24 h under normoxic (21% partial pressure of O2) and hypoxic (1% partial pressure of O2) conditions, respectively. Subsequently, to determine the in vivo effects of HCM, each secretome was injected into 70% partially hepatectomized mice, and liver specimens were obtained. HCM significantly reduced the apoptosis of thioacetamide-treated AML12 hepatocytes and promoted the autophagic processes of the cells (P < 0.05). Autophagy blockage by either bafilomycin A1 or ATG5 siRNA significantly abrogated the anti-apoptotic effect of HCM (P < 0.05), demonstrating that HCM exerts its anti-apoptotic effect by promoting autophagy. The effect of HCM — reduction of cell apoptosis and promotion of autophagic process — was also demonstrated in a mouse model. Conclusions HP appears to induce ASCs to release a secretome with enhanced anti-apoptotic effects by promoting the autophagic process of ASCs.
 
An outline of speed breeding protocol and its implication for accelerating breeding cycles for improving growth and yield as compared to the conventional breeding approach under regular photoperiod
An overview of the potential application of artificial intelligence in augmenting plant breeding technology for easy, precise, and early prediction of genotypes/parental combinations for varietal development
Article
Abstract Introduction In climate change, breeding crop plants with improved productivity, sustainability, and adaptability has become a daunting challenge to ensure global food security for the ever-growing global population. Correspondingly, climate-smart crops are also the need to regulate biomass production, which is imperative for the maintenance of ecosystem services world�wide. Since conventional breeding technologies for crop improvement are limited, time-consuming, and involve laborious selection processes to foster new and improved crop varieties. An urgent need is to accelerate the plant breeding cycle using artifcial intelligence (AI) to depict plant responses to environmental perturbations in real-time. Materials and methods The review is a collection of authorized information from various sources such as journals, books, book chapters, technical bulletins, conference papers, and verifed online contents. Conclusions Speed breeding has emerged as an essential strategy for accelerating the breeding cycles of crop plants by growing them under artifcial light and temperature conditions. Furthermore, speed breeding can also integrate marker�assisted selection and cutting-edged gene-editing tools for early selection and manipulation of essential crops with supe�rior agronomic traits. Scientists have recently applied next-generation AI to delve deeper into the complex biological and molecular mechanisms that govern plant functions under environmental cues. In addition, AIs can integrate, assimilate, and analyze complex OMICS data sets, an essential prerequisite for successful speed breeding protocol implementation to breed crop plants with superior yield and adaptability
 
Some pathogenic mechanisms are involved in BC occurrence and progression
The dual role of umbilical cord-derived mesenchymal stem cells against breast cancer
Article
Breast cancer (BC), as the most common cancer among women, afects a great number of subjects around the world. This heterogenic disease is divided into several types and subtypes, and each subtype has various phenotypes and genotypes. Against BC, several options have been proposed, such as surgery, radiotherapy, and chemotherapeutic agents. However, these approaches may have detrimental efects on health and life quality of patients. Hence, harnessing a therapeutic tool with high efectiveness and low side efects is required. Recently, mesenchymal stem cells (MSCs) have created a new window to treat various disorders, like cancer, and among these, umbilical cord (UC)-derived MSCs have acquired much interest due to their advantages. Therefore, in this narrative review, the infuences of UC-derived MSCs on BC were reviewed and summarized with a focus on the molecular mechanisms involved in its pathogenesis and treatment.
 
Appearance phenotypes of parents and their F1 and F2 progeny in the LWF2 population. LS1934 has purple fruit, with nasunin (type 1, Supplementary Table 2), whereas the WCGR112-8 fruit are anthocyanin deficient and characterized by a green color. The green stripe pattern observed on WCGR112-8 fruit is absent on LS1934 fruit. Fruit of F1 progeny of a cross of LS1934 and WCGR112-8 have the purple color of nasunin with the green stripe pattern. The presence of the green stripe pattern and the nasunin content segregate in the fruit of the F2 progeny
Appearance phenotypes of parents and their F1 and F2 progeny in the N28F2 population. Fruit colors in Nakate Shinkuro, PI 286106, and their F1 progeny were purple, pale purple, and purple, respectively. Anthocyanin profiles in the fruit and calyx of Nakate Shinkuro and the F1 are nasunin (type 1, Supplementary Table 2) accumulation, whereas those of PI 286106 are D3G (type 3, Supplementary Table 2) accumulation. The green stripe pattern is absent from the fruit of Nakate Shinkuro and PI 286106 and their progeny. The presence of nasunin/D3G and the content of nasunin/D3G segregate in the fruit of the F2 progeny
Comparison of the LWF2 map (left) and the N28F2 map (right). Two linkage maps were anchored using the common (anchor) markers. Four loci, gf, adf, wf, and SF, listed in Table 1, are shown on the LWF2 map. The locus for d3g for the fruit/calyx anthocyanin profile of Table 1 is shown on the N28F2 map
Article
Background The appearance quality of the eggplant (Solanum melongena L.) fruit is an important trait that influences its commercial value. It is known that quality traits such as anthocyanin composition and fruit surface pattern are categorical and are inherited simply. However, research examples of gene mapping for the composition (anthocyanin accumulation profile) and the surface pattern in eggplant fruit are limited. Methods and results To map loci for these traits including the accumulation profiles of two anthocyanins, a widely spreading anthocyanin, delphinidin 3-(p-coumaroyl) rutinoside-5-glucoside (nasunin), and the relatively rare delphinidin 3-glucoside (D3G), we used two F2 intracrossed populations (LWF2 and N28F2). For the LWF2 population, mapping was achieved by reconstructing the linkage map created by Fukuoka et al. [1]. In the case of the N28F2 population, we constructed a linkage map consisting of 13 linkage groups using 238 simple sequence repeats, 75 single-nucleotide polymorphisms. Using the two F2 populations, the nasunin accumulating profile, the striped pattern on the fruit surface, the colors of flowers, fruit, and calyxes, and the D3G accumulating profile were genetically mapped. Furthermore, by utilizing the eggplant reference genome information, mutations in the causative candidate genes for those loci were identified. Conclusion Overall, the results of this study suggest that inactivation of key enzymes of anthocyanin metabolism and the gene orthologous to the tomato u gene are potential causes of observed variety in eggplant appearance traits.
 
Expressions and comparisons of p97/ VCP, SVIP, StAR, CYP17A1, 3βHSD1, in TM3, MA-10 and HLC Leydig cells. p97/ VCP, SVIP, StAR, CYP17A1 and 3βHSD1 expressions were shown with immunofluorescence in the TM3, MA-10 and HLC (a-f, a’-f’). Western blotting was used to detect p97/ VCP, SVIP, StAR, CYP17A1, 3βHSD1 in extracts from three Leydig (TM3, MA-10, HLC) cell lines. β-actin was used as the loading control (g). The SVIP, StAR and CYP17A1 protein expressions were detected to be significantly higher in MA-10 Leydig cells, when compared to ones in TM3 and HLC (asterisk) (h).
Coexpression of SVIP and p97/VCP with steroidogenesis markers in TM3 and HLC cells. Detection of SVIP (green), 3βHSD1, CYP17A1, StAR (red), and double (yellow)-positive cells on TM3. Detection of p97/VCP (red), 3βHSD1, CYP17A1, StAR (green), and double (yellow)-positive cells on HLC. Merging of nuclear 4’,6-diamidino-2-phenylindole (DAPI) staining (blue) is also seen in colocalization. Scale bars: 20 µm.
Effect of p97/VCP and SVIP knockdown on the expression of StAR, CYP17A1, 3βHSD1 proteins in TM3, MA-10 and HLC cells. Cells were transfected with control siRNA (Csi), p97/VCP siRNA (p97/VCPsi) and SVIP siRNA (SVIPsi). The cells were then processed for the detection of p97/VCP, SVIP, StAR, 3βHSD1 and CYP17A1 (a-e) by Western blotting. p97/VCP, SVIP, StAR, 3βHSD1 and CYP17A1 expression was assessed by immunoblotting, with actin serving as the protein loading control (a-e). The knockdown of p97/VCP (p97/VCPsi) was almost 75% vs. (a), 55% vs. (c), 55% vs. (e) that of the nontargeting control si (Csi). The knockdown of SVIP (SVIPsi) was almost 60% vs. (b), 60% vs. (d), 70% vs. (f) that of the nontargeting control si (Csi). Each bar in the graphs is the mean ± SEM of three experiments. *p < 0.05.
Effect of p97/VCP and SVIP knockdown with hCG stimulation on the expression of StAR, CYP17A1, 3βHSD1 genes and testosterone synthesis in HLC cells. Knockdown efficiency of p97/VCP, SVIP with hCG-exposed were also demonstrated by q-RTPCR (a-b). Testosterone levels in cell media of siVCP-, siSVIP- and siControl transfected cells with hCG-exposed cells were determined (c-d). *p < 0.05.
Article
Background In recent studies, it was shown that Endoplasmic reticulum-associated degradation (ERAD) is regulated by androgens and small VCP-interacting protein (SVIP) is an ERAD inhibitor. There is no data available about the interactions of ERAD proteins with proteins involved in steroidogenesis. The aim of the study was to investigate the expressions of SVIP, p97/VCP, StAR, CYP17A1 and 3β-HSD in human and mouse. Methods and results HLC, TM3 and MA-10 Leydig cell lines were used to determine roles of ERAD proteins in steroidogenesis based on immunofluorescence, Western blot, qRT-PCR, ELISA. Findings showed that StAR, CYP17A1 and 3β-HSD were colocalized with SVIP and p97/VCP in Leydig cells. A decrease in CYP17A1, 3β-HSD and StAR expressions was observed as a result of suppression of SVIP siRNAs and p97/VCP siRNAs expressions in MA10, TM3 and HLC. When siSVIP transfected cells were compared with siSVIP transfected with hCG-exposed cells, SVIP protein expression was significantly increased as compared to the SVIP transfected group in human Leydig cells. Conclusion We suggest that the suppression of protein expressions by p97/VCP and SVIP siRNAs in Leydig cells, the effects of proteins involved in steroidogenesis (StAR, CYP17A1 and 3β-HSD) have proven to be originating from p97/VCP and SVIP which were playing a role in the steroidogenesis process. Additionally, it was demonstrated that testosterone levels decreased after transfection with p97/VCP siRNA and SVIP siRNA, p97/VCP and SVIP created an effect on testosterone synthesis while taking place in the steps of testosterone synthesis. Further, it was determined in the study that the SVIP was affected by hCG stimulations.
 
Flow cytometric DNA histogram of A549 cells incubated with three concentrations of A. nilotica extract (half IC50, IC50, and double IC50) for 72 h. Representative of one of three similar experiments
Article
Purpose In this study, two main research objectives were examined: (1) the cytotoxic and anticancer activities of the aqueous methanol extract from Acacia nilotica flowers on three human cancer cells, namely lung A549, breast MCF-7, and leukemia THP-1 cells, and (2) the genotoxic effects of A. nilotica extract and its influence on DNA damage induced by N-methyl-N-nitrosourea (MNU) in mice. Methods Mice were orally treated with A. nilotica extract (200, 500, and 800 mg/kg for 4 days) with or without MNU (80 mg/kg intraperitoneally for 24 h). Results In vitro experiments showed that A549 cells were the most sensitive to A. nilotica extract among the tested cell lines. A. nilotica extract inhibited A549 cell proliferation by blocking the cell cycle at the G 2 /M phase and accumulating apoptotic cells in the sub-G 0 /G 1 phase in A549 cells. In vivo experiments showed that MNU induced positive and negative genotoxicity in bone marrow cells and spermatocytes, respectively. Negative genotoxicity was observed in A. nilotica extract-treated groups only. However, A. nilotica extract (800 mg/kg) remarkably increased comet tail formation in bone marrow cells. Unexpectedly, the absence of antigenotoxicity was observed in three cotreated groups with A . nilotica extract and MNU compared with the MNU-treated group. Astonishingly, cotreatment with MNU and A. nilotica extract at a dose above 200 mg/kg remarkably increased micronucleus and comet tail formation in bone marrow cells compared with the MNU-treated group. Conclusions A. nilotica extract possessed anticancer activity with relative genotoxic effects at high doses.
 
Mutant IDH1 overexpression enhanced cell proliferation, invasion but not migration in U87-MG cells. (a) Cell proliferation, (b) cell invasion, (c) cell migration assay microscope photographs (d) cell migration assay analysis of IDH1 wild type and mutation transfected U87-MG cells. Data were obtained for cell proliferation and invasion assays for each 15 min via electrical impedance based measurement. Each line represents different mutation transfected glial cells. R132H (green), R132L (dark blue), R132S (pink), R132G (damson color), G394T (light purple), C394A (light blue), wild type (red). IDH1 mutations induce cell proliferation in U87-MG cell line. Cells were photographed at the beginning, 8. hour and 24. hour for cell migration assay. (*indicate statistical significance: *p < 0.05, **p < 0.001, ***p < 0.0001) Cells were photographed under 40X magnification. The size of scale bar is 100 μm
Overexpression of IDH1 mutation upregulated mTOR signaling pathway genes in U87-MG cells (a) WNT5A, (b) PRKAA2, (c) TSC1, (d) TSC2, (e) GSK3B, (f) MTOR quantitative gene expression results. (g) Western blot images of mTOR, phosphorylated mTOR (p-mTOR) and Actin (Housekeeping protein) proteins. (h) quantitative analysis of mTOR protein, (i) quantitative analysis of p-mTOR protein to total mTOR protein. Control refers to non-transfected, IDH1 WT refers to IDH1 wild type overexpressing, IDH1 R132H refers to R132H mutant IDH1 overexpressing U87-MG cells. (* indicate statistical significance: *p < 0.05, **p < 0.001, ***p < 0.0001, ns: non-significant)
mTOR signaling pathway associated genes upregulated in patient derived glioma cells and correlated with tumor histopathology and grades. Expression of IDH1, WNT5A, TSC1, TSC2, PRKAA2, GSK3B, MTOR genes between histological subtypes (a, c, e, g, i, k, m) and tumor grades (b, d, f, h, j, l, n). o) Gene expression correlation matrix. Colors indicate degree of correlation. Ast(IDH1 WT): IDH1 wild type astrocytoma, Ast(R132H): R132H IDH1 mutant astrocytoma, Olg(R132H): IDH mutant oligodendroglioma, GBM (IDH WT): IDH1 wild type glioblastoma
Interaction of IDH1 mutation and mTOR signaling pathway. Blue color indicates increased gene expression, red color indicates decreased gene expression
Article
Background Glioma is the most common type of brain tumors and isocitrate dehydrogenase (IDH1) gene is the most prominent molecular marker about the disease prognosis, response to therapy and patient survival. There are conflicting data about the effect of IDH1 mutation on glial cell proliferation, invasion and migration characteristics. The effect of IDH1 mutation on mTOR signaling pathway, which has key roles in tumorigenesis process, is limited and previous data is controversial. We aimed to explore the effect of wild type and mutant IDH1 overexpression on glioma cells and investigated the correlation with mTOR signaling pathway associated genes. Methods and Results U87-MG and A172 cells were transfected with different IDH1 mutant gene overexpressing (R132H, R132L, R132S, R132C) viral vectors. Cell proliferation, cell invasion and migration analysis as well as quantitative PCR analysis with the mutant glioma cell lines were performed. Forty-two patient derived glioma cells were obtained from patients with different glioma subtypes and cancer cells were enriched by culturing cells. Overexpression of both mutant and wild type IDH1 gene promoted the cell proliferation, but only IDH1 mutation increased cell invasion and migration. The expression of IDH1 mutation activated mTOR signaling via upregulation of WNTA, PRKAA2, GSK3B and MTOR genes as well as phosphorylated mTOR protein level. Conclusions Our results highlighted IDH1 mutation upregulate mTOR signaling pathway and promote cell proliferation, invasion and migration.
 
Article
The process of cell division plays a vital role in cancer progression. Cell proliferation and error-free chromosomes segregation during mitosis are central events in life cycle. Mistakes during cell division generate changes in chromosome content and alter the balances of chromosomes number. Any defects in expression of TIF1 family proteins, SAC proteins network, mitotic checkpoint proteins involved in chromosome mis-segregation and cancer development. Here we discuss the function of organelles deal with the chromosome segregation machinery, proteins and correction mechanisms involved in the accurate chromosome segregation during mitosis.
 
ENO1 and PGAM1 expression levels are increased in SKCM tissues and cell lines. A Pancreatic-cancer tissues vs. TCGA and GTEx-normal tissues. B SKCM tissues vs. TCGA and GTEx-normal tissues. C The discriminative ability of PAX3 in SKCM was evaluated by ROC curve analysis. D The PPI network related to ENO1, obtained from STRING datasets. E The correlation between ENO1 and PGAM1, obtained from the GEPIA datasets. F, G Protein levels of ENO1 and PGAM1 in SKCM, assessed using western blotting assays. T: SKCM tumor tissues; N: naevus tissues. H Protein levels of ENO1 in four SKCM cell lines (SK-MEL-28, HMCB, A375, and SK-MEL-19), assessed by western blotting. I Protein levels of ENO1 in A375 cells transfected with plasmids pCMV-SPORT6-ENO1 and pLENTI-CMV-GFP/Puro, assessed by western blotting. J Protein levels of ENO1 in SK-MEL-19 cells transfected with plasmids pET-28a-ENO1siRNA and pET-28a, assessed using western blotting assays. *P < 0.05, **P < 0.01. ENO1 α-Enolase; SKCM skin cutaneous melanoma; ROC Receiver Operating Characteristic; PGAM1 phosphoglycerate mutase 1; Protein–Protein Interaction
Overexpression of ENO1 inhibits apoptosis and promotes the proliferation, migration, and invasion of A375 cells. A CCK-8 assay showing that overexpression of ENO1 promotes the proliferation of A375 cells. B, C Wound-healing assay showing that overexpression of ENO1 increases the migration capabilities of A375 cells. D, E Transwell chamber assay showing that overexpression of ENO1 increases the invasion capabilities of A375 cells. *P < 0.05, **P < 0.01. ENO1 α-Enolase; CCK-8 cell counting kit-8
Downregulation of ENO1 induces apoptosis, and inhibits the proliferation, migration, and invasion of SK-MEL-19 cells. A CCK-8 assay showing that downregulation of ENO1 inhibits the proliferation of SK-MEL-19 cells. B, C Wound-healing assay showing that the downregulation of ENO1 inhibits the migration capabilities of SK-MEL-19 cells. D, E Transwell chamber assay showing that downregulation of ENO1 inhibits the invasion capabilities of SK-MEL-19 cells. *P < 0.05, **P < 0.01. ENO1 α-Enolase; CCK-8 cell counting kit-8
ENO1 Enhances Glycolysis and Promotes the Expression of β-catenin, c-Myc, MMP-9, and MMP-13 in SKCM cells. A, B Overexpression of ENO1 promotes the formation of lactate and pyruvate in A375 cells, measured using a micro pyruvate assay kit and lactic acid kit. C, D Downregulation of ENO1 inhibits the formation of lactate and pyruvate in SK-MEL-19 cells, assessed using a micro pyruvate assay kit and lactic acid kit. E, F Protein levels of β-catenin, c-Myc, MMP-9, and MMP-13 in A375 cells transfected with plasmid pCMV-SPORT6-ENO1 and SK-MEL-19 cells transfected with plasmid pET-28a-ENO1siRNA, assessed on western blotting assays. **P < 0.01. ENO1 α-Enolase; SKCM skin cutaneous melanoma; MMP-9 matrix metalloproteinase-9; MMP-13 matrix metalloproteinase-13
Article
Background The glycolytic enzyme, α-Enolase (ENO1), catalyzes the production of phosphoenolpyruvate from 2-phosphoglycerate, thereby enhancing glycolysis and contributing to tumor progression. In the present study, we aimed to determine the role of ENO1 in skin cutaneous melanoma (SKCM) and the potential underlying mechanism. Methods The Sangerbox database was used to analyze the mRNA expression of ENO1 in SKCM. Western blotting was used to assess the levels of ENO1, c-Myc, β-catenin, MMP-9, PGAM1, and MMP-13 in SKCM-derived cell lines or tumor tissues from patients with SKCM. The pCMV-SPORT6-ENO1 and pET-28a-ENO1siRNA plasmids were used to overexpress and knockdown ENO1 in SKCM cells, respectively. To determine the function of ENO1 in the malignant behavior of SKCM cells, we performed a wound-healing assay, cell counting kit 8 assay, and transwell chamber analyses. The production of pyruvate and lactic acid in tumor cells was evaluated using their respective kits. Results Compared with non-tumor tissues, ENO1 was found to be overexpressed in SKCM tissues. In SKCM cells, ENO1 overexpression promoted invasion, migration, and proliferation of tumor cells; increased pyruvate and lactate production; and increased β-catenin, MMP-9, MMP-13, and c-Myc levels. The opposite effects were observed in SKCM cells silenced for ENO1. Conclusions These results indicate that ENO1 is involved in SKCM progression by enhancing the invasion and proliferation of tumor cells. In addition, ENO1 might have an important function in tumor cell glycolysis. Therefore, ENO1 represents a potential therapeutic target for treatment of SKCM.
 
Oxidative stress and antioxidant enzyme biomarkers. Oxidative stress markers are measured in the terms of MDA represented in (A). Data are represented as mean ± SD of six independent experiments. *Represent significantly changed with respect to control group, #represent significantly changed when compared with HFD + Chitosan group. B–D represents the antioxidant enzyme activity SOD, Catalase, and GST. *Represent significantly changed with respect to control group, # represent significantly changed when compared with HFD + Chitosan group
Histopathological study of liver tissue. Photomicrograph liver tissue section was taken at 100X. (Dye: Hematoxylin&Eosin) A Control group; B Chitosan treated Group; C HFD treated group and D HFD + Chitosan treated group. CV is designated for central vein; BLACK arrow shows necrotic cells, and red arrow presents CV congestion and infiltration of inflammatory cells
Gene expression study. A–D represent the gene expression activity of Il-6, TNF-alpha, Leptin, and NFKB. Data are represented as mean ± SD of six independent experiments. *Represent significantly changed with respect to control group, #represent significantly changed when compared with HFD + Chitosan group
Article
Background An altered lipid profile may lead to the development of inflammation and NAFLD (Non-alcoholic fatty liver disease). Although statins have a positive effect on blood lipid levels their long-term use is known to cause adverse effects, in this backdrop there is an interest in natural compounds which may affect lipid metabolism and prevent NAFLD. We have examined the effect of Chitosan on rats subjected to a high-fat diet. Methods and results Male Wistar middle aged rats (12–16 months) were treated with high-fat diet orally for two months for creating a NAFLD model. Rats were also supplemented with Chitosan (2% chitosan daily) for 2 months. We assessed the activity of antioxidant enzymes, the histopathological profile of the liver. Inflammatory cytokines and adiponectin levels were also measured in serum. HFD induced significant changes in liver tissue and inflammatory markers (Il-6, TNF- alpha, NF-KB). Chitosan treatment protected rats from HFD induced alterations. Conclusions The findings suggest that Chitosan can effectively improve liver lipid metabolism by normalizing cholesterol, triglyceride, lowering NF-KB expression, and protecting the liver from oxidative stress by improving hepatic function. Chitosan also regulates genes related to lipidemic stress i,e leptin and adiponectin.
 
This figure shows the expression of caspase-8 in Muller cells treated with high glucose, xamoterol, salmeterol, isoproterenol and propranolol (A), the expression of caspase-8 in Muller cells treated with TNF-α (10 ng/ml), xamoterol, salmeterol, isoproterenol and propranolol (B), the expression of caspase-3 in Muller cells treated with high glucose, xamoterol, salmeterol, isoproterenol and propranolol (C) and TUNEL assay (apoptosis) in Muller cells treated with high glucose, xamoterol, salmeterol, isoproterenol and propranolol (D)
This figure shows the expression of BDNF in Muller cells treated with high glucose, xamoterol, salmeterol and isoproterenol (A), and the expression of BDNF treated with high glucose, xamoterol, salmeterol and isoproterenol after treating with CREB-shRNA (B)
This figure shows the DCFH-DA assay (generation of reactive oxygen species) in Muller cells in glucose and glucose + beta-adrenergic receptors agonists (A) and ELISA assay showing the effect of xamoterol, salmeterol, isoproterenol and propranolol on cytochrome c release in hyperglycemic Muller cells (B)
Article
Background: The current study aimed to investigate the stimulatory efect of beta-adrenergic receptors (β-ARs) on brain derived neurotropic factor (BDNF) and cAMP response element binding protein (CREB). Methods: Human Müller cells were cultured in low and high glucose conditions. Cells were treated with xamoterol (selective agonist for β1-AR), salmeterol (selective agonist for β2-AR), isoproterenol (β-ARs agonist) and propranolol (β-ARs antagonist), at 20 µM concentration for 24 h. Western Blotting assay was performed for the gene expression analysis. DNA damage was evaluated by TUNEL assay. DCFH-DA assay was used to check the level of reactive oxygen species (ROS). Cytochrome C release was measured by ELISA. Results: Xamoterol, salmeterol and isoproterenol showed no efect on Caspase-8 but it reduced the apoptosis and increased the expression of BDNF in Müller cells. A signifcant change in the expression of caspase-3 was observed in cells treated with xamoterol and salmeterol as compared to isoproterenol. Xamoterol, salmeterol and isoproterenol signifcantly decreased the reactive oxygen species (ROS) when treated for 24 hours. Glucose-induced cytochrome c release was disrupted in Müller cells. Conclusion: β-ARs, stimulated by agonist play a protective role in hyperglycemic Müller cells, with the suppression of glucose-induced caspase-3 and cytochrome c release. B-Ars may directly mediate the gene expression of BDNF.
 
Healthy and Phytoplasma infected Brassica rapa
Transcriptional regulation of developmental genes expression in Phytoplasma infected Brassica rapa through RT-PCR. ACTIN is used as controlled gene
Determination of Global Methylation level in healthy and phytoplasma infected Brassica through Southern Blot Hybridization. A. MspI and HpaII digested DNA gel pattern, B. Blotting pattern on PVDF membrane through Hybridization by using 5S probe. HFB = Healthy Flower Bud IFD = Infected Flower Bud, M = Molecular Marker
Determination of Methylation level through Bisulfite Sequencing in phytoplasma infected Brassica rapa. DNA was extracted from 3 mm flower buds of phytoplasma infected Brassica rapa. A. Graphical representation of gene sequence without bisulfite treatment. B and C Gene sequence after bisulfite treatment. Blue lines in graph represent the cytocine while red lines represent thymine
Genes expression in Azacitidine treated Brassica rapa. Azacitidine restore the expression of methylated genes (AGAMOUS and APETALA3). ACTIN is used as controlled gene. H = healthy, IA = Infected A, IB = Infected B
Article
Background The plants of B. rapa (syn. B. campestris) are the most important food crop of Pakistan for the production of cooking oil. Brassica plants infected by phytoplasma exhibit floral abnormalities including phyllody, virescence, hypertrophied sepal and aborted reproductive organs and affected flower developmental genes which reduces the yield manifold. Methods and results The expression level of flower developmental genes in healthy and phytoplasma infected brassica were compared by using semi-quantitative reverse transcription polymerase chain reaction and DNA hybridization. In infected brassica, LEAFY (LFY) gene, controlling the development and maintenance of floral organ, and directly involved in controlling the homeotic gene expression was affected, while APETALA2, regulate the production of sepals and petals, were not altered. Whereas the genes WUSCHEL, APETALA3 and AGAMOUS, were significantly down-regulated, that were responsible for the identity of shoot and central meristem, petals and stamens production, and stamens and carpels development, respectively. The GLUB gene, controlling the production of β-1,3-glucanases enzyme, was highly up-regulated. According to DNA hybridization results, AGAMOUS and APETALA3 were restricted to floral organs territories in healthy and phytoplasma infected brassica, indicating that their expression was tissue-specific. These outcomes indicated that flower abnormalities of phytoplasma infected B. rapa are linked with DNA methylation in the expression of homeotic genes regulating flower development. Conclusions Azacitidine act as a DNA demethylating reagent. By applying the foliar spray of azacitidine during the flower development, cells of Phytoplasma infected plants exhibits demethylation of DNA when treated with azacitidine chemical that incorporated as analogue of cytosine during the cell division stage. B. rapa showed the up-regulation of gene expression level significantly that restore the normal production of flowers, ultimately increase the oil production throughout the world.
 
Effects of TGF-β1 on BEAS-2B cells. TGF-β1 (5 ng/mL) was incubated with BEAS-2B cells for 24 h. a Morphological characteristics. b qRT–PCR. c Western blot showing EMT-related markers expression. d The relative expression of EMT-related markers was examined by Image J. The results are expressed as the means ± SD (n = 3), *P < 0.05 versus the control group
Effects of TGF-β1 and TNF-α on BEAS-2B cells. TGF-β1 (5 ng/mL) and TNF-α (20 ng/mL) were incdbated with BEAS-2B cells alone or in combination for 24 h. a Morphological characteristics. b RT–qPCR. c Western blot showing EMT-related markers levels. d The relative expression of EMT-related markers was examined by Image J. e Immunofluorescence staining of E-cadherin (green), vimentin (red) and α-SMA (red) is shown, and the nuclei were stained with DAPI (blue). (f) A wound healing assay was used to evaluate the cell migration rate. The cell migration rate (%) was determined by Image J software. The results are expressed as the means ± SD (n = 3), *P < 0.05 versus the control group. #P < 0.05 versus the TGF-β1 group. (Color figure online)
Effects of TGF-β1 and TNF-α on the NF-κB signaling pathway and NOX4 expression in BEAS-2B cells. BEAS-2B cells were cultured with 5 ng/mL TGF-β1, 20 ng/mL TNF-α or both for 24 h. a Western blot showing p-p65 and p65 levels after treatment with TGF-β1, TNF-α, or both. b The relative expression of p-p65/p65 was determined with Image J. c NOX4 protein expression was examined by western blotting. d The mRNA and protein expression of NOX4 was examined. e Immunofluorescence staining of p65 (red) is shown, and the nucleus was counterstained with DAPI (blue). The data are expressed as the means ± SD (n = 3). *P < 0.05 versus the control group. #P < 0.05 versus the TGF-β1 group. (Color figure online)
Inhibition of the NF-κB signaling pathway leads to NOX4 downregulation and attenuates the combined effect of TNF-α + TGF-β1 on EMT and migration in BEAS-2B cells. NF-κB p65 inhibition affects a EMT-related markers and NOX4 protein expression. b The expression of E-cadherin, vimentin, α-SMA and NOX4 were examined. c Cell migration rate. d Quantification of cell migration by Image J. The data are expressed as the means ± SD, and the experiments were repeated at least three times independently. *P < 0.05 versus the control group; #P < 0.05 versus the TGF-β1 group; ⁺P < 0.05 versus the TGF-β1 + TNF-α group
Schematic diagram of the relationship among TNF-α, TGF-β, the NF-κB p65/NOX4 pathway and EMT. TNF-α and TGF-β synergistically activate the NF-κB p65/NOX4 signaling pathway, which promotes EMT by increasing vimentin and α-SMA expression and decreasing E-cadherin expression, thus promoting cell migration
Article
Background Epithelial-to-mesenchymal transition (EMT) is the process by which epithelial cells transform into mesenchymal cells, which plays a significant role in lung fibrotic disease. Transforming growth factor-β1(TGF-β1) is considered to be the most effective EMT inducer. The purpose of this study was to investigate the effect of the proinflammatory cytokine tumor necrosis factor-α (TNF-α) on TGF-β1-induced EMT and the underlying mechanisms in the human bronchial epithelial cell line BEAS-2B. Methods Human bronchial epithelial BEAS-2B cells were treated with TGF-β1 and TNF-α separately or in combination for 24 h, and qRT–PCR, western blotting, immunofluorescence staining, and migration assays were used to investigate the EMT process. Moreover, to further explore the effect of the NF-κB pathway on the EMT process, inhibitor assays (BAY-117082, NF-κB inhibitor), wound healing assays, and western blotting were performed. Results The results showed that both cytokines enhanced the transformation of BEAS-2B cells from epithelial to mesenchymal cells. In addition, combined treatment with TNF-α and TGF-β1 further reduced E-cadherin expression, which conversely elevated α-SMA and vimentin mRNA and protein levels. Correspondingly, the migration rate of BEAS-2B cells was also increased. Furthermore, inhibiting the NF-κB signaling pathway blocked the expression of EMT-related markers and NOX4 induced by TGF-β1 and TNF-α, as well as cell migration. Conclusion Taken together, TNF-α and TGF-β1 cooperatively promoted EMT and cell migration in BEAS-2B cells through the NF-κB/NOX4 signaling pathway.
 
Dendrogram based on SSR marker data of 319 Turkish Oriental tobaccos
Article
Background Turkey is one of the traditional oriental tobacco producing countries. Oriental tobaccos produced in Turkey are ecotypes specific to certain regions, local varieties and landraces, and they have unique characteristics. The present study was conducted to reveal the general and interregional genetic diversity levels of tobacco genotypes collected from different regions of Turkey based on DNA markers. Methods and Results A total of 319 ecotypes/lines of Turkish Oriental Tobaccos collected from different regions (Aegean Region, Marmara Region, Karadeniz Region, and East and Southeast Anatolia Regions) of Turkey and cultivars Xanthi 81, Xanthi 2A, Canik Sitmasuyu, Katerini, Canik 190-5 and NC 55 were subjected to SSR marker analysis. Eighty-nine alleles were obtained from 21 markers examined, and the average number of alleles per marker was 4.05. A total of 314 unique tobacco genotypes were found among 319 plants evaluated using 19 polymorphic SSR markers. In the dendrogram constructed using SSR marker data, genotypes were divided into six clades based on geographic regions and levels of genetic diversity. Conclusions The presence of 314 unique genotypes among 319 tobacco plants evaluated indicated the high level of genetic diversity among Turkish tobaccos. While some of them were N. rustica tobaccos, some others were reminiscent of semi-oriental tobaccos. These genotypes merit further agronomic and technological characterizations, which could allow their utilization in future tobacco breeding.
 
Influence of surface angiotensin-converting enzyme 2 (ACE2) on heart rate variation. Sensitivity of heart rate control reflexes affected by ACE2 receptor in brainstem, where it converts Ag II to Ag-(1-7). Variations in the time interval between successive heartbeats in milliseconds are shown by the R-R interval difference on ECG.
Potential mechanisms of SARS-CoV-2-induced changes in heart rate variability. SARS-CoV-2 can directly invade the brain via angiotensin-converting enzyme 2 (ACE2) in the brainstem. These would lead to the local accumulation of angiotensin II (Ag II) and a decrease in angiotensin (1?7), which reduced baroreceptor sensitivity in heart rate control. The resulting autonomic system dysfunction from disequilibrium Ang 1?7/Ang II ratios may cause a reduction in RR interval fluctuations.
Article
Increasing evidence strongly support that the newly identified severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) leads to the development of COVID-19-associated central nervous system (CNS) manifestations. The presence of SARS-CoV-2 viral protein in the brainstem, which includes cardiovascular control centers, has been documented previously. Given the changes in autonomic nervous system function evaluated by heart rate variability (HRV) metrics, which are observed even prior to clinical signs, the potential effect of SARS-CoV-2 on the autonomic nervous system (ANS) center is likely. The integral parts of the brain renin-angiotensin system, as ACE2 enzyme, are highly expressed in the brainstem, which may also be involved in baroreflex sensitivity, playing an important role in HRV. SARS-CoV-2 may bind to ACE2 in order to enter the host brainstem cell and change baroreflex sensitivity due to the altered ratio of the concentration of angiotensin II (Ag II) to angiotensin (1–7). In this article, we discussed the information on the possibility that the SARS-CoV-2 viral particle by disrupting the homeostasis of the brain renin-angiotensin system even without brainstem neuropathological changes, may affect the function of the ANS center in the brainstem. SARS-CoV-2 could influence ANS function before affecting the immune system. It is possible that the altered HRV parameters imply the potential neurotropic characteristics of SARS-CoV-2. Therefore, this potential feature should be taken into account in diagnostic and therapeutic approaches for COVID-19 patients.
 
Schematic representation of the experimental design and schedule of the protocols. Total population involving 32 male rats, including 8 control, 12 rats exposed with white noise (WN) and 12 exposed with purple noise (PN). Animal divided into five main groups: (1) control group (n = 8), without noise exposure; (2) WN-3D (n = 6), animals exposed to white noise for 3 days with a frequency range of 100-20000 Hz; (3) WN-6D (n = 6) animals exposed to white noise for 6 days with the same frequency; (4) PN-3D (n = 6), animals exposed to purple noise for 3 days with a frequency range of 4–20 kHz; (5) PN-6D (n = 6), animals exposed to purple noise for 6 days with the same frequency range. All experimental groups were exposed to a sound pressure level (SPL) of 118–120 dB for 8 h per day. At two time points, 1 h and 1 week after termination of noise exposure, 3 rats of each experimental group and 4 rats of the control group were anesthetized, and the samples were collected for pathologic and gene expression analysis
Hematoxylin and Eosin staining of cochlea tissue of different exposure group samples, 1 h and 1 week after cessation of noise exposure. WN-3D, group were exposed to white noise for 3 days; WN-6D, exposed to white noise for 6 days; PN-3D, group exposed to purple noise for 3 days; PN-6D; exposed to purple noise for 6 days. Abnormal morphology of auditory nerve ganglion and Reisner membrane was observed following noise exposure
Expression analysis of GJB2 gene using real-time RT-PCR after white & purple noise exposure in cochlea tissue of rat. The expression level of target genes is reported as the gene fold in the noise-exposure samples normalized to the internal control gene (GAPDH) and relative to the control group using the 2−∆∆CT method. In all experiment group the expression of GJB2 is significantly downregulated at 1 week post noise- exposure compared to 1 h. Data are expressed as mean ± SD of three independent experiments. * p < 0.05; ** p < 0.01, ***p, 0.001, Two-way ANOVA followed by the Tukey multiple-comparison test
Expression analysis of SLC26A4 gene after white & purple noise exposure in cochlea tissue of rat. The transcript level of target genes is reported as the gene fold in the noise-exposure samples normalized to the internal control gene (GAPDH) and relative to the control group using the 2−∆∆CT method. In all exposed-group the RNA level of GJB2 is significantly downregulated at 1 week post noise- exposure compared to 1 h. Data are expressed as mean ± SD of three independent experiments. Asterisks indicate significant differences between groups (* p < 0.05; ** p < 0.01, ***p < 0.001, Two-way ANOVA followed by Tukey’s Multiple Comparison Test)
Article
Background Noise-induced hearing loss (NIHL) is one the major causes of acquired hearing loss in developed countries. Noise can change the pattern of gene expression, inducing sensorineural hearing impairment. There is no investigation on the effects of noise frequency on the expression of GJB2 and SLC26A4 genes involved in congenital hearing impairment in cochlear tissue. Here we investigated the impacts of white and purple noise on gene expression and pathologic changes of cochlear tissue. Methods In this study, 32 adult male Westar rats were randomly divided into experimental groups: WN, animals exposed to white noise with a frequency range of 100-20000 Hz; PN, animals exposed to purple noise with a frequency range of 4–20 kHz, and control group, without noise. The experimental groups were exposed to a 118–120 dB sound pressure level for 8 h per 3 days and 6 days. 1 h and 1 week after termination of noise exposure, cochlear tissue was prepared for pathology and gene expression analysis. Results Both white and purple noises caused permanent damage to the cortical, estrosilica systems of hair cells and ganglion of the hearing nerve. GJB2 and SLC26A4 were downregulated in both groups exposed with white and purple noise by increasing the time of noise exposure. However, differences are notably more significant in purple noise, which is more intensified. Also, 1 weak post noise exposure, the downregulation is remarkably higher than 1 h. Conclusions Our findings suggest that downregulation of GJB2 and SLC26A4 genes are associated with pathological injury in response to noise exposure in cochlear tissue. It would be suggested the demand for assessment of RNA and protein expression of genes involved in noise-induced hearing loss and subsequently the practice of hearing protection programs.
 
Morphology of BMSCs during differentiation and transfection efficiency of KDM5B. A. Morphology of P3 MSCs induced with 5‑AZA for 21 days (× 100, bar = 10 um). B. Immunofluorescence was used to detect transfection of lentivirus in the cells. Green fluorescence indicates positive transfection. (× 200, bar = 20 um); C. Transfection efficiency calculated according to DAPI and EGFP green fluorescence. There was no significant difference among three groups
Induction of myocardial differentiation into BMSCs in vitro. A. Immunofluorescence staining was used to detect cardiomyocyte marker proteins α-Actin and cTnT expression in cells; green fluorescence indicated the positive expression of α-actin while red fluorescence indicated the positive expression of cTnT (× 100, bar = 10 um). B. Fluorescence intensity was detected by means of flow cytometry on day 21, which demonstrated that the proportion of α-Actin- and cTnT‑positive cells rate was ~ 80% in KDM5B-ov group, ~ 50% in the KDM5B-nc group and ~ 20% in the KDM5B-si group. C. Flow cytometric analysis demonstrated the percentages of α-actin- and cTnT-positive cells on different days, which demonstrated that α-Actin- and cTnT‑positive cell rate in the KDM5B-ov group was the highest and the KDM5B-si group was the lowest. The mean ± standard deviation is shown; n = 3 independent experiments
Gene expression levels in BMSCs differentiated into cardiomyocytes. Expression of A KDM5B, B Nanog and C HCN4 was detected via qRT-PCR. The gene expression levels of each group (KDM5B-ov, KDM5B-si and KDM5B-nc) were compared to the blank group (level of significance *p < 0:05; **p < 0:01)
Relative expression levels of protein in BMSCs differentiated into cardiomyocytes. A. Following the differentiation of BMSCs, western blotting demonstrated the expression of KDM5B, H3K4me3, H3K4 and HCN4 protein expression, which demonstrated H3K4me3 was downregulated while HCN4 was upregulated following KDM5B upregulation. H3K4 and β-actin was used as an internal control. B. Cardiomyocyte differentiation of the BMSCs was detected by RT-qPCR, which demonstrated that KDM5B upregulation increased the expression of HCN4 by 2 times compared with the control group. The opposite result was found when KDM5B was downregulated. *p < 0.05 and ** p < 0.01 vs. the KDM5B-nc group
Density of INa current in each group of cells. Whole-cell patch clamp was used to detect Na current density on cell membrane surface and the results showed that KDM5B overexpression (B, E) increased the sodium current density compared with the control (A, C, E). The opposite result was found when KDM5B was downregulated (D, E)
Article
Background Studies have shown that histone H3 methylation is involved in regulating the differentiation of Bone Marrow Mesenchymal Stem Cells (BMSCs). KDM5B can specifically reduce the level of histone 3 lysine 4 trimethylation (H3K4me3), thereby activating the expression of related genes and participating in biological processes such as cell differentiation, embryonic development and tumor formation. Whether KDM5B is involved in the regulation of BMSCs differentiation into cardiomyocytes through the above manner has not been reported. Objective To investigate the effect of KDM5B on the induction and differentiation of swine BMSCs into myocardial cells in vitro. Methods Swine bone marrow BMSCs were isolated and cultured, and the overexpression, interference expression and blank vector of KMD5B were constructed and transfected by lentivirus. BMSCs was induced to differentiate into cardiomyocytes by 5-azacytidine (5-AZA) in vitro, and the differentiation efficiency was compared by immunofluorescence, RT-PCR, Western Blot and whole-cell patch clamp detection. Result Compared with the control group, the expression levels of histone H3K4me3 and pluripotency gene Nanog in KDM5B overexpression group were significantly decreased, while the expression level of key myocardial gene HCN4 and myocardial marker gene α-Actin and cTNT were significantly increased, and the Na⁺ current density on the surface of differentiated myocardial cell membrane was significantly increased. Meanwhile, the corresponding results of the KDM5B silent expression group were just opposite. Conclusions It indicated that enhanced KDM5B expression could promote the differentiation of BMSCs into cardiomyocytes and improve the differentiation efficiency by controlling H3K4 methylation levels.
 
Contribution of enteric methane and manure methane by different livestock in India
Article
In livestock sector, dairy animals alone produce 18% of the total greenhouse gas emissions globally as methane (CH4). This Enteric methane is the largest component of total carbon footprints produced by livestock production system and its reduction is today's new challenge to make livestock farming sustainable for earth’s environment. The production of enteric methane in ruminants is a complex phenomena involving different host factors like host genotype, rumen microbiome, host physiology along with dietary factors. Efforts have been made to reduce methane emissions largely through nutritional interventions and dietary supplements, but permanent reductions can be obtained through genetic means by selecting and breeding of low methane emitting animals. From genome-wide association studies, many important genomic QTL regions and single nucleotide polymorphisms involved in shaping the composition of the ruminal microbiome and thus their carbon footprints have been recognised, implying that methane emission traits are quantitative traits. The major bottleneck in implementation of reduced methane emission traits in the breeding programs is wide variation at phenotypic level, lack of precise methane measurements at individual level. Overall, the heritability for CH4 production traits is moderate, and it can be used in breeding programmes to target changes in microbial composition to reduce CH4 emission in the dairy industry for far-reaching environmental benefits at the cost of a minor reduction in genetic gain in production traits.
 
FGL1 expression of HCC tissues was downregulated compared with paired normal tissues, and the downregulation of FGL1 was correlated with poor HCC prognosis. A FGL1 expression profile across all cancer samples and paired normal tissues on the basis of GEPIA. B The differential expression level of FGL1 in tumor and non-tumorous liver tissues. C FGL1 expression in LIHC based on individual pathological stages by UALCAN online website. D Kaplan–Meier survival curves of LIHC patients with low and high FGL1 expression based on the OncoLnc Database. “*” signifies P value < 0.05
FGL1 expression showed obvious downregulation in several human HCC cell lines and HCC tissues. A Determination of FGL1 expression in several human HCC cell lines and the normal liver cell line via western blot analysis. B Determination of FGL1 expression with 3 pairs of HCC tissues and peri-tumor tissues and paired normal liver tissues via western blot analysis. C Determination of FGL1 expression with 8 pairs of HCC tissues and paired normal liver tissues via western blot analysis. D IHC staining for tumor tissues and adjacent normal liver tissues from HCC patients in the TMA. N normal liver tissue, P peri-tumor tissue, C cancer tissue; the numbers before N, P and C represent the group number
Kaplan–Meier survival analysis of HCC patients with different expression levels of FGL1 in TMA
Article
Background Fibrinogen-like-protein 1 (FGL1), a member of the fibrinogen-related protein (FREP) family, is a major ligand of the immune inhibitory receptor lymphocyte-activation gene 3 (LAG-3). While FGL1 is strongly implicated in the development and prognosis of a variety of diseases, its role in hepatocellular carcinoma (HCC) is still disputed. Therefore, the role of FGL1 expression in the progression and prognosis of HCC was investigated. Methods and results In the present study, bioinformatics analysis was first used to probe the expression profile of FGL1 in multiple malignant tumor tissues and paired normal tissues, and to explore the possible relationship between FGL1 and prognosis of HCC patients. Thereafter, the expression levels of FGL1 were determined and compared in human HCC cell lines, HCC tissues, peri-tumor tissues and normal liver tissues by western blot analysis. Furthermore, tissue microarrays were used to detect the expression of FGL1 through immunohistochemical staining and to verify whether the FGL1 expression level was associated with clinicopathological features and the prognosis of HCC patients. The results showed that FGL1 was downregulated significantly in most of the HCC cells lines and HCC tissues, corresponding to the results of the bioinformatics and western blot analyses. FGL1 expression level in HCC was found to be correlated to Edmondson grade and metastasis of the HCC. Additionally, high FGL1 expression was associated with better overall survival in HCC patients, suggesting that FGL1 could function as a tumor suppressor. Conclusions The expression level of FGL1 can be correlated with the progression and prognosis of HCC, suggesting its potential as a prognostic biomarker.
 
Phylogenetic tree analysis of tsSEC14L2 from indicated organisms. Note Self-development value: 1000 times. Method: Neighbor-Joining (NJ) method
The structural analysis of tsSEC14L2 protein. A Amino acid content map of tsSEC14L2 protein. B Analysis of the hydrophobicity of tsSEC14L2 protein. C Secondary structure prediction of tsSEC14L2 protein. D The protein domain prediction of tsSEC14L2
The spatial structure of tsSEC14L2 predicted using Swiss-Model. Tree shrew (green) and human (red) SEC14L2 proteins. SEC14 and CRAL-TRIO domains are indicated. N-terminal (N); C-terminal (C). (Color figure online)
Expression of SEC14L2 mRNA in different tissues of various mammalian species. A Analysis of relative expression levels of SEC14L2 mRNA in 11 tree shrew tissue. B Heat map cluster of SEC14L2 expression in different tissues of tree shrews and human, mouse, and rat. Note Black box indicates that the information is missing. (Color figure online)
The protein expressions of SEC14L2 in the tree shrew. A Expression of SEC14L2 protein in 14 tissues of tree shrew. B Quantitative analysis of SEC14L2 protein in 14 tree shrew tissues
Article
Background The product of the SEC14L2 (SEC14 Like Lipid Binding 2) gene belongs to a family of lipid-binding proteins including Sec14p, alpha-tocopherol transfer protein, and cellular retinol-binding protein. SEC14L2 expression enables replication of clinical hepatitis C virus (HCV) isolates in several hepatoma cell lines, and mutations in SEC14L2 may enhance HCV replication in vitro. The Chinese tree shrew (Tupaia belangeri chinensis) is a potential animal model for studying HCV replication, however, the cDNA sequence, protein structure, and expression of the Chinese tree shrew SEC14L2 gene have yet to be characterized. Methods and results In the present study, we cloned the full-length cDNA sequence of the SEC14L2 in the Chinese tree shrew by using rapid amplification of cDNA ends technology. This led us to determine that, this is 2539 base pairs (bp) in length, the open reading frame sequence is 1212 bp, and encodes 403 amino acids. Following this, we constructed a phylogenetic tree based on SEC14L2 molecules from various species and compared SEC14L2 amino acid sequence with other species. This analysis indicated that the Chinese tree shrew SEC14L2 protein (tsSEC14L2) has 96.28% amino acid similarity to the human protein, and is more closely related to the human protein than either mouse or rat protein. The Chinese tree shrew SEC14L2 mRNA was detected in all tissues, and showed highest expression levels in the pancreas, small intestine and trachea, however the tsSEC14L2 protein abundance was highest in the liver and small intestine. Conclusion The Chinese tree shrew SEC14L2 gene was closer in evolutionary relation to humans and non-human primates and expression of the tsSEC14L2 protein was highest in the liver and small intestine. These results may provide useful information for tsSEC14L2 function in HCV infection.
 
Distribution of PTGS2 promoter methylation in patients affected with AML and the control group
A: Different patterns of PTGS2 and VEGF-C expression in AML patients compared to the normal controls. These groups consist of Upregulated for fold changes of expression equal or more than 2, unchanged for fold changes of expression more than 0.5 and less than 2, and downregulated for fold changes of expression equal or less than 0.5 B: PTGS2 and VEGF-C mRNA mean fold changes of expression in PBMCs of AML patients and normal controls (** for p = 0.0024 and *** for p = 0.0001), C: Correlation between PTGS2 and VEGF-C expression in AML patients and normal controls (Linear regression)
Kaplan–Meier survival curves for different patterns of PTGS2 (A) and VEGF-C (B) expression (upregulated, unchanged and downregulated), and (C) PTGS2 non-methylated and methylated groups
Receiver operating characteristic (ROC), (A) ROC curves for PTGS2 methylation level of PTGS2. (B) ROC curves for PTGS2 mRNA level and (C) ROC curve for VEGF-C mRNA level differentiating AML patients and healthy controls
Article
Hereby, we aimed to investigate the expression of prostaglandin-endoperoxide synthase 2 (PTGS2) and Vascular Endothelial Factor-C (VEGF-C) besides the methylation of PTGS2 in AML patients. VEGF-C and PTGS2 expression analysis were evaluated in newly diagnosed AML patients and healthy controls by quantitative Reverse Transcriptase PCR method. Also, PTGS2 methylation status was evaluated by Methylation-Sensitive High-Resolution Melting Curve Analysis (MS-HRM). While 34% of patients were female, the mean age of the patients was 43.41 ± 17.60 years suffering mostly from M4 (48.21%) type of AML. Although methylation level between patients and controls was not significantly different, none of the normal controls showed methylation in the PTGS2 promoter. PTGS2 and VEGF-C levels were elevated in AML cases and correlated with WBC, Platelet, and Hemoglobin levels. The survival of patients with overexpressed VEGF-C and PTGS2 was poorer than others. It can be concluded that PTGS2 and especially VEGF-C expression but not PTGS2 methylation can be considered as diagnostic biomarkers for AML.
 
Primary cultured NFs and OLP AFs are of fibroblast origin Immunofluorescence for keratin and vimentin in primary cultured cells isolated from healthy controls and OLP. Scale bar: 100 μm
Expression of miR-155-5p and FAP-α is upregulated and positively correlated in OLP AFs (a, b) The relative expression of miR-155-5p and FAP-α were in NFs and OLP AFs (NEOLP and EOLP groups) (n = 15 each group) detected by qRT-PCR, and results were represented as box plots (box, 25–75%; whisker, 10–90%; line, median). (c) Pearson’s correlation test was applied to determine the correlation between FAP-α mRNA expression and miR-155-5p expression. All experiments were performed in three independent replicates. *p < 0.05; **p < 0.01; ***p < 0.001
MiR-155-5p promotes cytokine production in OLP AFs (a) NFs and OLP AFs (NEOLP and EOLP groups) (n = 15 each group) were incubated in serum-free DMEM without antibiotics, and the supernatants were collected after 24 h. (b) Transfection efficiency was detected by qRT-PCR. (c) OLP AFs (n = 3) were transfected with miR-155-5p mimic, and the culture media was collected after 48 h. (d) OLP AFs (n = 3) were transfected with miR-155-5p inhibitor and the culture media was collected after 48 h. IL-6 and IL-8 levels in the supernatants were determined by ELISA. All experiments were performed in three independent replicates. *p < 0.05; **p < 0.01; ***p < 0.001
MiR-155-5p inhibits SOCS1 expression by directly targeting its 3′-UTR (a) Schematic representation of the putative miR-155-5p-binding site in the 3′-UTR of SOCS1. Western blotting assay was used to analyze the protein level of SOCS1 in NFs and OLP AFs (n = 15 each group). β-actin was used as an endogenous control. (b) Luciferase activity was detected after co-transfection with reporter plasmid (pmiR-RB-SOCS1 3′-UTR wild or pmiR-RB-SOCS1 3′-UTR mut) and miR-155-5p transfection reagent (miR-155-5p mimic or miR-155-5p mimic NC), respectively. (c) Expression of SOCS1 in OLP AFs following miR-155-5p mimic transfection was analyzed by western blotting assay (n = 3). (d) Expression of SOCS1 in OLP AFs following transfection with miR-155-5p inhibitors or miR-155-5p inhibitors NC was analyzed by western blotting assay (n = 3). All experiments were performed in three independent replicates. *p < 0.05; **p < 0.01; ***p < 0.001
SOCS1 inhibits pro-inflammatory cytokine release in OLP AFs (a-b) Expression of SOCS1 was analyzed using western blotting assay in OLP AFs transfected with si-SOCS1 (siRNA#1 and siRNA#2) or siRNA NC (n = 3). (c-d) IL-6 and IL-8 secretion in the supernatants of OLP AFs transfected with si-SOCS1 (siRNA#1 and siRNA#2) or siRNA NC was determined by ELISA (n = 3). All experiments were performed in three independent replicates. *p < 0.05; **p < 0.01
Article
Background Oral lichen planus (OLP) is a chronic inflammatory oral mucosal disease. Cytokines are closely associated with OLP development. In addition to immune cells, fibroblasts have been reported to induce regional inflammation. MicroRNA(miR)-155-5p is reportedly increased significantly in OLP and is known to regulate inflammation. This study aimed to investigate the role of miR-155-5p in fibroblasts of OLP lesions. Methods and results Normal mucosal fibroblasts (NFs) and OLP associated-fibroblasts (OLP AFs) were isolated from the oral mucosa of 15 healthy controls and 30 OLP patients. We detected the expression of miR-155-5p and fibroblast activation protein alpha (FAP-α) using quantitative RT-PCR and analyzed their correlation. Interleukin (IL)-6 and IL-8 levels were determined using ELISA. Expression of suppressor of cytokine signaling (SOCS) 1 was analyzed by western blotting. A dual-luciferase reporter assay was performed to investigate the interaction between miR-155-5p and SOCS1. MiR-155-5p and FAP-α were significantly increased and positively correlated in OLP AFs. Overexpression of miR-155-5p in OLP AFs augmented IL-6 and IL-8 release and decreased SOCS1 expression, whereas knockdown of miR-155-5p in OLP AFs decreased IL-6 and IL-8 release. The expression of SOCS1 was downregulated in OLP AFs, and SOCS1 silencing augmented IL-6 and IL-8 production in OLP AFs. Furthermore, miR-155-5p inhibited SOCS1 expression by directly targeting its 3′-UTR in OLP AFs. Conclusions MiR-155-5p regulates the secretion of IL-6 and IL-8 by downregulating the expression of SOCS1 in activated OLP AFs. Our results provide novel insights into the pathogenesis of OLP and identify a potential new target for OLP therapy.
 
aIn vitro soybean (Glycine max L. Merr.) plants, b 14 day-old callus obtained from hypocotyls of in vitro soybean plant, c 2-mount-old soybean callus obtained from hypocotyls of in vitro soybean plant, d Soybean suspension culture obtained from hypocotyl callus
Absorbances of soybean cell culture extracts (10, 50, 70 and 96% ethanol) and concentrations (0–600 μM) of gallic acid at 765 nm. 10 EtOH 10% ethanol, 50 EtOH 50% ethanol, 70 EtOH 70% ethanol and 96 EtOH 96% ethanol extract of soybean cell culture. Total phenolic content of soybean cell culture extracts at 1 mg/ml was determined using the Folin-Ciocalteu method. Each bar represents the mean ±SD (n:3). ∗: indicates a significant difference compared to the blank. #: indicates a significant difference compared to the 10% ethanol extract (One-Way ANOVA and Tukey’s test, P value <0.05)
a Effect of soybean cell culture extracts (SCE) on cell viability of murine melanoma (B16-F10) cells. The cells were incubated with concentrations of SCE (0, 0.1, 0.3, 0.5 and 1 mg/mL), b Effects of SCE and kojic acid on melanin content in B16-F10 cells. c Effects of SCE and kojic acid on tyrosinase activity in B16-F10 murine melanoma cells, B16-F10 melanoma cells in all groups were treated with α-MSH. The cells, except for the control group, were incubated with SCE (0.3 and 1 mg/mL) and kojic acid (200 and 400 μM) in the melanin content and tyrosinase activity experiments. Each bar represents the mean ±SD (n:3). ∗: indicates a significant difference compared to the control group (One-Way ANOVA and Tukey’s test, P value <0.05)
Effect of soybean cell culture extract (SCE) on expression level of melanogenic genes, a TYR, b TRP1 c TRP2, d MITF in murine melanoma (B16-F10) cells. B16-F10 melanoma cells except for the control group were treated with α-MSH. The cells, except for the control group and group that was treated only with α-MSH were incubated with SCE (0.5 and 1 mg/mL). Data were analyzed using the 2[-ΔΔct] method and presented as the means ±SD normalized to beta-actin (β-Actin). #: indicates a significant difference compared to the control, ∗: indicates a significant difference compared to the group that was treated only with α-MSH. (One-Way ANOVA and Tukey’s test, P value <0.05)
Effects of soybean cell culture extract (SCE) and kojic acid on protein expression of TYR, TRP1, TRP2 and MITF in murine melanoma (B16-F10) cells. The cells, except for the control group were treated with 100 nm α-MSH. The cells, except for the control group and and the group that was treated only with α-MSH were incubated with SCE (0.5 and 1 mg/mL) and kojic acid (200 and 400 μM). Expression of beta-actin (β-Actin) was used as an internal control
Article
Background Hyperpigmentation, which causes excessive melanin synthesis and accumulation, is an important issue in the cosmetic industry. Since compounds developed against hyperpigmentation often come with side effects such as skin irritation and contact dermatitis, new studies focus on the use of natural agents that have no side effects. Methods and Results In this study, it was found that the effects of soybean cell culture extract (SCE) on alpha-melanocyte-stimulating hormone (α-MSH) induced melanogenesis in B16-F10 murine melanoma cells. The cells were incubated with SCE for 48 h after treatment with α‑MSH for 24 h to analysis the melanin content, cellular tyrosinase activity, and gene and protein expression. SCE at 1 mg/mL decreased melanin content and tyrosinase activity by 34% and 24%, respectively, compared to the α-MSH-treated group, which did not decrease cell viability. In addition, SCE (1 mg/mL) downregulated the expression of tyrosinase (TYR), tyrosinase-related protein (TRP)-1, tyrosinase-related protein (TRP)-2, and microphthalmia-associated transcription factor (MITF) genes 1.5-, 1.5-, 2-, and 2-fold, respectively. Furthermore, SCE inhibited the expression of TYR, TRP1, and TRP2 by decreasing the expression of MITF, as shown in a western blot assay. Conclusions This study suggests that SCE reveals dose-dependent inhibition of melanin synthesis through the suppression of tyrosinase activity as well as molecular levels of TYR, TRP1, TRP2, and MITF in B16-F10 murine melanoma cells. Therefore, SCE has the potential to be an effective and natural skin-whitening agent for application in the cosmetic industry.
 
Article
Background Hepatic lipid accumulation is one of the main pathological features of alcoholic liver disease (ALD). Metformin serves as an AMPK activator and has been shown to have lipids lowering effects in non-alcoholic fatty liver disease (NAFLD), but its role in ALD remains unclear. The purpose of this study was to explore the potential mechanism of metformin regulating lipid metabolism in ALD. Methods and results In vitro and in vivo ALD models were established using AML12 cells and C57BL/6 mice, respectively. To determine the effect of metformin on ALD in vitro, the concentration of cellular triglyceride was examined by Nile red staining and a biochemical kit. To further reveal the role of metformin on ALD in vivo, liver tissues were examined by HE and oil red O staining, and the levels of ALT and AST in serum were determined via an automatic biochemical analyzer. The expression of mRNA and proteins were measured using qRT-PCR and Western blot, respectively. The role of the LKB1/AMPK/ACC axis on metformin regulating ethanol-induced lipid accumulation was evaluated by siRNA and AAV-shRNA interference. The results showed metformin reduced the ethanol-induced lipid accumulation in AML12 cells through activating AMPK, inhibiting ACC, reducing SREBP1c, and increasing PPARα. In addition, compared with control mice, metformin treatment inhibited ethanol-induced liver triglyceride accumulation and the increase of ALT and AST in serum. Interference with LKB1 attenuated the effect of metformin on ethanol-induced lipid accumulation both in vitro and in vivo. Conclusion Metformin protects against lipid formation in ALD by activating the LKB1/AMPK/ACC axis.
 
Top-cited authors
Young Ho Lee
  • Chungnam National University
Jong Dae Ji
  • Korea University
Hong Chen
  • Northwest A & F University
Sungjae Jae Choi
  • Korea University
Li Zhang