Journal of Biomedicine and Biotechnology

Published by Hindawi Publishing Corporation
Online ISSN: 1110-7251
Strain Hhs.015T (Saccharothrix yanglingensis sp. nov.), an antagonistic endophytic Saccharothrix actinomycete isolated from roots of cucumber plants, exhibited a broad antimicrobial spectrum in vitro and was active as a biocontrol against plant diseases in field trials. The SSY medium was used for production of antimicrobial metabolites by strain Hhs.015T. However, this medium is too expensive for large-scale production. In this study, an alternative culture medium, based on agricultural waste products (e.g., apple pomace), was optimized. The results showed that the alternative medium contained 15 g apple pomace, 4 g rapeseed meal, 0.1 g KH2PO4, and 0.6 g MgSO4·7H2O in 1 L distilled water. This medium reduced the material costs by 91.5% compared to SSY medium. Response surface methodology (RSM) was used to investigate the influence of environmental variables on production of compounds of antimicrobial metabolites. The optimal conditions achieved were initial pH 7.0, medium volume of 90 mL in 250 mL flasks, rotary speed of 100 rpm, temperature 25°C, and inoculation volume of 15.8%. The antimicrobial activity was increased by 20% by optimizing the environmental parameters. The results obtained allow an efficient production of components with antimicrobial activity by strain Hhs.015T on a large scale at low costs.
Signaling pathways involved in the development of colon carcinogenesis in the DMH/AOM rat model. 
Phenotypic, genetic, and epigenetic alterations involved in multistep development of colon carcinogenesis in the DMH/AOM rat model.
Signaling pathways involved in the development of colon carcinogenesis in the DMH/AOM rat model.
The dimethyhydrazine (DMH) or azoxymethane (AOM) model is a well-established, well-appreciated, and widely used model of experimental colon carcinogenesis. It has many morphological as well as molecular similarities to human sporadic colorectal cancer (CC), which are summarized and discussed in this paper. In addition, the paper combines present knowledge of morphological and molecular features in the multistep development of CC recognized in the DMH/AOM rat model. This understanding is necessary in order to accurately identify and interpret alterations that occur in the colonic mucosa when evaluating natural or pharmacological compounds in DMH/AOM rat colon carcinogenesis. The DMH/AOM model provides a wide range of options for investigating various initiating and environmental factors, the role of specific dietary and genetic factors, and therapeutic options in CC. The limitations of this model and suggested areas in which more research is required are also discussed.
We examined whether deficiency of the GGTA1 gene in pigs altered the expression of several glycosyltransferase genes. Real-time RT-PCR and glycosyltransferase activity showed that 2 sialyltransferases [α2,3-sialyltransferase (α2,3ST) and α2,6-sialyltransferase (α2,6ST)] in the heterozygote GalT KO liver have higher expression levels and activities compared to controls. Enzyme-linked lectin assays indicated that there were also more sialic acid-containing glycoconjugate epitopes in GalT KO livers than in controls. The elevated level of sialic-acid-containing glycoconjugate epitopes was due to the low level of α-Gal in heterozygote GalT KO livers. Furthermore, proteomics analysis showed that heterozygote GalT KO pigs had a higher expression of NAD+-isocitrate dehydrogenase (IDH), which is related to the CMP-N-acetylneuraminic acid hydroxylase (CMAH) enzyme reaction. These findings suggest the deficiency of GGTA1 gene in pigs results in increased production of N-glycolylneuraminic acid (Neu5Gc) due to an increase of α2,6-sialyltransferase and a CMAH cofactor, NAD+-IDH. This indicates that Neu5Gc may be a critical xenoantigen. The deletion of the CMAH gene in the GalT KO background is expected to further prolong xenograft survival.
The major cell wall constituent of Ganoderma lucidum (G. lucidum) is β-1,3-glucan. This study examined the polysaccharide from the residues of alkaline-extracted fruiting bodies using high-performance anion-exchange chromatography (HPAEC), and it employed nuclear magnetic resonance (NMR) and mass spectrometry (MS) to confirm the structures. We have successfully isolated low-molecular-weight β-1,3-glucan (LMG), in high yields, from the waste residue of extracted fruiting bodies of G. lucidum. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay evaluated the capability of LMG to suppress H₂O₂-induced cell death in RAW264.7 cells, identifying that LMG protected cells from H₂O₂-induced damage. LMG treatment decreased H₂O₂-induced intracellular reactive oxygen species (ROS) production. LMG also influenced sphingomyelinase (SMase) activity, stimulated by cell death to induce ceramide formation, and then increase cell ROS production. Estimation of the activities of neutral and acid SMases in vitro showed that LMG suppressed the activities of both neutral and acid SMases in a concentration-dependent manner. These results suggest that LMG, a water-soluble β-1,3-glucan recycled from extracted residue of G. lucidum, possesses antioxidant capability against H₂O₂-induced cell death by attenuating intracellular ROS and inhibiting SMase activity.
Induction of second-degree thermal burns in male Wistar rats. (a) Back trichotomy by direct hair tension, (b) depth second-degree thermal burn with r = 10 mm, (c) treatment of thermal burn using 100 μL hydrogel.
Evaluation of hemagglutinating activity of 1,4 Cramoll isolectin combined to the hydrogel excipient. Cra 50: Pure Cramoll 1,4 isolectin at 50 μg/mL. Cra 50 I: Pure Cramoll 1,4 isolectin at 50 μg/mL irradiated (15 k Gy h−2). Cra IG: Pure Cramoll 1,4 isolectin at 50 μg/mL associated with hydrogel excipient and irradiated (15 k Gy h−2). Cra 100: Pure Cramoll 1,4 isolectin at 100 μg/mL. Cra 100 I: Pure Cramoll 1,4 isolectin at 100 μg/mL irradiated (15 k Gy h−2). Cra IG 100: Pure Cramoll 1,4 isolectin at 100 μg/mL associated with hydrogel excipient and irradiated (15 k Gy h−2). Hydrogel irradiated without lectin. The title was expressed as the highest dilution showing hemagglutinating activity. Values are mean ± SEM.
Clinical evaluation of second-degree burn healing in Wistar male rats. G1: experimental group treated with hydrogel containing isolectin Cramoll 1,4 at 100 μg/mL. (a) Thermal lesion aspect after 7 days: macroscopically shows thin and dry crust with detachment of edges. (b) Thermal lesion aspect after 14 days of treatment: absence of crust and the presence of scar tissue. (c) Thermal lesion aspect after 21 days of treatment: presence of scar tissue and a small detachment point of the crust. (d) Thermal lesion aspect after 28 days of treatment: presence of scar tissue only. (e) Thermal lesion aspect after 35 days of treatment: view of a discrete scar tissue. G2: control group treated by topical application of hydrogel excipient. (f) Thermal lesion aspect in control animals after 7 days: view of thin and dry crust with detachment of edges. (g) Thermal lesion aspect in control animals after 14 days: absence of crust and the presence of scar tissue. (h) Thermal lesion aspect in control animals after 21 days: presence of scar tissue, with the point of detachment of the crust. (i) Thermal lesion aspect in control animals after 28 days: presence of scar tissue and a second crust. (j) Thermal lesion aspect in control animals after 35 days: view of scar tissue.
Effect of hydrogel topical application on the burn wound expressed as percentage of wound contraction. G1 = Treatment, G2 = Control. n = 2. Values are mean ± SEM. *P < 0.05.
Epithelial tissue of rats in group 1 subjected to second-degree thermal burns. Masson's trichrome staining. 100x Magnification. (a) Normal epithelial tissue with all skin appendages. (b) Animal presenting epithelial tissue with complete destruction of the dermis and epidermis showing exudates albumin/leukocyte/macrophage intense, necrosis, edema, and crust at the 7th day after injury induction. (c) Animal at the 14th day with tissue reepithelialization, moderate autolysis, moderate exudate albumin/leukocyte/macrophage, intense neovascularization, and discrete fibroblast proliferation with the presence of loose collagen and mild fibrosis. (d) Animal at the 21st day with incomplete tissue reepithelialization, mild exudate albumin/leukocyte/macrophage, moderate neovascularization, intense fibroblastic proliferation, and presence of dense collagen, not modeled and moderate fibrosis. (e) Animal at the 28th day with complete tissue epithelialization, exudate albumin/leukocyte/macrophage discrete in the epidermis, moderate fibroblastic proliferation, presence of modeled dense collagen mesh and moderate fibrosis. (f) Animal at the 35th day with complete reepithelialization, mild fibroblastic proliferation, and presence of modeled dense collagen mesh and moderate fibrosis.
This study aimed at evaluating the use of hydrogel isolectin in the treatment of second-degree burns. Twenty male rats were randomly divided into two groups (G1 = treatment with hydrogel containing 100 μg/mL Cramoll 1,4 and G2 = Control, hydrogel). After 7, 14, 21, 28, and 35 days, animals were euthanized. On the 7th day, G1 showed intense exudates, necrosis and edema. On the 14th day, G1 showed tissue reepithelialization and moderate autolysis. On the 21st day, G1 showed intense fibroblastic proliferation, presence of dense collagen, and moderate fibrosis. On the 28th day, G1 showed complete tissue epithelialization. On the 35th day, G1 showed modeled dense collagen. The significant wound contraction was initiated from day, 14 in the G1. There were no significant differences in biochemical and hematological parameters analyzed. These results extend the potential of therapeutic applications for Cramoll 1,4 in the treatment of thermal burns.
Effect of weight of H-MCM-22 on the synthesis of 1,5- benzodiazepine.
Synthesis of 1,5-benzodiazepines using H-MCM-22 catalysts at room temperature.
Reaction mechanism of the synthesis of 1,5-benzodiazepines using H-MCM-22 catalyst at room temperature [29].
Scheme 1: Synthesis of 1,5-benzodiazepines using H-MCM-22 catalysts at room temperature.
A simple and versatile method for the synthesis of 1,5-benzodiazepines is via condensation of o-phenylenediamines (OPDA) and ketones in the presence of catalytic amount of H-MCM-22 using acetonitrile as solvent at room temperature. In all the cases, the reactions are highly selective and are completed within 1-3 h. The method is applicable to both cyclic and acyclic ketones without significant differences. The reaction proceeds efficiently under ambient conditions with good-to-excellent yields.
A naturally occurring BHT was identified in the leaves of the halophyte plant Mesembryanthemum crystallinum . This phenol was extracted in this study by two methods at the different plant growth stages. One of the methods was better for BHT extraction; the concentration of this phenol is plant growth stage dependent. In this study, the floraison stage has the highest BHT concentration. The antioxidant activity of the plant extract was not related to BHT concentration. The higher antioxidant activity is obtained at seedlings stage.
To perform cardiac imaging in mice without having to invest in expensive dedicated equipment, we adapted a clinical 1.5 Tesla (T) magnetic resonance imaging (MRI) scanner for use in a murine ischemia/reperfusion model. Phase-sensitive inversion recovery (PSIR) sequence facilitated the determination of infarct sizes in vivo by late gadolinium enhancement. Results were compared to histological infarct areas in mice after ischemia/reperfusion procedure with a good correlation (r = 0.807, P < .001). In addition, fractional area change (FAC) was assessed with single slice cine MRI and was matched to infarct size (r = -0.837) and fractional shortening (FS) measured with echocardiography (r = 0.860); both P < .001. Here, we demonstrate the use of clinical 1.5 MRI scanners as a feasible method for basic phenotyping in mice. These widely available scanners are capable of investigating in vivo infarct dimensions as well as assessment of cardiac functional parameters in mice with reasonable throughput.
Sampling process of the study subjects.
Hair mercury content and frequency of neurological signs. Dis.dom.bil. S.D.: distal dominant bilateral Sensory Disturbance S.D.: sensory disturbance.
Large-scale poisonings caused by methyl mercury (MeHg) have occurred in Japan (Minamata in the 1950s and Niigata in the 1960s) and Iraq (in the 1970s). The current WHO neurological risk standard for adult exposure (hair level: 50 μg/g) was based partly on evidence from Niigata which did not consider any cases who were diagnosed later and/or exposed to low level of MeHg (hair mercury level less than 50 μg/g). Early in the Niigata epidemic in June 1965 there were two extensive surveys. From these two surveys, we examined 103 adults with hair mercury measurement who consulted two medical institutions. We compared the prevalence and the distribution of neurological signs related to MeHg poisoning between exposure categories. We found 48 subjects with neurological signs related to MeHg poisoning who had hair mercury concentration less than 50 μg/g. Among the neurological signs, sensory disturbance of the bilateral distal extremities was observed more frequently, followed by disequilibrium, hearing impairment, and ataxia, in groups with hair MeHg concentration both below 50 μg/g and over 50 μg/g. The present study suggests the possibility that exposure to MeHg at levels below the current WHO limits could cause neurologic signs, in particular, sensory disturbance.
DNA amplification fingerprintings of breast cancer cell (MCF-7) and human mammary epithelial cell (MCF-10A). Three µL of the DAF PCR amplification reaction mixture was loaded with 3 µL of loading buffer. Electrophoresis was continued at 100 V until the dye front was approximately 1 cm from the end of the gel. The amplification fragments were separated by polyacrylamide gel (5%) electrophoresis. DNA was visualized using a fast and sensitive silver staining procedure that detects 1 pg DNA/mm band cross-section. The polymorphic marker was found at 262 bps. These results were confirmed by three additional experiments. 
Restriction mapping of the plasmid containing the expected polymorphic marker. The DNA of 30 individual colonies having the expected polymorphic marker was extracted by the DNAzol extraction method. The DNA (4 µL), restriction enzyme (EcoRI, 20 U/mL) (1 µL), buffer (10X)(2 µL), and water (13 µL) were mixed together. The reaction mixture was incubated at a 37 • C for 2 hours in a water bath. 15 µL of reaction mixture with 1 µL of loading buffer was loaded into a 1.0% agarose gel, stained with ethidium bromide (0.5 µg/mL), and viewed under UV light. These results were reproduced in three supplementary experiments. 
This study investigated the use of DNA amplification fingerprinting (DAF) to identify biomarkers useful in the elucidating genetic factors that lead to carcinogenesis. The DNA amplification fingerprinting (DAF) technique was used to generate fingerprint profiles of a normal human mammary epithelial cell line (MCF-10A) and a human breast cancer cell line (MCF-7). When compared with one another, a polymorphic biomarker gene (262 base pairs (bps)) was identified in MCF-10A but was not present in MCF-7. This gene was cloned from the genomic DNA of the MCF-10A cell line, and subjected to Genbank database analysis. The analysis of the nucleotide sequence polymorphic marker (Genbank account: AC079630) shows that this biomarker has 100% homology with the nucleotide sequence of human chromosome 12 BAC RP11-476D10 (bps 19612-19353). The nucleotide sequence was used for possible protein translation product and the result obtained indicated that the gene codes for hypothetical protein XF2620. In order to evaluate the effects that the 262 bps biomarker would have on the morphology of MCF-7 cells, it was transfected into MCF-7 cells. There were observable changes in the morphology of the transfected cells. These changes included an increase in cell elongation and a decrease in cell aggregation.
Accuracy of HPLC-UV method for canrenone.
11-α-hydroxylation of canrenone by microbial transformation.
LC-MS chromatograms of mixed standards (a) and (b) in negative-ion mode. HPLC was performed on a ZORBAX Eclipse XDB-C18 column (150 mm × 4.6 mm, 5 μm) at the column temperature of 30°C. The separation was achieved using the following gradient program: 0–40 min (10%~50% methanol), 40–60 min (50%~100% methanol). The flowrate was at 0.8 mL/min and the sample injection volume was 5.0 μL. Peak assignments: (a) canrenone; (b) 11-α-hydroxy-canrenone. MS spectrograms (A) and (B) stand for the molecular weight of the peak (a) and (b) at 357.44 and 341.40 amu, respectively.
Typical HPLC-UV chromatogram (λ = 280 nm) of (C) mixed standards of canrenone and 11-α-hydroxy-canrenone and (D) samples extracted from microbial transformed fluid. The quantitative gradient elution system consist of methanol and water. The gradient program: 0–40 min, 10%~100% methanol, and 90%~0% water. For other chromatographic conditions were the same as those in Section 3.3. Peak assignments: (a) canrenone; (b) 11-α-hydroxy-canrenone.
Representative chromatograms of substrate canrenone before transformation (α), and of bioconversion broth obtained after conversion for 48 h (β), and of blank (γ) peaks: a = canrenone, b = 11-α-hydroxy-canrenone.
A procedure for simultaneous identification and quantification of canrenone and its biotransformed product 11-α-hydroxy-canrenone by high-performance liquid chromatography with ultraviolet detector (HPLC-UVD) and mass spectrometry (LC-MS) methods was proposed. The optimal determination variables on the HPLC-UVD or LC-MS coupled with a ZORBAX Eclipse XDB-C18 column (150 mm × 4.6 mm, 5 μm) were set as follows: detection wavelength of 280 nm, mobile phase of water and methanol gradient elution, temperature for the chromatographic column of 30°C, flow rate of mobile phase of 0.8 mL/min, sample injection volume of 5 μL, and elution time of 40 min. The MS conditions were set as follows: the flow rate of sheath gas, aux gas, and sweep gas were kept at 35 arb, 5 arb, and 0 arb, respectively. The temperature of capillary was held at 300°C, and capillary voltage was set at 30.00 V. Tube lens were performed at 100.00 V. The proposed method was validated by linearity (r2 ≥ 0.9910), average recovery (94.93%, RSD1.21%), precision (RSD ≤ 1.31%), limit of detection, and limit of quantification (LOD 0.1~0.12 mg/L, LOQ 0.5~0.67 mg/L), which proved to be affordable for simultaneously determining canrenone and its bio-transformed product 11-α-hydroxy-canrenone.
The Tumor inhibition of the Hca/FAP solid tumor after treatment.
The chemical structure of CH used in the present study.
Mean fluorescence intensities of CD8+ cells were linearly correlated to the dose of Rho123 administered (r2 = 0.9942). The treatment protocol and experimental procedures are described in the text. Responses to treatments were significantly different from each other (P < .05).
Accumulation of Rho123 in CD8+ cells by different doses of reversors with or without Rho123 at a dose of 2.5 mg/kg in vivo. The increase of the mean fluorescence intensity that reflects the efflux is inhibited. *P < 0.05, different from the control. The results are the mean ± SD of at least 4 independent experiments.
Rho123 accumulations in CD8+ cells ex vivo, where the grey lines stand for cells treated with Rho123 plus the vehicle and it was set as the control in the four sub-figures and the black lines represent cells treated with Rho123 in the presence of various concentrations reversors: A. VER 5.0 μM, B. CH 2.5 μM, C. CH 5.0 μM, D. CH 10.0 μM. This figure is representative of at least four independent experiments.
The purpose of this study was the use of rhodamine 123 (Rho123) accumulation in peripheral blood CD8(+)cells as a surrogate indicator to evaluate the modulating effect of P-glycoprotein (P-gp) inhibitors in the multidrug resistance (MDR) tumor-bearing mouse model. Rho123 was administered to mice, and the fluorescence level in CD8(+) cells was measured. Cepharanthine hydrochloride (CH) and verapamil (VER), two P-gp inhibitors, were administered to mice 1 hour prior to Rho123 administration in vivo or added to peripheral blood 1 hour prior to Rho123 addition ex vivo. The tumor inhibition effect of 5-fluorouracil/adriamycin/cisplatin (FAP) protocol plus CH was also investigated. A concentration- or dose-response relationship was shown between the concentration and dose of CH and Rho123 accumulation or the antitumor activity. In conclusion, the measurement of Rho123 accumulation in CD8(+) cells provides a surrogate assay for the screening of candidate P-gp inhibitors in preclinical trials, and CH is effective in modulating P-gp-mediated MDR in vivo.
Activation of hedgehog (HH) pathway signaling is observed in many tumors. Due to a feedback loop, the HH receptor Patched (PTCH-1) is overexpressed in tumors with activated HH signaling. Therefore, we sought to radiolabel the PTCH-1 ligand sonic (SHH) for detection of cancer cells with canonical HH activity. Receptor binding of ¹³¹I-SHH was increased in cell lines with high HH pathway activation. Our findings also show that PTCH-1 receptor expression is decreased upon treatment with HH signaling inhibitors, and receptor binding of ¹³¹I-SHH is significantly decreased following treatment with cyclopamine. In vivo imaging and biodistribution studies revealed significant accumulation of ¹³¹I-SHH within tumor tissue as compared to normal organs. Tumor-to-muscle ratios were approximately 8 : 1 at 5 hours, while tumor to blood and tumor to bone were 2 : 1 and 5 : 1, respectively. Significant uptake was also observed in liver and gastrointestinal tissue. These studies show that ¹³¹I-SHH is capable of in vivo detection of breast tumors with high HH signaling. We further demonstrate that the hedgehog receptor PTCH-1 is downregulated upon treatment with hedgehog inhibitors. Our data suggests that radiolabeled SHH derivatives may provide a method to determine response to SHH-targeted therapies.
Demographic and clinical data of study subjects.
Qualitative analysis of Tables 1 and 2.
A 27-year-old woman (patient 15) who had stage 4 CKD presented with stage 1 hypertension alone due to bilateral adrenal hyperplasia, whose PAC was elevated, but whose serum potassium level was normal, whose PRA was nonsuppressive, whose ARR was negative, whose confirmatory testing was negative, whose bilateral adrenal lesions had normal appearing on CT (a) and faint uptakes on planar imaging (b) but true positive on SPECT (c) and coronal SPECT/CT (d) imaging. After treatment with 25 mg of spironolactone, her BP and PAC were normalized.
Accumulating evidence has shown the adverse effect of long-term hyperaldosteronism on cardiovascular morbidity that is independent of blood pressure. However, the diagnosis of primary aldosteronism (PA) remains a challenge for patients who present with subtle or atypical features or have chronic kidney disease (CKD). SPECT/CT has proven valuable in the diagnosis of a number of conditions. The aim of this study was to determine the usefulness of I-131 NP-59 SPECT/CT in patients with atypical presentations of PA and in those with CKD. The records of 15 patients with PA were retrospectively analyzed. NP-59 SPECT/CT was able to identify adrenal lesion(s) in CKD patients with suspected PA. Patients using NP-59 SPECT/CT imaging, compared with those not performing this procedure, significantly featured nearly normal serum potassium levels, normal aldosterone-renin ratio, and smaller adrenal size on CT and pathological examination and tended to feature stage 1 hypertension and non-suppressed plasma renin activity. These findings show that noninvasive NP-59 SPECT/CT is a useful tool for diagnosis in patients with subclinical or atypical features of PA and those with CKD.
Regional distribution of the transversal stiffness along the sarcomere of isolated Triton-treated Sol muscle fibers in liquid in relaxed (1), calcium activated (2), and rigor (3) states after 14-day gravitational unloading and 3-day reloading; A: group “Control,” B: group “14-HS,” C: group “14-HS + 3-R.”
Protein content in the rat's Sol after 14-day gravitational unloading and 3-day reloading; (a) beta-actin, (b) alpha-actinin-4; *P < 0.05 as compared to the group “Control,” $P < 0.05 as compared to the group “14-HS.”
The aim of the work was to analyze the structural changes in different parts of the sarcolemma and contractile apparatus of muscle fibers by measuring their transversal stiffness by atomic force microscopy in a three-day reloading after a 14-day gravity disuse, which was carried out by hind-limbs suspension. The object of the study was the soleus muscle of the Wistar rat. It was shown that after 14 days of disuse, there was a reduction of transversal stiffness of all points of the sarcolemma and contractile apparatus. Readaptation for 3 days leads to complete recovery of the values of the transversal stiffness of the sarcolemma and to partial value recovery of the contractile apparatus. The changes in transversal stiffness of sarcolemma correlate with beta-actin and alpha-actinin-4 in membrane protein fractions.
Clinical, morphologic, and cytogenetic characteristics of patients with myelodysplastic syndrome and isolated trisomy 14.
Clinical, morphologic, and cytogenetic characteristics of patients with acute myeloid leukemia and isolated trisomy 14.
Clinical data for patients with myelodysplastic syndrome and isolated trisomy 14 or diploid karyotype.
Overall survival of patients with myeloid neoplasms associated with isolated trisomy 14.
Overall survival of patients with myelodysplastic syndrome and trisomy 14 versus diploid karyotype.
Trisomy 14 is a rare recurrent cytogenetic abnormality in myeloid neoplasms; however, its clinicopathologic features have not been well described. We report the clinicopathologic, immunophenotypic, and molecular genetic features of 16 cases of myeloid neoplasms with isolated trisomy 14. Our results show that cases with isolated trisomy 14 encompass a heterogeneous group of myeloid neoplasms including myelodysplastic syndrome (MDS, 44%), myelodysplastic/myeloproliferative neoplasms (31%), and acute myeloid leukemia (25%). The patients are usually elder (median age 71 years), and there is a male predominance (82%). Multilineage dysplasia is noted in all cases. Oncogenic mutations of genes involved in cell proliferation and/or survival rarely occur. Compared with cases of MDS with diploid karyotype, patients of MDS with isolated trisomy 14 demonstrate a similar overall survival and rate of leukemia transformation.
The selected fermentation factors and their assigned levels.
Impact of selected fermentation-factor-assigned level on 2,3-butanediol yield by K. oxytoca. Impact of selected-factor-assigned levels on 2,3-butanediol yield by K. oxytoca. X-axis represents assigned levels of selected factor and Y-axis represents 2,3-butanediol yield. (a) Glucose, (b) acetic acid, (c) succinic acid, (d) pH, (e) temperature, (f)  mixing intensity, and (g) inoculum size G (- - -) indicates average 2,3-butanediol yield during experimentation and (—) indicates individual factors contribution 2,3-butanediol yield during experimentation.
The relative influence of factors and interaction.
Optimal operating parameters of 2,3-Butanediol production using Klebsiella oxytoca under submerged culture conditions are determined by using Taguchi method. The effect of different factors including medium composition, pH, temperature, mixing intensity, and inoculum size on 2,3-butanediol production was analyzed using the Taguchi method in three levels. Based on these analyses the optimum concentrations of glucose, acetic acid, and succinic acid were found to be 6, 0.5, and 1.0 (% w/v), respectively. Furthermore, optimum values for temperature, inoculum size, pH, and the shaking speed were determined as 37°C, 8 (g/L), 6.1, and 150 rpm, respectively. The optimal combinations of factors obtained from the proposed DOE methodology was further validated by conducting fermentation experiments and the obtained results revealed an enhanced 2,3-Butanediol yield of 44%.
The main objective of this investigation was to determine the absorption, distribution, excretion, and pharmacokinetics of the antimalarial drug pyronaridine tetraphosphate (PNDP) in Sprague-Dawley rats. Following oral administration of a single dose (10 mg/Kg) of 14C-PNDP, it was observed that the drug was readily absorbed from the small intestine within 1 hour following oral administration and was widely distributed in most of the tissues investigated as determined from the observed radioactivity in the tissues. The peak value of the drug in the blood was reached at around 8 hours postadministration, and radioactivity was detected in most of the tissues from 4 hours onwards. 14C-PNDP showed a poor permeability across the blood-brain barrier, and the absorption, distribution, and excretion of 14C-PNDP were found to be gender-independent as both male and female rats showed a similar pattern of radioactivity. Excretion of the drug was predominantly through the urine with a peak excretion post 24 hours of administration. A small amount of the drug was also excreted in the feces and also in the breath. It was found that the Cmax, AUC (0-inf), and Tmax values were similar to those observed in the Phase II clinical trials of pyronaridine/artesunate (Pyramax) conducted in Uganda.
The phenotype of the patient.
(a) A partial R-banding karyotype; (b) a partial GTG-banding karyotype.
Metaphases hybridized (a) with WCP15; (b) with
D15S10/PLM. Two normal copies of chromosome 15 displayed the
expected centromeric signals and signals mapped at q11-q13 and
q22. The der(15) has two copies of the centromeric signal and a
duplicated signal for D15S10, suggesting tetrasomy 15q11-q13.
We report the case of a Moroccan boy with mental retardation, hyperactivity, epilepsy, developmental problems and behavioural disorders. Cytogenetic analysis showed the presence of a supernumerary marker chromosome. Molecular cytogenetics allowed us to determine the marker as an inverted duplication of chromosome 15. It is the first case of a Moroccan patient with tetrasomy 15q in which fluorescence in situ hybridization (FISH) enabled us to specify the diagnosis. Interestingly, this patient has an infantile autism with cytogenetic abnormalities on chromosomal region 15q11-q13 as reported in patients with Autistic Disorder.
Coupling ratio optimization for HPV L1 VLPs and microspheres. (a) HPV-16 L1 VLP and #38 microspheres. (b) HPV-18 L1 VLP and #53 microspheres.
Coupling ratio optimization for neutralizing mAbs biotinylation. (a) H16.V5 biotinylation. (b) H18.J4 biotinylation.
Cross-neutralizing reactivity investigation.
2-plex standard curve for the Luminex-based immunoassay.
Human papillomavirus (HPV) L1 virus-like particles (VLPs) were proven an effective vaccine candidate to prevent against HPV-16 and -18 infections. In order to evaluate the potency of our produced HPV-16 and -18 L1 VLPs-based vaccine candidates, also to quantify neutralizing antibodies induced by them, a 2-plex Luminex-based competitive immunoassay was developed. Unlike the published paper, the no-biotin conjugated neutralizing mAbs spiked normal human serum (NHS) was used for standard curve preparation, while phycoerythrin (PE) was not labeled directly to neutralizing mAbs for signaling. After the coupling optimization of VLPs to microspheres and the neutralizing mAbs biotinylation, the 2-plex standard curve was prepared with good fit and high dynamic range. In addition, no cross-reactivity was also confirmed. The 2-plex Luminex-based immunoassay represents good potential not only for vaccine candidate's evaluation but also for its further clinical use.
Demographic characteristics of preeclamptic patients and controls. Preeclampsia N = 125 Controls N = 132 Odds ratio (95% CI) P value χ 2 
Multiple logistic regression analysis with forward stepwise selection (Wald). 
Some evidence suggests that a variety of genetic factors contributed in pathogenesis of the preeclampsia. The aim of this study was to assess the association between the angiotensin-converting enzyme (ACE) I/D and angiotensin II type1 receptor A1166C polymorphisms with preeclampsia. This study was performed in 125 preeclamptic pregnant women and 132 controls. The I/D Polymorphism of the ACE gene was assessed by polymerase chain reaction and the A1166C Polymorphism of the AT1R gene was determined by restriction fragment length polymorphism. The genotype and allele frequencies of I/D polymorphism differed between two groups. The risk of preeclampsia was 3.2-fold in pregnant women with D allele (OR, 3.2 [95% CI, 1.1 to 3.8]; P = 0.01). The distribution of the AT1R gene A1166C polymorphism was similar in affected and control groups. Our results supported that presence of the I/D polymorphism of ACE gene is a marker for the increased risk of preeclampsia.
Schematic diagram of the synthesis of the nucleolin aptamer containing 5-(N-benzylcarboxyamide)-2′-deoxyuridine. Z indicates where the thymidines in AS1411 oligonucleotides were substituted with 5-(N-benzylcarboxyamide)-2′-deoxyuridine.
Fluorescence analysis of Cy3-(5-BzdU)-modified-AS1411 compounds targeting C6 cells. The fluorescence of the 47 different Cy3-(5-BzdU)-modified-AS1411 compounds that were bound and targeted the nucleolin protein in the C6 cells was quantified. The X-axis indicates numbers of compounds. AS represents the Cy3-labeled AS1411 and Mt the mutant form of the original AS1411 labeled with Cy3. These data are presented as the means ± SD calculated from quadruple wells. All fluorescence data were obtained at an excitation of 535 nm and emission of 570 nm.
Confocal microscopy imaging of the Cy3-(5-BzdU)-modified-AS1411 targeting the C6 cells. numbers 29, 41, 12, and 26, Cy3-AS1411, and the mutant form of AS1411 labeled with Cy3 were visualized in the C6 cells. The confocal images were magnified to 200x. The nucleus was stained by DAPI (emission: 460 nm, blue color) and fluorescent imaging of the targeting of the C6 cells was visualized by a red color. Fluorescence images were acquired at an excitation of 535 nm and emission of 570 nm.
Fluorescence analysis of numbers 29, 41, 12, and 26, Cy3-AS1411, and the mutant form of AS1411 labeled with Cy3. (a) Quantitative fluorescence intensity in HeLa and CHO cells. Data are represented as means ± standard error of means (*P < .05, **P < .005 unpaired t-test). (b) Confocal microscopy imaging in HeLa cells. (c) Confocal microscopy imaging in CHO cells. (d) Antiproliferation effects were measured by MTT assay. 4 μM of each compounds was treated at 2 × 105 C6 cells per well. Data are represented as means ± standard error of means (  **  P < .005 unpaired t-test).
Correlation between the structure and activity of the 5-BzdU-modified-AS1411. The position and structure of the chemical modification in numbers 12, 29, and 26 and the original sequence of AS1411 were drawn in G-quadruplex structure that normally formed by dimerization of AS1411 aptamers to bind to nucleolin protein.
Chemically modified nucleotides have been developed and applied into SELEX procedure to find a novel type of aptamers to fit with targets of interest. In this study, we directly performed chemical modification of 5-(N-benzylcarboxyamide)-2'-deoxyuridine (called 5-BzdU) in the AS1411 aptamer, which binds to the nucleolin protein expressed in cancer cells. Forty-seven compounds of AS1411-containing Cy3-labeled 5-BzdU (called Cy3-(5-BzdU)-modified-AS1411) were synthesized by randomly substituting thymidines one to twelve in AS1411 with Cy3-labeled 5-BzdU. Both statistically quantified fluorescence measurements and confocal imaging analysis demonstrated at least three potential compounds of interest: number 12, 29 and 41 that significantly increased the targeting affinity to cancer cells but no significant activity from normal healthy cells. These results suggest that the position and number of substituents in AS1411 are critical parameters to improve the aptamer function. In this study, we demonstrated that chemical modification of the existing aptamers enhanced the binding and targeting affinity to targets of interest without additional SELEX procedures.
FISH analysis of MYC copy number in gastric cancer cell lines, in their parental primary tumors and in control samples.
Distribution of cells according to (a) MYC signals in AGP01 parental tumor and cell line passages; (b) MYC signals in ACP02 parental tumor and cell line passages; (c) MYC signals in ACP03 parental tumor and cell line passages; (d) mean of MYC signals of AGP01, ACP02, and ACP03 parental tumor and cell line passages; (e) TP53/chr17 signals in AGP01 parental tumor and cell line passages; (f) TP53/chr17 signals in ACP02 parental tumor and cell line passages; (g) TP53/chr17 signals in ACP03 parental tumor and cell line passages; (h) mean of TP53/chr17 signals of AGP01, ACP02, and ACP03 parental tumor and cell line passages.
Fluorescence in situ hybridization assay. (a) Interphase nuclei presenting two MYC signals from normal gastric mucosa; (b) interphase nuclei presenting 2–5 MYC signals from ACP02 parental primary tumor; (c) interphase nuclei presenting MYC signal number alterations, including high amplification, from the 85th passage of AGP01 cell line; (d) interphase and metaphase cells presenting two copies of chr17/TP53 from lymphocytes control, with the green spots representing the 17 centromere probe and the red representing the TP53 gene probe; (e) interphase nuclei presenting two signals of chr17 and two or one TP53 signal(s) from ACP02 parental primary tumor; (f) interphase nuclei presenting three signals for chr17 and two TP53 signals from the 85th passage of ACP03 cell line.
We evaluated whether MYC, TP53, and chromosome 17 copy-number alterations occur in ACP02, ACP03, and AGP01 gastric cancer cell lines and in their tumor counterpart. Fluorescence in situ hybridization for MYC and TP53 genes and for chromosome 17 was applied in the 6th, 12th, 60th, and 85th passages of the cell lines and in their parental primary tumors. We observed that three and four MYC signals were the most common alterations in gastric cell lines and tumors. ACP02 presented cells with two copies of chr17 and loss of one copy of TP53 more frequently than ACP03 and AGP01. Only ACP03 and AGP01 presented clonal chr17 trisomy with three or two TP53 copies. The frequency of MYC gain, TP53 loss, and chromosome 17 trisomy seems to increase in gastric cell lines compared to their parental tumors. Our findings reveal that these cell lines retain, in vitro, the genetic alterations presented in their parental primary tumors.
Flower-like collagen fibrils (▸) and irregular interfibrillar spaces.
Calcified microcyst in an elastic fiber.
Globules of presumably hyaluronic acid and granulofilamentous meshwork inside the collagen bundles.
The distinction between the Ehlers-Danlos syndrome hypermobile type (EDSH) and the benign joint hypermobility syndrome (BJHS) is unclear. The aim of the present study was to compare skin ultrastructural abnormalities of EDSH and BJHS among different families. Skin of 23 EDSH, 27 BJHS, and 41 asymptomatic subjects from 17 families was examined using transmission electron microscopy. Similar ultrastructural abnormalities were found irrespective of the Beighton score. Flower-like collagen fibrils represented the key change and elastic fibers were altered as well. Beighton score is a clinical parameter rating joint mobility that appeared unrelated to quantitative and qualitative collagen ultrastructural alterations in the skin. Some EDSH family members fit with BJHS diagnosis. BJHS possibly represents a mild variant of EDSH.
The IL-17 response is amplified in SLE. Nucleic acid-containing immune complexes and other inflammatory stimuli (e.g., cytokines) induce the production of pro-inflammatory cytokines by dendritic cells. These factors, along with others not yet identified, favor the generation of pro-inflammatory IL-17-producing T cell subsets (i.e., TH17 and DN T cells) able to migrate to target organs and inflict damage.  Produced IL-17 amplifies the inflammatory response and stimulates other cell types (e.g., B cells).
IL-17 is a cytokine with powerful proinflammatory activity. Production of IL-17 is abnormally increased in patients with systemic lupus erythematosus (SLE), a multiorgan chronic autoimmune disease. In patients with SLE, CD3+CD4(-)CD8(-) (double negative) T cells are an important source of IL-17. IL-17 produced by double negative and CD4 T cells participates in the pathogenesis of the disease. IL-17-producing T cells are present in the kidneys of patients with lupus nephritis. IL-17 increased production in patients with SLE can amplify the immune response by increasing target organ inflammation and damage and by augmenting the production of antibodies by B cells.
Association of miR-17 expression in human glioma tissues with different clinicopathological features.
Univariate and multivariate analyses of different prognostic parameters in patients with gliomas by Cox regression analysis.
Subgroup log-rank analysis of miR-17 expression and prognosis in patients with different pathological grades.
microRNA-17 (miR-17) expression in 108 glioma and 20 normal brain tissues detected by quantitative real-time polymerase chain reaction (qRT-PCR) analysis. Compared with normal brain tissues, miR-17 expression was significantly higher in glioma tissues (mean ± SD: 5.1 ± 2.5 versus 0.9 ± 0.6, P < 0.001, Figure 1), corresponding to the glioma WHO grades. The statistic results showed that its expression in high-grade (III-IV; mean ± SD: 6.2 ± 2.2) and low-grade (I-II; mean ± SD: 2.4 ± 0.8) gliomas were both significantly higher than that in normal brains tissues (both P < 0.001). Moreover, there was also a significant difference in miR-17 expression between high-grade (III-IV) and low-grade (I-II) glioma tissue specimens (P < 0.001).
Kaplan-Meier survival curves for glioma patients in high and low microRNA-17 (miR-17) expression groups. (a) The 5-year overall survival rate of glioma patients with high miR-17 expression was significantly lower than those with low miR-17 expression (P = 0.001). (b) The 5-year overall survival rate of grade I~II glioma patients with high miR-17 expression had no significant differences from those with low miR-17 expression (P = 0.1). (c) The 5-year overall survival rate of grade III~IV glioma patients with high miR-17 expression was significantly lower than those with low miR-17 expression (P < 0.001).
Aim: To investigate the clinical significance of microRNA-17 (miR-17) expression in human gliomas. Methods: Quantitative real-time polymerase chain reaction (qRT-PCR) analysis was used to characterize the expression patterns of miR-17 in 108 glioma and 20 normal brain tissues. The associations of miR-17 expression with clinicopathological factors and prognosis of glioma patients were also statistically analyzed. Results: Compared with normal brain tissues, miR-17 expression was significantly higher in glioma tissues (P < 0.001). In addition, the increased expression of miR-17 in glioma was significantly associated with advanced pathological grade (P = 0.006) and low Karnofsky performance score (KPS, P = 0.01). Moreover, Kaplan-Meier survival and Cox regression analyses showed that miR-17 overexpression (P = 0.008) and advanced pathological grade (P = 0.02) were independent factors predicting poor prognosis for gliomas. Furthermore, subgroup analyses showed that miR-17 expression was significantly associated with poor overall survival in glioma patients with high pathological grades (for grade III~IV: P < 0.001). Conclusions: Our data offer the convinced evidence that the increased expression of miR-17 may have potential value for predicting poor prognosis in glioma patients with high pathological grades, indicating that miR-17 may contribute to glioma progression and be a candidate therapeutic target for this disease.
In the present study it is shown that poloxamer 188, added before or immediately after an electrical pulse used for electroporation, decreases the number of dead cells and at the same time does not reduce the number of reversible electropores through which small molecules (cisplatin, bleomycin, or propidium iodide) can pass/diffuse. It was suggested that hydrophobic sections of poloxamer 188 molecules are incorporated into the edges of pores and that their hydrophilic parts act as brushy pore structures. The formation of brushy pores may reduce the expansion of pores and delay the irreversible electropermeability. Tumors were implanted subcutaneously in both flanks of nude mice using HeLa cells, transfected with genes for red fluorescent protein and luciferase. The volume of tumors stopped to grow after electrochemotherapy and the use of poloxamer 188 reduced the edema near the electrode and around the subcutaneously growing tumors.
Comparison of interscapular BAT uptake in mice receiving different interventions from microPET sagittal images. For visual analysis, microPET images showed that mouse interscapular BAT 18F-FDG uptake (arrow) varied under different interventions. (c) Cold plus NE interventions showed highest BAT uptake (R = 15.64 ± 5.58, P = 2.19 × 10−6) compared to (b) cold exposure (R = 10.22 ± 4.13, P = 1.96 × 10−4) and (a) control (R = 4.08 ± 1.32), while (d) warming (R = 2.13 ± 0.43, P = .001) and (e) propranolol (R = 1.30 ± 0.16, P = .027) showed decreased BAT uptake in cold pre-exposure mice.
Comparison of the effects of different interventions to stimulate interscapular BAT in mice. (a) BAT FDG uptake ratio in cold, cold + NE and control group. Cold together with NE intervention showed the highest uptake in BAT. (b) BAT FDG uptake ratio in NE, epinephrine, isoprenaline, and control group. All these pharmaceutics increase BAT activity, but only NE showed significant difference (P = .002).
Comparison of the effects of warming and propranolol to inhibit interscapular BAT in mice. (a) BAT FDG uptake ratio under warm intervention. Warming could significantly decrease BAT metabolism in mice with or without cold pre-exposure (P = .001 and .017, resp.). (b) BAT FDG uptake ratio under propranolol intervention. Propranolol could significantly decrease BAT activity in cold pre-exposure mice (P = .027).
Brown adipose tissue (BAT) is emerging as a potential target for treating human obesity. It has been indicated that BAT is rich in innervations of sympathetic nerve control. Using (18)F-FDG microPET imaging, this study aims at evaluating how factors related to sympathetic activation/inhibition changed BAT metabolism of mice. BAT (18)F-FDG uptake were semiquantitatively evaluated in different groups of mice under temperature (cold or warm stimulus) or pharmacological interventions (norepinephrine, epinephrine, isoprenaline, or propranolol) and were compared with the corresponding controls. It was found that BAT activation can be stimulated by cold exposure (P = 1.96 × 10(-4)), norepinephrine (P = .002), or both (P = 2.19 × 10(-6)) within an hour before (18)F-FDG injection and can also be alleviated by warming up (P = .001) or propranolol lavage (P = .027). This preliminary study indicated that BAT function could be evaluated by (18)F-FDG PET imaging through short-term interventions, which paved the way for further investigation of the relationship between human obesity and BAT dysfunction.
Capture efficiency of 18S and 28S ribosomal RNA and recovery of E. coli RNA using a single-step capture protocol on a selective solid phase.
The abundance of mammalian 18S and 28S ribosomal RNA can decrease the detection sensitivity of bacterial or viral targets in complex host-pathogen mixtures. A method to capture human RNA in a single step was developed and characterized to address this issue. For this purpose, capture probes were covalently attached to magnetic microbeads using a dendrimer linker and the solid phase was tested using rat thymus RNA (mammalian components) with Escherichia coli RNA (bacterial target) as a model system. Our results indicated that random capture probes demonstrated better performance than specific ones presumably by increasing the number of possible binding sites, and the use of a tetrame-thylammonium-chloride (TMA-Cl-) based buffer for the hybridization showed a beneficial effect in the selectivity. The subtraction efficiency determined through real-time RT-PCR revealed capture-efficiencies comparable with commercially available enrichment kits. The performance of the solid phase can be further fine tuned by modifying the annealing time and temperature.
In skeletal muscle fibers, forces must be transmitted between the plasma membrane and the intracellular contractile lattice, and within this lattice between adjacent myofibrils. Based on their prevalence, biomechanical properties and localization, desmin and keratin intermediate filaments (IFs) are likely to participate in structural connectivity and force transmission. We examined the passive load-bearing response of single fibers from the extensor digitorum longus (EDL) muscles of young (3 months) and aged (10 months) wild-type, desmin-null, K19-null, and desmin/K19 double-null mice. Though fibers are more compliant in all mutant genotypes compared to wild-type, the structural response of each genotype is distinct, suggesting multiple mechanisms by which desmin and keratin influence the biomechanical properties of myofibers. This work provides additional insight into the influences of IFs on structure-function relationships in skeletal muscle. It may also have implications for understanding the progression of desminopathies and other IF-related myopathies.
Accumulation coefficient and translocation coefficient of inbred lines.
In the last two decades, the accumulation of heavy metal in crop grains has become the study hotspot. In this study, 19 representative elite maize inbred lines and 3 hybrid varieties were investigated at the seedling stage, which can accumulate Pb and Cd in the stems and leaves, respectively. The results demonstrated that significant differences are among inbred lines for accumulation of heavy metals, implying that the Cd accumulation is significant correlation between the male parents and their hybrids and some inbred lines have been selected for cross-breeding with low Pb or Cd accumulation, such as S37, 9782, and ES40; Moreover, some inbred lines could be suitable for phytoremediation species for soil bioremediation with high levels of Pb and Cd accumulation, including 178, R08, 48-2, and Mo17ht.
ORFs identified on plasmid pYe4449-2.
Yersinia enterocolitica biotype 1A strain 07-04449 adhering to eukaryotic cells. Rat intestinal IEC-6 cells (a) and human intestinal Int-407 cells (b) were infected with strain 07-04449 at a multiplicity of infection (MOI) of 50, stained with Giemsa and subjected to microscopy.
Maps of plasmids pYe4449-1 and pYe4449-2 
					. Open reading frames (ORFs) with attributable functions and e-values below e-20 are indicated by black arrows, ORFs with significant homology to database entries (e-values below e-10) but unclear functions are indicated by grey arrows, and ORFs without significant homology (e-values above 0.1) to database entries are indicated by white arrows. Details about identified ORFs are given in Tables 1  and 2.
ORF7 of plasmid pYe4449-2 encodes a putative new repeat protein with a high content of phosphorylatable amino acids. (a) Scheme of the arrangement of the ORF7-encoded hypothetical protein. The nonameric repeat module “RTDSLGNTY” occurring twice in each repeat is indicated by dark grey boxes. (b) Alignment of the protein repeat segments encoded by ORF7, residues identical in all three repeats are highlighted against a dark grey background and residues identical in two of the three repeats are highlighted against a light grey background. The repeat module “RTDSLGNTY” occurring twice in each repeat is indicated by a bar below the consensus sequence. Below these bars, the interspecies repeat motif “DxxGN(x)nDxxGN” is given as deduced from the closest homologues of ORF 7 (BACOVA_04330; NE2449; MPG2080_02246). Serine and threonine residues most likely to be phosphorylated in a eukaryotic background according to NetPhos 2.0, server predictions (score 0.968 and above) were indicated by circles.
Detection of epitope-tagged ORF3 expressed at 4°C. A transposon mutant derivative of isolate 07-04449 carrying plasmid pYe4449-2/ORF3::Tn5 was transformed with plasmid pACYC184/ORF3-RGS(H)6 and pACYC184, respectively, and cultured over night at 4°C in LB broth. Bacterial pellet fractions “P” and TCA-precipitated supernatant fractions “S” were subjected to denaturing SDS-PAGE. Subsequently, the gel was Western-blotted followed by chromogenic detection using a monoclonal antibody directed against the RGS(H)4 epitope. The arrow indicates the ORF3-RGS(H)6 product.
We report the nucleotide sequence of two novel cryptic plasmids (4357 and 14 662 base pairs) carried by a Yersinia enterocolitica biotype 1A strain isolated from pork. As distinguished from most biotype 1A strains, this isolate, designated 07-04449, exhibited adherence to eukaryotic cells. The smaller plasmid pYe4449-1 carries five attributable open reading frames (ORFs) encoding the first CcdA/CcdB-like antitoxin/toxin system described for a Yersinia plasmid, a RepA-like replication initiation protein, and mobilizing factors MobA and MobC. The deduced amino acid sequences showed highest similarity to proteins described in Salmonella (CcdA/B), Klebsiella (RepA), and Plesiomonas (MobA/C) indicating genomic fluidity among members of the Enterobacteriaceae. One additional ORF with unknown function, termed ORF5, was identified with an ancestry distinct from the rest of the plasmid. While the C+G content of ORF5 is 38.3%, the rest of pYe4449-1 shows a C+G content of 55.7%. The C+G content of the larger plasmid pYe4449-2 (54.9%) was similar to that of pYe4449-1 (53.7%) and differed from that of the Y. enterocolitica genome (47.3%). Of the 14 ORFs identified on pYe4449-2, only six ORFs showed significant similarity to database entries. For three of these ORFs likely functions could be ascribed: a TnpR-like resolvase and a phage replication protein, localized each on a low C+G island, and DNA primase TraC. Two ORFs of pYe4449-2, ORF3 and ORF7, seem to encode secretable proteins. Epitope-tagging of ORF3 revealed protein expression at 4 degrees C but not at or above 27 degrees C suggesting adaptation to a habitat outside swine. The hypothetical protein encoded by ORF7 is the member of a novel repeat protein family sharing the DxxGN(x)(n)DxxGN motif. Our findings illustrate the exceptional gene pool diversity within the species Y. enterocolitica driven by horizontal gene transfer events.
A serum ELISA using a monoclonal antibody that detects a MUC5AC-related antigen (NPC-1C antigen) expressed by pancreatic and colorectal cancer was developed. The NPC-1C antibody reacts with specific epitopes expressed by tumor-associated MUC5AC that does not appear on MUC5AC from normal tissues. Based on observations of a highly specific antibody, we tested the ELISA to differentiate serum from healthy blood donors compared to serum from patients with colorectal or pancreatic cancer. Additionally, patient tumor tissue was stained to examine the expression pattern of MUC5AC-related antigen in pancreatic and colorectal cancers. The results indicate the NPC-1C antibody ELISA distinguished serum of cancer patients from normal donors with very good sensitivity and specificity. Most patient's tumor biopsy exhibited NPC-1C antibody reactivity, indicating that tumor-associated MUC5AC antigen from tumor is shed into blood, where it can be detected by the NPC-1C antibody ELISA. This serum test provides a new tool to aid in the diagnosis of these cancers and immune monitoring of cancer treatment regimens.
There is a traditional belief in the Middle East that camel milk may aid in prevention and treatment of numerous cases of cancer yet, the exact mechanism was not investigated. Therefore, we examined the ability of camel milk to modulate the expression of a well-known cancer-activating gene, Cytochrome P450 1a1 (Cyp1a1), and cancer-protective genes, NAD(P)H:quinone oxidoreductase 1 (Nqo1) and glutathione S-transferase a1 (Gsta1), in murine hepatoma Hepa 1c1c7 cell line. Our results showed that camel milk significantly inhibited the induction of Cyp1a1 gene expression by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the most potent Cyp1a1 inducer and known carcinogenic chemical, at mRNA, protein, and activity levels in a concentration-dependent manner. In addition, camel milk significantly decreased the xenobiotic responsive element (XRE)-dependent luciferase activity, suggesting a transcriptional mechanism is involved. Furthermore, this inhibitory effect of camel milk was associated with a proportional increase in heme oxygenase 1. On the other hand, camel milk significantly induced Nqo1 and Gsta1 mRNA expression level in a concentration-dependent fashion. The RNA synthesis inhibitor, actinomycin D, completely blocked the induction of Nqo1 mRNA by camel milk suggesting the requirement of de novo RNA synthesis through a transcriptional mechanism. In conclusion, camel milk modulates the expression of Cyp1a1, Nqo1, and Gsta1 at the transcriptional and posttranscriptional levels.
Mycobacterium bovis Bacillus Calmette-Guérin (BCG) as a live vector of recombinant bacterial vaccine is a promising system to be used. In this study, we evaluate the disrupted expression of heterologous HIV-1gp120 gene in BCG Pasteur host strain using replicative vectors pMV261 and pJH222. pJH222 carries a lysine complementing gene in BCG lysine auxotrophs. The HIV-1 gp120 gene expression was regulated by BCG hsp60 promoter (in plasmid pMV261) and Mycobacteria spp. alpha-antigen promoter (in plasmid pJH222). Among 14 rBCG:HIV-1gp120 (pMV261) colonies screened, 12 showed a partial deletion and two showed a complete deletion. However, deletion was not observed in all 10 rBCG:HIV-1gp120 (pJH222) colonies screened. In this study, we demonstrated that E. coli/Mycobacterial expression vectors bearing a weak promoter and lysine complementing gene in a recombinant lysine auxotroph of BCG could prevent genetic rearrangements and disruption of HIV 1gp120 gene expression, a key issue for engineering Mycobacterial based vaccine vectors.
We report the successful classification, by artificial neural networks (ANNs), of (1)H NMR spectroscopic data recorded on whole-cell culture samples of four different lung carcinoma cell lines, which display different drug resistance patterns. The robustness of the approach was demonstrated by its ability to classify the cell line correctly in 100% of cases, despite the demonstrated presence of operator-induced sources of variation, and irrespective of which spectra are used for training and for validation. The study demonstrates the potential of ANN for lung carcinoma classification in realistic situations.
Virotherapy using oncolytic vaccinia virus strains is one of the most promising new strategies for cancer therapy. In the current study, we analyzed the therapeutic efficacy of the oncolytic vaccinia virus GLV-1h68 against two human prostate cancer cell lines DU-145 and PC-3 in cell culture and in tumor xenograft models. By viral proliferation assays and cell survival tests, we demonstrated that GLV-1h68 was able to infect, replicate in, and lyse these prostate cancer cells in culture. In DU-145 and PC-3 tumor xenograft models, a single intravenous injection with GLV-1h68 resulted in a significant reduction of primary tumor size. In addition, the GLV-1h68-infection led to strong inflammatory and oncolytic effects resulting in drastic reduction of regional lymph nodes with PC-3 metastases. Our data documented that the GLV-1h68 virus has a great potential for treatment of human prostate carcinoma.
Research by other investigators has established that insulin-like growth factor-1 receptor (IGF-1R) is a key oncological target, and that derivatives of 1, 3-disubstituted-imidazo[1,5-alpha] pyrazine are potent IGF-1R inhibitors. In this paper, we report on our three-dimensional quantitative structure activity relationship (3D-QSAR) studies for this series of compounds. We validated the 3D-QSAR models by the comparison of two major alignment schemes, namely, ligand-based (LB) and receptor-guided (RG) alignment schemes. The latter scheme yielded better 3D-QSAR models for both comparative molecular field analysis (CoMFA) (q(2) = 0.35, r(2) = 0.95) and comparative molecular similarity indices analysis (CoMSIA) (q(2) = 0.51, r(2) = 0.86). We submit that this might arise from the more accurate inhibitor alignment that results from using the structural information of the active site. We conclude that the receptor-guided 3D-QSAR may be helpful to design more potent IGF-1R inhibitors, as well as to understand their binding affinity with the receptor.
High glucose stimulated the proliferation, migration, and inflammatory gene expression of VSMCs. VSMCs cultured in low-glucose (5.5 mmol/L) or high-glucose (25 mmol/L) complete media were deprived of serum for 24 h. In proliferation and inflammatory gene expression assays, VSMCs were stimulated by complete media for 6 h, respectively. In migration assays, VSMCs were suspended, and then low-glucose or high-glucose complete media were added to the lower compartment respectively. (a) proliferation of VSMCs, (b) migration of VSMCs, and (c) inflammatory gene expression of VSMCs (*P < 0.05).
Overexpression of PGC-1α inhibited proliferation, migration, and inflammatory gene expression of VSMCs in high glucose. VSMCs cultured in high-glucose (25 mmol/L) complete media were deprived of serum for 24 h. The cells were then treated with adenovirus infection for 48 h. (a) Proliferation of VSMCs, (b) migration of VSMCs, (c) inflammatory gene expression of VSMCs (*P < 0.05), and (d) Western blotting analysis for PGC-1α in VSMCs.
Overexpression of PGC-1α downregulated VSMCs-specific genes and upregulated macrophage-specific genes. (a) Downregulation of VSMCs-specific genes (*P < 0.05), (b) upregulation of macrophage-specific genes (*P < 0.05), (c) Western blotting analysis for α-actin in VSMCs, and (d) Western blotting analysis for CD68 in VSMCs.
Phagocytosis of VSMCs. VSMCs deprived of serum for 24 h were infected by Ad-PGC-1α or Ad-GFP. After 48 h, VSMCs were incubated with 1 μm microspheres (2.7 × 106 beads per mL) for 10 h. 27.36% of VSMCs infected by Ad-GFP had phagocytotic activity. However, the proportion of VSMCs having phagocytotic activity increased to 77.45% after overexpression of PGC-1α.
We assessed the role of PGC-1α (PPARγ coactivator-1 alpha) in glucose-induced proliferation, migration, and inflammatory gene expression of vascular smooth muscle cells (VSMCs). We carried out phagocytosis studies to assess the role of PGC-1α in transdifferentiation of VSMCs by flow cytometry. We found that high glucose stimulated proliferation, migration and inflammatory gene expression of VSMCs, but overexpression of PGC-1α attenuated the effects of glucose. In addition, overexpression of PGC-1α decreased mRNA and protein level of VSMCs-related genes, and induced macrophage-related gene expression, as well as phagocytosis of VSMCs. Therefore, PGC-1α inhibited glucose-induced proliferation, migration and inflammatory gene expression of VSMCs, which are key features in the pathology of atherosclerosis. More importantly, PGC-1α transdifferentiated VSMCs to a macrophage-like state. Such transdifferentiation possibly increased the portion of VSMCs-derived foam cells in the plaque and favored plaque stability.
Localization of Flag-NT-PGC-1α, GFP-PGC-1α, and Flag-NFATc1 in muscle fibers. (a) A representative image of a muscle fiber expressing Flag-NT-PGC-1α detected by anti-Flag antibody staining shows a predominantly cytoplasmic locatiozation of Flag-NT-PGC-1α in cultured adult skeletal muscle fibers. (b) A different fiber expressing the full-length PGC-1α-GFP fusion protein shows that full length PGC-1α is mainly located in the fiber nucleus. (c) A third muscle fiber expressing nuclear-targeted Flag-tagged NFATc1 detected by the same anti-Flag antibody procedure as in (a) has a strong nuclear staining, thus establishing nuclear penetration of the anti-Flag antibody. Scale bar, 10 μm.
Activation of PKA increases nuclear Flag-NT-PGC-1α. (a) Representative images of Flag-NT-PGC-1α in a control fiber (control) and in another fiber after 1 h treatment with 1 mM dbcAMP in culture medium in the tissue culture incubator (dbcAMP). Scale bar, 10 μm. (b) The n/c fluorescence ratio of Flag-NT-PGC-1α in muscle fibers with (dbcAMP) or without (control) dbcAMP treatment. n/c values from 20 nuclei from 20 randomly selected fibers were averaged to give the mean value for each group. Asterisk indicates statistical significance between groups at P < 0.05.
Muscle activity does not alter the subcellular distribution of Flag-NT-PGC-1α. (a) Representative images of Flag-NT-PGC-1α in a control fiber (control) and in another fiber stimulated continuously at 10 Hz for 4 h (stimulation). (b) The n/c fluorescence ratio of Flag-NT-PGC-1α in muscle fibers with (stimulation) or without (control) electrical stimulation. n/c values from 20 nuclei from 20 randomly selected different fibers were averaged to give the mean value for each group. (c) Representative images of NFATc1-GFP in a control fiber (control) and in another fiber stimulated continuously at 10 Hz for 4 h (stimulation). Scale bars, 10 μm for (a) and (c). (d) Representative Western blots showing the effect of muscle activity on p38 MAPK activation. Protein expression was quantified and averaged from 3 independent treatments. Phos p38, phospho-p38 MAPK; total p38, total p38 MAPK. Asterisk indicates statistical significance between groups at P < 0.05.
Activation of AMPK does not alter the subcellular distribution of Flag- NT-PGC-1α in muscle fibers. (a) The n/c fluorescence ratio of Flag-NT-PGC-1α in muscle fibers with (AICAR) or without (control) 2 mM AICAR treatment for 5 h in culture medium in the tissue culture incubator. n/c values from 20 nuclei from 20 randomly selected different fibers were averaged to give the mean value for each group. (b) Representative Western blots showing the effect of AICAR treatment on AMPK activation. Protein expression was quantified and averaged from 5 independent treatments. Phos AMPK, phospho-AMPK. Asterisk indicates statistical significance between groups at P < 0.05.
Inhibition of CRIM1 increases nuclear accumulation of Flag-NT-PGC-1α. (a) Representative images of Flag-NT-PGC-1α in a control fiber (control) and that of another fiber treated with 40 nM CRM1 inhibitor leptomycin B (LMB) for 20 h in culture medium in the tissue culture incubator. Scale bar, 10 μm. (b) The n/c fluorescence ratio of Flag-NT-PGC-1α in muscle fibers with (LMB) or without (control) leptomycin B treatment. n/c values from 40 nuclei from 40 randomly selected different fibers were averaged to give the mean value for each group. Asterisk indicates statistically significant difference between groups at P < 0.05.
The transcriptional coactivator peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) regulates expression of genes for metabolism and muscle fiber type. Recently, a novel splice variant of PGC-1α (NT-PGC-1α, amino acids 1-270) was cloned and found to be expressed in muscle. Here we use Flag-tagged NT-PGC-1α to examine the subcellular localization and regulation of NT-PGC-1α in skeletal muscle fibers. Flag-NT-PGC-1α is located predominantly in the myoplasm. Nuclear NT-PGC-1α can be increased by activation of protein kinase A. Activation of p38 MAPK by muscle activity or of AMPK had no effect on the subcellular distribution of NT-PGC-1α. Inhibition of CRM1-mediated export only caused relatively slow nuclear accumulation of NT-PGC-1α, indicating that nuclear export of NT-PGC-1α may be mediated by both CRM1-dependent and -independent pathways. Together these results suggest that the regulation of NT-PGC-1α in muscle fibers may be very different from that of the full-length PGC-1α, which is exclusively nuclear.
Biodistribution of 125 I-POS in FM3A-bearing mice. Administration dose of 125 I-POS 2.0 MBq, POS: 30 μg, tumor weight: 0.3-2.3 g, tumor uptake 4.3% AD/g, T/B: 13.1%(n = 5).
Biodistribution of 125I-POS in FM3A-bearing mice.  Administration dose of 125I-POS 2.0 MBq, POS: 30 μg, tumor weight: 0.3–2.3 g, tumor uptake 4.3% AD/g, T/B: 13.1% (n = 5).
Intratumoral distribution of 125I-POS, pimonidazole. Administration dose of 125I-POS 2.0 MBq, POS: 30 μg, tumor weight: 0.3 g, tumor uptake 5.2%, slice thickness for ARG 20 μm, slice thickness for staining 6 μm.
In vivo visualization of heterogeneous intratumoral distribution of POS by SPECT/CT. The comparison of in vivo SPECT images and ex vivo images. Upper raw: in vivo SPECT images, lower raw: ex vivo SPECT images, left column: sagittal sectional images, right column: axial sectional images. Arrows 1: markers for coregistration, arrows 2: vertebrae, and arrowheads: the tumor.
In vivo fusion imaging of intratumoral distribution of 125I-POS and MR images. A representative image is enlarged. Arrows 1: markers, arrows 2: urinary bladder, and arrowheads: the tumor.
We aimed to clearly visualize heterogeneous distribution of hypoxia-inducible factor 1α (HIF) activity in tumor tissues in vivo. We synthesized of (125)I-IPOS, a (125)I labeled chimeric protein probe, that would visualize HIF activity. The biodistribution of (125)I-IPOS in FM3A tumor-bearing mice was evaluated. Then, the intratumoral localization of this probe was observed by autoradiography, and it was compared with histopathological findings. The distribution of (125)I-IPOS in tumors was imaged by a small animal SPECT/CT scanner. The obtained in vivo SPECT-CT fusion images were compared with ex vivo images of excised tumors. Fusion imaging with MRI was also examined. (125)I-IPOS well accumulated in FM3A tumors. The intratumoral distribution of (125)I-IPOS by autoradiography was quite heterogeneous, and it partially overlapped with that of pimonidazole. High-resolution SPECT-CT fusion images successfully demonstrated the heterogeneity of (125)I-IPOS distribution inside tumors. SPECT-MRI fusion images could give more detailed information about the intratumoral distribution of (125)I-IPOS. High-resolution SPECT images successfully demonstrated heterogeneous intratumoral distribution of (125)I-IPOS. SPECT-CT fusion images, more favorably SPECT-MRI fusion images, would be useful to understand the features of heterogeneous intratumoral expression of HIF activity in vivo.
Demographics, CD34 + Cells, telomerase activity, and IGF1 concentration in 20070721-GX subjects. 
Regression analysis for CD34 + cells. 
Regression analysis for telomerase activity. 
Few rejuvenation and antiaging markers are used to evaluate food supplements. We measured three markers in peripheral blood to evaluate the antiaging effects of a food supplement containing placental extract. Samples were evaluated for CD34(+) cells, insulin-like growth factor 1 (IGF1), and telomerase activity, which are all markers related to aging. To control the quality of this food supplement, five active components were monitored. In total, we examined 44 individuals who took the food supplement from 1.2 months to 23 months; the average number of CD34(+) cells was almost 6-fold higher in the experimental group compared with the control group. Food supplement intake did not change serum IGF1 levels significantly. Finally, the average telomerase activity was 30% higher in the subjects taking this food supplement. In summary, our results suggest that the placental extract in the food supplement might contribute to rejuvenation and antiaging.
With the BLyS inhibitor Belimumab recently approved by the United States Food and Drug administration as the first treatment for Systemic Lupus Erythematosus in decades, and the ongoing trials of other cytokine inhibitors as potential therapeutic agents for lupus, the potential of cytokines in the realm of therapeutic targets for lupus is beginning to reach its promise. These fascinating molecules regulate multiple facets of the immune system and, as such, have been extensively examined for a role in many different aspects of lupus etiology, pathogenesis, and regulation. This special issue contains novel research about three cytokines and their roles in lupus. Alleles of Osteopontin were found to be associated with multiple clinical manifestations of lupus (T. Trivedi et al.). The feasibility of Neutrophil gelatinase-associated lipocalin as a biomarker for kidney damage was assessed (Y.-L. Yang et al.). The lupus risk haplotype of the Interferon regulatory factor five gene was found to influence gene expression in three key pathways that have the potential to influence the development or pathogenesis of lupus (J. M. Guthridge et al.). The reviews included in this issue summarize the most current research on the multiple roles of cytokines in lupus (K. Ohl and K. Tenbrock). The influences of cytokines on T cell subsets in lupus and how those subsets relate to lupus are discussed (A. Okamoto et al., K. Miyake et al.). The roles of cytokines in clinical manifestations, such as nephritis (Y. Iwata et al.), neuropsychiatric manifestations, cutaneous lupus, and premature atherosclerosis (T. P. Karageorgas et al.), are a major focus of this issue. Cytokine research holds tremendous promise for understanding and treating lupus. We thank all those who contributed articles for this special issue and those who reviewed and in other ways contributed. We hope that this avenue of research will continue to be supported and be a benefit to the lives of those suffering with this complex and difficult disease. Brian D. Poole Timothy B. Niewold George C. Tsokos
Viability of four CRC cell lines in response to treatment with dichloroacetate (DCA) and 5-fluorouracil (5-FU). The viability of SW620, LoVo, LS174t, and HT29 was decreased significantly with different concentration of 5-FU and DCA alone or in combination. Each experiment was performed in triplicate; *P < .05.
Mean values of the combination index at the affected fractions of 50% (IC50) and 80% (IC80) when 5-fluoruracil (5-FU) was combined with dichloroacetate (DCA) in HT-29, LoVo, LS174t, and SW620 cells. Mean IC50 and IC80 ± SD (n = 3) are shown. A CI value significantly less than 1 indicates synergism, a CI not significantly different from 1 indicates addition, and a CI significantly higher than 1 indicates antagonism; *P < .05.
Changes in cell cycle progression in SW620, LoVo, LS174t, and HT29 cells after 48-hour treatment with 5-fluoruracil (5-FU) and dichloroacetate (DCA) applied alone or in combination. Each bar represents the mean ± SD (n = 3). The data obtained from FACS were analyzed using SPSS13.0; *P < .05.
Induction of apoptosis in SW620, LoVo, LS174t, and HT29 cells after 48-hour treatment with 5-fluoruracil (5-FU) and dichloroacetate (DCA) alone or in combination. Each bar represents the mean ± SD (n = 3); *P < .05.
Effects of 5-fluorouracil (5-FU) and dichloroacetate (DCA) on apoptosis-associated molecules expression. Bcl-2 expression was significantly decreased by DCA in SW620, LoVo, and LS174t and HT29 cells. Bax and caspase-3 expression levels were higher after exposure to 5-FU and DCA compared with control.
Dichloroacetate (DCA), an inhibitor of pyruvate dehydrogenase kinase (PDK), has been recently demonstrated as a promising nontoxic antineoplastic agent that promotes apoptosis of cancer cells. In the present study, we aimed to investigate the antitumor effect of DCA combined with 5-Fluorouracil (5-FU) on colorectal cancer (CRC) cells. Four human CRC cell lines were treated with DCA or 5-FU, or a combination of DCA and 5-FU. The cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The interaction between DCA and 5-FU was evaluated by the median effect principle. Immunocytochemistry with bromodeoxyuridine (BrdU) was carried out to determine the proliferation of CRC cells. Cell cycle and apoptosis were measured by flow cytometry, and the expression of apoptosis-related molecules was assessed by western blot. Our results demonstrated that DCA inhibited the viability of CRC cells and had synergistic antiproliferation in combination with 5-FU. Moreover, compared with 5-FU alone, the apoptosis of CRC cells treated with DCA and 5-FU was enhanced and demonstrated with the changes of Bcl-2, Bax, and caspase-3 proteins. Our results suggest that DCA has a synergistic antitumor effect with 5-FU on CRC cell lines in vitro.
In closing, we wish to thank all of the authors for their high level of enthusiasm in providing high quality manuscripts to support this special issue and appreciate their effort and time spent processing their manuscripts. We also extend much gratitude to all of the reviewers, most of whom reviewed at least two versions of the same manuscript, for their time and effort. We hope the scientific information presented within this special issue will be helpful to both CTL aficionados and the broader immunological community and promote new avenues of exploration for scientists studying CTLs in the context of vaccine development.
The authors sincerely give thanks to Consejo Mexiquense de Ciencia y Tecnología (COMECYT) and Instituto de Ciencia y Tecnología del Distrito Federal (ICyTDF) for their support in the organization of the IV Mexican Immunoparasitology Meeting held in November 2010, which was the platform for this special issue.
We sincerely thank all the contributors, the reviewers, and the publishing team for their enthusiasm and hard work.
We here identified human leukocyte antigen-(HLA-)A(∗)2402-restricted epitope peptides from Cadherin 3, type 1, P-cadherin (CDH3) and kinesin family member 20A (KIF20A) that were found to be specifically expressed in cancer cells through genome-wide expression profile analysis. CDH3-10-807 peptide and KIF20A-10-66 peptide successfully induced specific CTL clones, and these selectively responded to COS7 cells expressing both HLA-A(∗)2402 and respective protein while did not respond to parental cells or COS7 cells expressing either HLA-A(∗)2402 or respective protein. Furthermore, CTL clones responded to cancer cells that endogenously express HLA-A(∗)2402 and respective protein, suggesting that CDH3-10-807 peptide and KIF20A-10-66 peptide are naturally presented on HLA-A(∗)2402 molecule of human cancer cells. Our results demonstrated that CDH3-10-807 peptide and KIF20A-10-66 peptide are novel HLA-A24-restricted tumor-associated antigens and would be applicable for CTL-inducing cancer therapies.
Systemic Lupus Erythematosus (SLE) is an autoimmune disorder characterized by excessive production of a variety of autoantibodies and a wide range of clinical manifestations. Pathogenesis of SLE is complex and not fully understood. There is however evidence that B and T cells are critical to the development of disease, and that T cell-derived cytokines are involved in the SLE-associated inflammatory response. One such cytokine seems to be interleukin (IL)-21, the latest identified member of the gamma-chain-related cytokine family. IL-21 has an important role in the control of the growth, survival, differentiation, and function of both T and B cells, and excessive production of IL-21 has been associated with the development of multiple immune-mediated diseases. Here we review data supporting the involvement of IL-21 in the pathogenesis of SLE.
Top-cited authors
Shiliang Li
  • Universität Heidelberg
Yinhu Li
  • City University of Hong Kong
Linda Liu
  • Tenth Affiliated Hospital of Tongji University
Martina Perse
  • University of Ljubljana
Anton Cerar
  • University of Ljubljana