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Molecular cloning: A laboratory manual, second ed
... Total DNA from Rhodococcus aetherivorans BCP1 and the transposon mutants was extracted from cells grown in 50 mL liquid LB cultures incubated at 30°C for 48 h (up to OD 600 of 1-1.2). After cell harvesting, BCP1 WT and mutant cells were lysed using the protocol reported by Cappelletti et al. (2011), while standard molecular techniques were used for DNA manipulation and cloning (Sambrook et al., 1989). Shortly, for molecular cloning 1.5-3 μg of DNA were digested with appropriate restriction endonucleases (1 U) (Roche) for 3-4 h at 37°C. ...
... Ligation reactions were performed overnight in a final volume of 15 μL using the T4 ligase (Roche) based on the manufacturer instructions except for the use of a temperature gradient ranging from 37°C to 4°C. Recombinant plasmids were introduced into E. coli DH5α using chemical transformation standard protocol (Sambrook et al., 1989). PCR and plasmid purifications were carried out using the Plasmid Mini Kit and PCR purification kit (Qiagen) according to the manufacturer's instructions. ...
... 5 μg of DNA was then subjected to enzymatic restriction with the enzyme PstI and ran on electrophoresis gel. The chromosomal DNAs from the agarose gels were then transferred to nylon membrane filters and Southern hybridization was performed according to the previously described protocol (Sambrook et al., 1989). The probe was made through PCR amplification using pTNR-TA as template together with the primers TioR-For (5′-GGATCCGCCAGAGAGCGACGAC-3′) and TioR-Rev (5′-CGCCTTCGAGGAGTGCCCG-3′) to amplify part of the thiostrepton resistance gene that is comprised within the transposable region of the integrative vector. ...
Soluble di‐iron monooxygenases (SDIMOs) are multi‐component enzymes catalysing the oxidation of various substrates. These enzymes are characterized by high sequence and functional diversity that is still not well understood despite their key role in biotechnological processes including contaminant biodegradation. In this study, we analysed a mutant of Rhodoccocus aetherivorans BCP1 (BCP1‐2.10) characterized by a transposon insertion in the gene smoA encoding the alpha subunit of the plasmid‐located SDIMO SmoABCD. The mutant BCP1‐2.10 showed a reduced capacity to grow on propane, lost the ability to grow on butane, pentane and n‐hexane and was heavily impaired in the capacity to degrade chloroform and trichloroethane. The expression of the additional SDIMO prmABCD in BCP1‐2.10 probably allowed the mutant to partially grow on propane and to degrade it, to some extent, together with the other short‐chain n‐alkanes. The complementation of the mutant, conducted by introducing smoABCD in the genome as a single copy under a constitutive promoter or within a plasmid under a thiostreptone‐inducible promoter, allowed the recovery of the alkanotrophic phenotype as well as the capacity to degrade chlorinated n‐alkanes. The heterologous expression of smoABCD allowed a non‐alkanotrophic Rhodococcus strain to grow on pentane and n‐hexane when the gene cluster was introduced together with the downstream genes encoding alcohol and aldehyde dehydrogenases and a GroEL chaperon. BCP1 smoA gene was shown to belong to the group 6 SDIMOs, which is a rare group of monooxygenases mostly present in Mycobacterium genus and in a few Rhodococcus strains. SmoABCD originally evolved in Mycobacterium and was then acquired by Rhodococcus through horizontal gene transfer events. This work extends the knowledge of the biotechnologically relevant SDIMOs by providing functional and evolutionary insights into a group 6 SDIMO in Rhodococcus and demonstrating its key role in the metabolism of short‐chain alkanes and degradation of chlorinated n‐alkanes.
... Cuando se añade etanol, esta capa se rompe y los grupos fosfato quedan expuestos. De esta forma se favorece la unión con cationes como el Na + , reduciendo las fuerzas de repulsión entre las dos cadenas de nucleótidos y permiten que el ADN precipite (Sambrook et al., 1989). Además, la carga negativa del ADN puede favorecer que se una a matrices inorgánicas con carga positiva (Sambrook et al., 1989), esto es el principio que emplean muchos kits de extracción de ADN, aunque también se puede realizar una extracción de manera tradicional. ...
... De esta forma se favorece la unión con cationes como el Na + , reduciendo las fuerzas de repulsión entre las dos cadenas de nucleótidos y permiten que el ADN precipite (Sambrook et al., 1989). Además, la carga negativa del ADN puede favorecer que se una a matrices inorgánicas con carga positiva (Sambrook et al., 1989), esto es el principio que emplean muchos kits de extracción de ADN, aunque también se puede realizar una extracción de manera tradicional. Los pasos, junto con su explicación, de un protocolo típico, serían los siguientes: ...
... Para ello, se emplean soluciones básicas, detergentes o agentes caotrópicos que permiten disolver dichas membranas, junto con inhibidores que inactiven las enzimas que degradan el ADN. Es por ello que muchas soluciones de lisis contienen también EDTA, que forma un complejo con los iones de Mg 2+ e impide el funcionamiento de las DNasas (Sambrook et al., 1989). En el caso de tener centrífuga, se puede emplear para separar del ADN los componentes celulares no solubles como el material fibroso y proteínas que permanecen en solución. ...
La sociedad actual está ampliamente influenciada por los grandes adelantos científicos y tecnológicos que se han desarrollado desde el siglo pasado. Esto implica que la ciudadanía se enfrenta diariamente a situaciones problemáticas que requieren la puesta en marcha de competencias científicas, estableciendo la importancia del desarrollo de la alfabetización científica, que permite la reflexión, el razonamiento y el establecimiento de conexiones para resolverlas de manera satisfactoria. En este sentido, los trabajos de tipo práctico suponen una herramienta óptima en las aulas, puesto que sitúan al alumnado en el centro del proceso de enseñanza-aprendizaje, buscando soluciones a hechos que forman parte de su vida cotidiana mediante la observación y la manipulación, transformándolos en hechos científicos escolares y adquiriendo así nuevos conocimientos. Por todo ello, en este trabajo se analiza una experiencia práctica: la extracción de ADN. A tal efecto, se presenta la estructura de ADN, se analizan los pasos que suelen llevarse a cabo en cualquier proceso de extracción de esta macromolécula y se diseña una propuesta para Educación Primaria y otra para Educación Secundaria Obligatoria en las que los estudiantes extraen ADN de material cotidiano y adquieren conocimientos sobre su estructura siguiendo el método científico. Como resultados, ambas propuestas han sido validadas a través de un juicio de expertos destacando que son innovadoras y atractivas para los alumnos. Como conclusión se recomienda implementar las propuestas, así como tener en cuenta el tiempo necesario para su desarrollo y la importancia del número de alumnos presentes en el aula.
... The genetic constructs used in this study were replicated in Escherichia coli One Shot TOP10 Electrocomp cells (Invitrogen, Brazil), cultivated at 37 °C in Luria-Bertani (LB) medium [36], supplemented with 100 μg/mL of spectinomycin and agitated at 220 rpm. The cultivation of Synechococcus elongatus PCC 7942 (Thermo Fisher Scientific) was carried out in BG-11 medium [37] at 34 °C under cool fluorescent light at 100 μmol m −2 s −1 . ...
... Exposure to MF was performed using ferrite magnets with an average intensity of 30 mT (MF30) (150 × 50 × 10 mm). This intensity was chosen as it has been observed to have a positive influence on Photosystem 2 [36] for fluorescence and gene expression assays. The exposure was conducted for two days at the beginning of the stationary phase of the culture when the OD 750 reached 1. MF intensity was measured at various points in the flasks used in the assays using a magnetic field meter (Global Mag, TLMP-HALL 05 K, Brazil). ...
The cyanobacterium Synechococcus elongatus PCC 7942 holds significant potential as a biofactory for recombinant protein (RP) production due to its capacity to harness light energy and utilize CO2. This study aimed to enhance RP production by integration of native promoters and magnetic field application (MF) in S. elongatus PCC 7942. The psbA2 promoter, which responds to stress conditions, was chosen for the integration of the ZsGreen1 gene. Results indicated successful gene integration, affirming prior studies that showed no growth alterations in transgenic strains. Interestingly, exposure to 30 mT (MF30) demonstrated a increase in ZsGreen1 transcription under the psbA2 promoter, revealing the influence of MF on cyanobacterial photosynthetic machinery. This enhancement is likely attributed to stress-induced shifts in gene expression and enzyme activity. MF30 positively impacted photosystem II (PSII) without disrupting the electron transport chain, aligning with the "quantum–mechanical mechanism" theory. Notably, fluorescence levels and gene expression with application of 30 mT were significantly different from control conditions. This study showcases the efficacy of utilizing native promoters and MF for enhancing RP production in S. elongatus PCC 7942. Native promoters eliminate the need for costly exogenous inducers and potential cell stress. Moreover, the study expands the scope of optimizing RP production in photoautotrophic microorganisms, providing valuable insights for biotechnological applications.
... Their growth was monitored by measuring the protein concentration in the cultures using the BCA Protein Assay Kit (Beyotime, China). E. coli strains were grown in lysogeny broth (LB) (35) supplemented with 50 µg/mL kanamycin or 100 µg/mL ampicillin at 37°C and 180 rpm. ...
... The almA tru5aa gene was amplified and fused into pET-28a (+) (digested with NdeI and BamHI) using a One Step cloning kit (Vazyme Biotech Co., Ltd.). The resultant plasmids pET-almA tru5aa were transformed into E. coli BL21 (DE3) using a standard procedure (35). For protein expression and purification, the strain was grown in LB supplemented with 50 µg/mL kanamycin at 37°C and 180 rpm. ...
Many Acinetobacter species can grow on n-alkanes of varying lengths (≤C40). AlmA, a unique flavoprotein in these Acinetobacter strains, is the only enzyme proven to be required for the degradation of long-chain (LC) n-alkanes, including C32 and C36 alkanes. Although it is commonly presumed to be a terminal hydroxylase, its role in n-alkane degradation remains elusive. In this study, we conducted physiological, biochemical, and bioinformatics analyses of AlmA to determine its role in n-alkane degradation by Acinetobacter baylyi ADP1. Consistent with previous reports, gene deletion analysis showed that almA was vital for the degradation of LC n-alkanes (C26–C36). Additionally, enzymatic analysis revealed that AlmA catalyzed the conversion of aliphatic 2-ketones (C10–C16) to their corresponding esters, but it did not conduct n-alkane hydroxylation under the same conditions, thus suggesting that AlmA in strain ADP1 possesses Baeyer–Villiger monooxygenase (BVMO) activity. These results were further confirmed by bioinformatics analysis, which revealed that AlmA was closer to functionally identified BVMOs than to hydroxylases. Altogether, the results of our study suggest that LC n-alkane degradation by strain ADP1 possibly follows a novel subterminal oxidation pathway that is distinct from the terminal oxidation pathway followed for short-chain n-alkane degradation. Furthermore, our findings suggest that AlmA catalyzes the third reaction in the LC n-alkane degradation pathway.
IMPORTANCE
Many microbial studies on n-alkane degradation are focused on the genes involved in short-chain n-alkane (≤C16) degradation; however, reports on the genes involved in long-chain (LC) n-alkane (>C20) degradation are limited. Thus far, only AlmA has been reported to be involved in LC n-alkane degradation by Acinetobacter spp.; however, its role in the n-alkane degradation pathway remains elusive. In this study, we conducted a detailed characterization of AlmA in A. baylyi ADP1 and found that AlmA exhibits Baeyer–Villiger monooxygenase activity, thus indicating the presence of a novel LC n-alkane biodegradation mechanism in strain ADP1.
... DNA extraction and PCR. DNA was extracted from the pectoral-fin tissues using a modified Phenolchloroform method (Sambrook et al. 1989). The COI gene was amplified using primers FishF1 (5'-TCAACCAACCACAAAGACATTGGCAC-3') and FishR1 (5'-TAGACTTCTGGGTGGCCAAAGAATCA-3') (Ward et al. 2005). ...
Cobitis feroniae, new species, is described from central Italy. It is distinguished from C. zanandreai, its putatively closest relative, by having several, small, black dots below Z4; minute, black spot at the upper caudal peduncle, and the pigmentation in Z2 separated from pigmentation in Z1 anterior to the dorsal-fin origin. It is further distinguished from C. zanandreai by having 13 diagnostic nucleotide substitutions in the mtDNA COI barcode region, and a K2P nearest-neighbour distance of 2.9%.
... For individuals for whom skin samples were available, total-cell DNA was extracted from tissue samples either by standard phenol: chloroform extractions [48] or using the Qiagen dNeasy™ Blood and Tissue Kit (Qiagen Inc.) according to the manufacturer's instructions. Extracted DNA was re-suspended in 1x TE buffer (10mM Tris-HCl, 1mM EDTA, pH 8.0). ...
Background
The life cycle of most baleen whales involves annual migrations from low-latitude breeding grounds to high latitude feeding grounds. In most species, these migrations are traditionally considered to be carried out according to information acquired through vertical social learning during the first months of life and made individually. However, some recent studies have suggested a more complex scenario, particularly for the species of the Balaenoptera genus.
Methods
Here, we studied the variation of δ¹⁵N and δ¹³C values along the growth axis of the baleen plate from 24 fin whales feeding off western Iceland to delve into their pattern of movements and to identify potential associations between individuals. The segment of baleen plate analyzed informed about at least two complete migratory cycles. We performed cluster analyses through two different methodologies and, whenever possible, we genotyped 20 microsatellite loci to determine potential existence of kinship.
Results
Results of the of δ¹⁵N and δ¹³C values agree with a dispersion strategy in the winter breeding grounds. However, and despite the overall large variability, several pairs or groups of individuals with no kinship showed highly similar isotopic patterns for two consecutive years for both δ¹⁵N and δ¹³C values.
Conclusions
Our results suggest that, notably, some whales without kinship share the same migratory regime and destinations. We hypothesize that this could reflect either: (i) the sharing of particularly beneficial migratory regimes, and/or (ii) long-term association between individuals.
... The invA, Class 1 integron, blaTEM, qnrS, and aadA1 genes were amplified as designed, and cycling conditions are shown in Table 1. According to Sambrook et al. (1989). In a 25-µl reaction, the primers were used along with 12.5 µl of EmeraldAmp Max PCR Master Mix (Takara, Japan), 1 µl of each primer at a concentration of 20 pmol, 5.5 µl of water, and 5 µl of the DNA template. ...
... The invA, Class 1 integron, blaTEM, qnrS, and aadA1 genes were amplified as designed, and cycling conditions are shown in Table 1. According to Sambrook et al. (1989). In a 25-µl reaction, the primers were used along with 12.5 µl of EmeraldAmp Max PCR Master Mix (Takara, Japan), 1 µl of each primer at a concentration of 20 pmol, 5.5 µl of water, and 5 µl of the DNA template. ...
... Initially, the codon-optimized synthetically PhySc gene in ampicillin-resistant pUCIDT vector was double digested by MscI and Bpu1102I restriction endonucleases and ligated into double digested pET-32b (+) vector by the T4 DNA ligase. Then, the transformation into E.coli Top10 competent cells prepared by CaCl 2 as the cloning host were performed through the heat shock method (Sambrook and Maniatis 1989). The extraction of the recombinant pET-32b(+)-PhySc plasmid from an overnight culture of LB broth consisting of 100 μg/mL ampicillin concentration was carried out using the FavorPrep™ Plasmid DNA Extraction Mini Kit. ...
A histidine acid phosphatase (HAP) (PhySc) with 99.50% protein sequence similarity with PHO5 from Saccharomyces cerevisiae was expressed functionally with the molecular mass of ∼110 kDa through co-expression along with the set of molecular chaperones dnaK, dnaJ, GroESL. The purified HAP illustrated the optimum activity of 28.75 ± 0.39 U/mg at pH 5.5 and 40 ˚C. The Km and Kcat values towards calcium phytate were 0.608 ± 0.09 mM and 650.89 ± 3.6 s− 1. The half-lives (T1/2) at 55 and 60 ˚C were 2.75 min and 55 s, respectively. The circular dichroism (CD) demonstrated that PhySc includes 30.5, 28.1, 21.3, and 20.1% of random coils, α-Helix, β-Turns, and β-Sheet, respectively. The Tm recorded by CD for PhySc was 56.5 ± 0.34˚C. The molecular docking illustrated that His59 and Asp322 act as catalytic residues in the PhySc. MD simulation showed that PhySc at 40 ˚C has higher structural stability over those of the temperatures 60 and 80 ˚C that support the thermodynamic in vitro investigations. Secondary structure content results obtained from MD simulation indicated that PhySc consists of 34.03, 33.09, 17.5, 12.31, and 3.05% of coil, helix, turn, sheet, and helix310, respectively, which is almost consistent with the experimental results.
... Standard molecular genetic techniques were used (72,73). For conjugative gene transfer, overnight cultures of donor strain E. coli S17/1 (74) and of the C. metallidurans recipient strains grown at 30°C in Tris-buffered medium were mixed (1:1) and plated onto nutrient broth agar. ...
The metal-resistant bacterium Cupriavidus metallidurans occurs in metal-rich environments. In auriferous soils, the bacterium is challenged by a mixture of copper ions and gold complexes, which exert synergistic toxicity. The previously used, self-made Au(III) solution caused a synergistic toxicity of copper and gold that was based on the inhibition of the CupA-mediated efflux of cytoplasmic Cu(I) by Au(I) in this cellular compartment. In this publication, the response of the bacterium to gold and copper was investigated by using a commercially available Au(III) solution instead of the self-made solution. The new solution was five times more toxic than the previously used one. Increased toxicity was accompanied by greater accumulation of gold atoms by the cells. The contribution of copper resistance determinants to the commercially available Au(III) solution and synergistic gold-copper toxicity was studied using single- and multiple-deletion mutants. The commercially available Au(III) solution inhibited periplasmic Cu(I) homeostasis, which is required for the allocation of copper ions to copper-dependent proteins in this compartment. The presence of the gene for the periplasmic Cu(I) and Au(I) oxidase, CopA, decreased the cellular copper and gold content. Transcriptional reporter gene fusions showed that up-regulation of gig, encoding a minor contributor to copper resistance, was strictly glutathione dependent. Glutathione was also required to resist synergistic gold-copper toxicity. The new data indicated a second layer of synergistic copper-gold toxicity caused by the commercial Au(III) solution, inhibition of the periplasmic copper homeostasis in addition to the cytoplasmic one.
IMPORTANCE
When living in auriferous soils, Cupriavidus metallidurans is not only confronted with synergistic toxicity of copper ions and gold complexes but also by different gold species. A previously used gold solution made by using aqua regia resulted in the formation of periplasmic gold nanoparticles, and the cells were protected against gold toxicity by the periplasmic Cu(I) and Au(I) oxidase CopA. To understand the role of different gold species in the environment, another Au(III) solution was commercially acquired. This compound was more toxic due to a higher accumulation of gold atoms by the cells and inhibition of periplasmic Cu(I) homeostasis. Thus, the geo-biochemical conditions might influence Au(III) speciation. The resulting Au(III) species may subsequently interact in different ways with C. metallidurans and its copper homeostasis system in the cytoplasm and periplasm. This study reveals that the geochemical conditions may decide whether bacteria are able to form gold nanoparticles or not.
... The standard phenol: chloroform: isoamyl alcohol process was implemented with minor modifications for DNA isolation from the muscles of all fish samples (Sambrook et al., 1989). To analyze genetic variation, a partial mitochondrial Cyt b gene was amplified by Polymerase Chain Reaction (PCR) with universal primers L14724 (5'-CGAAGCTTGATATGAAAAACCATCGTTG-3') and H15149 (5'-AAACTGCAGCCCCTCAGAATGATATTTGTCCTCA-3') (Kocher et al., 1989). ...
Genetic structure and some bio-ecological characteristics of lessepsian lizardfish Saurida lessepsianus populations collected from the Iskenderun, Mersin, and Antalya Bays were revealed by mtDNA sequencing of Cyt b dataset and length-weight relationships and the parameters of von Bertalanffy growth function. The highest genetic divergence was detected between the Mersin and Antalya populations (0.0026), whereas the lowest was between the Iskenderun and Antalya populations (0.0014). The length-weight relationships for all individuals of Iskenderun, Mersin, and Antalya populations were calculated as W = 0.0042 x L 3.2207 , W = 0.0039 x L 3.1627 and W = 0.0196 x L 2.6515 , respectively. The von Bertalanffy growth parameters (L∞, k, and to) were estimated for all individuals as L∞ = 47.33 cm, k = 0.024, and to =-1.28 for Iskenderun; L∞ = 41.74 cm, k = 0.085 and to =-1.72 for Mersin and L∞ = 50.99 cm, k = 0.154 and to =-2.41 for Antalya. This study examines the genetic structure and bio-ecological characteristics of the S. lessepsianus populations on the Mediterranean coast of Türkiye together for the first time. Genetic findings and bio-ecological parameters of populations generally support each other and can provide support for future studies.
... Frontiers in Microbiology 07 frontiersin.org Salmonella-specific hilA PCR (Cheng et al., 2015) was used to confirm that RNA was free of contaminating DNA, and the quality and integrity of the RNA were evaluated by gel electrophoresis (Sambrook et al., 1989). Genomic Salmonella DNA served as a labeling and hybridization reference control. ...
Baby chicks administered a fecal transplant from adult chickens are resistant to Salmonella colonization by competitive exclusion. A two-pronged approach was used to investigate the mechanism of this process. First, Salmonella response to an exclusive (Salmonella competitive exclusion product, Aviguard®) or permissive microbial community (chicken cecal contents from colonized birds containing 7.85 Log10Salmonella genomes/gram) was assessed ex vivo using a S. typhimurium reporter strain with fluorescent YFP and CFP gene fusions to rrn and hilA operon, respectively. Second, cecal transcriptome analysis was used to assess the cecal communities’ response to Salmonella in chickens with low (≤5.85 Log10 genomes/g) or high (≥6.00 Log10 genomes/g) Salmonella colonization. The ex vivo experiment revealed a reduction in Salmonella growth and hilA expression following co-culture with the exclusive community. The exclusive community also repressed Salmonella’s SPI-1 virulence genes and LPS modification, while the anti-virulence/inflammatory gene avrA was upregulated. Salmonella transcriptome analysis revealed significant metabolic disparities in Salmonella grown with the two different communities. Propanediol utilization and vitamin B12 synthesis were central to Salmonella metabolism co-cultured with either community, and mutations in propanediol and vitamin B12 metabolism altered Salmonella growth in the exclusive community. There were significant differences in the cecal community’s stress response to Salmonella colonization. Cecal community transcripts indicated that antimicrobials were central to the type of stress response detected in the low Salmonella abundance community, suggesting antagonism involved in Salmonella exclusion. This study indicates complex community interactions that modulate Salmonella metabolism and pathogenic behavior and reduce growth through antagonism may be key to exclusion.
... Dòng vi khuẩn có khả năng hấp thu ammonium hiệu quả nhất được tuyển chọn để giải trình tự gen 16S-rRNA. ADN của vi khuẩn được ly trích dựa theo quy trình trích ADN của Sambrook et al. (1989) và giải trình tự tại Công ty TNHH Dịch vụ và Thương mại Nam Khoa, Thành phố Hồ Chí Minh. Trình tự gen 16S-rRNA của dòng vi khuẩn tiềm năng được khuếch đại bằng kỹ thuật PCR sử dụng cặp mồi tổng 27F (5'-AGAGGTTTGATCC-TGGCTC-3') và 1492R (5'-TACGGTTACCTT-GTTAACGACT-3') (Frank et al., 2008). ...
Nước thải chế biến thủy sản có chứa protein nên khi bị phân hủy tạo amine, ammonia có mùi hôi gây ảnh hưởng đến sức khỏe cộng đồng. Trong nước, ammonia được chuyển thành ammonium và được vi khuẩn hấp thu cho sự tăng trưởng. Nghiên cứu được thực hiện nhằm phân lập vi khuẩn bản địa trong nước thải chế biến thủy sản có khả năng hấp thu ammonium. Từ 4 mẫu nước thải, 24 dòng vi khuẩn có khả năng hấp thu ammonium đã được phân lập. Kết quả khảo sát chứng tỏ dòng WY3.3 có khả năng hấp thu ammonium (200 ppm) hiệu quả nhất, đạt 94,6% ở thời điểm 24 giờ nuôi cấy. Nuôi cấy thông khí và môi trường có pH = 7 là điều kiện tối ưu cho sự hấp thu ammonium của dòng vi khuẩn WY3.3, đạt hiệu suất lần lượt là 91,9 % và 91,7%. Khi môi trường có bổ sung NaCl 1% và 2%, dòng WY3.3 hấp thu ammonium cao, đạt lần lượt là 99,1% và 97%. Phân tích trình tự gen 16S-rRNA cho thấy dòng WY3.3 tương đồng 99,44% với loài Bacillus funiculus nên được định danh là Bacillus sp. WY3.3.
... Agarose gel electrophoresis is a common technique used in molecular biology to separate and analyze DNA fragments based on their size. (Maniatis, 1982). ...
This study, which was conducted between October 20, 2021, and May 15, 2022, aims to assess the genetic diversity of three breeds of Iraqi goats using two molecular markers. A total of 60 blood samples of 3 goat breeds from different goats in color, age and sex were collected, the 30 Black (Rashoky) and 10 Hybrid goats were used in the center of Sulaimani governorate, Garmian Administration, Raparin Administration, and from different villages within those three areas. 20 blood samples of Meriz (Kurdi) goats were taken from Hajiawa area, Saruchawa area, and
Daraban village in Raparin Administration. Whole blood (5 ml) was collected from each animal from jugular vein into 10 ml vacutainer tubes containing the anticoagulant, ethylenediamine tetraacetic acid (EDTA) and these samples were
stored at –20 °C until DNA extractions. At the University of Sulaimani's College of Agricultural Engineering Sciences, the Biotechnology lab was applied all the laboratory works. Each blood sample was used to extract DNA, which was then quantified using a spectrophotometer after the DNA quantity was verified. Using 1.5% agarose gel electrophoresis, the DNA's quality was evaluated. Two of the most effective markers (SSR and RAPD markers) were used by 11 SSR and 15 RAPD primers, PCR amplification was conducted. The genetic analysis of molecular variance (AMOVA) and pairwise estimates across goat breeds were performed using the GenALEx ver. 6.51 software, POPGENE 32 software package, XLSTAT software, and program STRUCTURE were used for data analysis. The results revealed that 11 SSR primers were amplified and show 847 total bands, 53 are polymorphic with
6.58 percentage of polymorphic bands. All the 15 RAPD primers amplify 6085 total bands, in which 273 are polymorphic bands with 4.33 percentage of polymorphic bands. Different unique bands have been detected for each breed. Both SSR and RAPD give moderate polymorphism 66.67% and 61.52%, respectively. Besides, this value is consistent with the moderate value of the mean of polymorphism information content (PIC) 0.19 and 0.28, respectively. Meriz and Hybrid breeds revealed the longest genetic distance (0.114 and 0.316). While, Hybrid and Black breeds revealed the highest closeness (0.956 and 0.831) for both SSR and RAPD markers, respectively.
Furthermore, the UPGMA dendrogram for both of SSR and RAPD markers classified the three goat breeds into two main clusters. The first one contained Black and Hybrid breeds. While, the second one contained only Meriz breed. The results of the current study will be helpful for future researchers as a key guide to better understanding the genetic relationships and breed differences in Iraqi goat breeds for planning strategies for the future genetic improvement program.
... Obtained consortia genome DNA extracts were directly used for qPCR. For calibration standards preparation, 10 −1 to 10 −5 dilutions were performed for individual strain genome DNA extracted from 100 mL of axenic cultures for algae (7 days culture) and 100 mL of each bacteria (overnight culture) following the same DNA extraction process and additional cleaning step using ethanol acetate precipitation method specified by Sambrook et al. (1989). The initial numbers of DNA molecules in qPCR calibration standards were determined using Equation 5 (Karunakaran et al., 2016) based on known DNA genome concentrations obtained using a microvolume spectrophotometer (NanoDrop™, Thermo Scientific). ...
Microbial communities have been used as important biological tools for a variety of purposes associated with agriculture, the food industry and human health. Artificial engineering of microbial communities is an emerging field of research motivated by finding stable and efficient microbial systems. However, the successful design of microbial communities with desirable functions not only requires profound understanding of microbial activities, but also needs efficient approaches to piece together the known microbial traits to give rise to more complex systems. This study demonstrates the bottom-up integration of environmentally isolated phototrophic microalgae and chemotrophic bacteria as artificial consortia to bio-degrade selected volatile organic compounds (VOCs). A high throughput screening method based on 96-well plate format was developed for discovering consortia with bioremediation potential. Screened exemplar consortia were verified for VOCs degradation performance, among these, certain robust consortia were estimated to have achieved efficiencies of 95.72% and 92.70% and near 100% removal (7 days) of benzene, toluene, and phenol, respectively, with initial concentrations of 100 mg/L. VOCs degradation by consortia was mainly attributed to certain bacteria including Rhodococcus erythropolis, and Cupriavidus metallidurans, and directly contributed to the growth of microalgae Coelastrella terrestris (R = 0.82, p < 0.001). This work revealed the potential of converting VOCs waste into algal biomass by algae-bacteria consortia constructed through a bottom-up approach. The screening method enables rapid shortlisting of consortia combinatorial scenarios without prior knowledge about the individual strains or the need for interpreting complex microbial interactions.
... DNA was isolated from the selected isolate according to Sambrook et al. [56]. The 16s rDNA was amplified by polymerase chain reaction (PCR), using primers designed to amplify 1500 bp fragment of the 16s rDNA region. ...
... DNA substrate χ 0 was prepared from circular plasmid pBR322, while χ + DNA was prepared from pBR322 3F3H, (a pBR322 derivative with two sets of three tandem χ-sequences) (21). These circular plasmid DNA were purified by cesium chloride density gradient centrifugation (30). The molar concentration of DNA was determined using an extinction coefficient of 6290 M −1 cm −1 nt −1 at 260 nm. ...
The loading of RecA onto ssDNA by RecBCD is an essential step of RecBCD-mediated homologous recombination. RecBCD facilitates RecA-loading onto ssDNA in a χ-dependent manner via its RecB nuclease domain (RecBn). Before recognition of χ, RecBn is sequestered through interactions with RecBCD. It was proposed that upon χ-recognition, RecBn undocks, allowing RecBn to swing out via a contiguous 70 amino acid linker to reveal the RecA-loading surface, and then recruit and load RecA onto ssDNA. We tested this hypothesis by examining the interactions between RecBn (RecB928–1180) and truncated RecBCD (RecB1–927CD) lacking the nuclease domain. The reconstituted complex of RecB1–927CD and RecBn is functional in vitro and in vivo. Our results indicate that despite being covalently severed from RecB1–927CD, RecBn can still load RecA onto ssDNA, establishing that RecBn does not function while only remaining tethered to the RecBCD complex via the linker. Instead, RecBCD undergoes a χ-induced intramolecular rearrangement to reveal the RecA-loading surface.
... Standard molecular genetic techniques were used (64,65). For conjugative gene transfer, overnight cultures of donor strain E. coli S17/1 (66) and of the C. metallidurans-recipient strains grown at 30°C in Tris-buffered medium were mixed (1:1) and plated onto nutrient broth agar. ...
In Cupriavidus metallidurans and other bacteria, biosynthesis of the essential biochemical cofactor tetrahydrofolate (THF) initiates from guanosine triphosphate (GTP). This step is catalyzed by FolE_I-type GTP cyclohydrolases, which are either zinc-dependent FolE_IA-type or metal-promiscuous FolE_IB-type enzymes. As THF is also essential for GTP biosynthesis, GTP and THF synthesis form a cooperative cycle, which may be influenced by the cellular homeostasis of zinc and other metal cations. Metal-resistant C. metallidurans harbors one FolE_IA-type and two FolE_IB-type enzymes. All three proteins were produced in Escherichia coli . FolE_IA was indeed zinc dependent and the two FolE_IB enzymes metal-promiscuous GTP cyclohydrolases in vitro , the latter, for example, functioning with iron, manganese, or cobalt. Single and double mutants of C. metallidurans with deletions in the folE_I genes were constructed to analyze the contribution of the individual FolE_I-type enzymes under various conditions. FolE_IA was required in the presence of cadmium, hydrogen peroxide, metal chelators, and under general metal starvation conditions. FolE_IB1 was important when zinc uptake was impaired in cells without the zinc importer ZupT (ZIP family) and in the presence of trimethoprim, an inhibitor of THF biosynthesis. FolE_IB2 was needed under conditions of low zinc and cobalt but high magnesium availability. Together, these data demonstrate that C. metallidurans requires all three enzymes to allow efficient growth under a variety of conditions.
IMPORTANCE
Tetrahydrofolate (THF) is an important cofactor in microbial biochemistry. This “Achilles heel” of metabolism has been exploited by anti-metabolites and antibiotics such as sulfonamide and trimethoprim. Since THF is essential for the synthesis of guanosine triphosphate (GTP) and THF biosynthesis starts from GTP, synthesis of both compounds forms a cooperative cycle. The first step of THF synthesis by GTP cyclohydrolases (FolEs) is metal dependent and catalyzed by zinc- or metal-promiscuous enzymes, so that the cooperative THF and GTP synthesis cycle may be influenced by the homeostasis of several metal cations, especially that of zinc. The metal-resistant bacterium C. metallidurans needs three FolEs to grow in environments with both high and low zinc and cadmium content. Consequently, bacterial metal homeostasis is required to guarantee THF biosynthesis.
... Fifty ml of pure E.coli culture, strain JM109 was cultivated in liquid LB medium (Sambrook et al., 1989). The culture was incubated overnight at 37 °C in shaking incubator (150 rpm). ...
Dietary fibre has positive effects on digestion and on gastrointestinal tract of pigs. Pigs do not possess indigenous enzymes for its digestion. Fibre is fermented by gastrointestinal microflora. The influence of different fibre sources on quantity of microbial biomass in gastrointestinal tract (GIT) of weaned piglets was studied in our experiment. First we introduced ATP-luciferase assay with pure E. coli culture, mixture of E. coli and somatic cells and samples from small and large intestine. The proper protocol for preparation of samples from GIT for ATP-luciferase assay was established. Twenty four weaned piglets, divided into four groups, were fed the same feed except the included fibre: carboxymethylcellulose (group CMC), lignified cellulose (group LIG) and pure cellulose (group CEL). The control group received starch instead of fibre (group KON). Piglets were sacrificed after 13 days of feeding and digesta samples were taken from: stomach, proximal part of small intestine, distal part of small intestine, caecum, colon and rectum together. The quantity of microorganisms in different parts of GIT was estimated by ATP-luciferase assay. The highest concentration (conc.) of microbial ATP was found in large intestine in all groups. Statistically significant effect of different fibres on conc. of microbial ATP in different parts of GIT was not demonstrated. The experiment did not provide evidence that the soluble dietary fibre (carboxymethylcellulose) would enable more intensive microbial fermentation in weaned pigs than insoluble fiber. The reason was probably in big variability and nonhomogenity of samples from GIT.
... Ligation of the FbpA to the pET-32a expression vector with the composition of the ligation reaction consisted of 1 µl T4 DNA Ligase buffer, 50 ng/µl pET-32a expression vector, 18 ng inserted DNA, 1 µl T4 DNA ligase (3u/µl), 4 µl Nuclease Free Water and stored in the refrigerator for 24 hours. Transformation of Recombinant Plasmids to Escherichia coli BL21 with the method is heat shock based on Sambrook et al. 1989. ...
The main challenge in the management of Tuberculosis (TB) is diagnosing quickly and accurately, especially Latent Tuberculosis Infection (LTBI). LTBI detection was carried out using the Tuberculin Skin Test (TST) and Interferon Gamma Release Assay (IGRA). In TB endemic areas, these two examinations have limitations, so current research is directed at finding specific antigens for the diagnosis of LTBI. One of the potential proteins is Antigen 85A (Ag85A) Mycobacterium tuberculosis (Mtb) encoded by Fibronectin-binding protein A (FbpA). The Ag85 complex induces the proliferation of T-cells and interferon-gamma in most healthy individuals infected with Mycobacterium tuberculosis, Mycobacterium leprae, and BCG-vaccinated mice, making it a potential antigen. This study aims to clone and produce recombinant protein Ag85A from Mtb in Escherichia coli BL21. The methods used were ligation to the pET-32a expression vector, transformation to Escherichia coli BL21, and production of protein by IPTG induction. Characterization of recombinant clones by colony PCR and sequencing. The results obtained were that the fbpA gene isolated from Mtb clinical isolate had been amplified, and the PCR product was 900 bp. The production of Antigen 85A has been successfully carried out and produces 44 kDa.
... The bacterial strains and plasmids used or constructed in this study are summarized and listed in Table 1, and all oligonucleotides are described in Table 2. Escherichia coli was grown and transformed according to the standard procedures (26), with E. coli JM109 serving as the host for routine cloning, and E. coli ET12567 (27) used for the isolation of non-methylated DNA for transformation into Streptomyces. For protein heterologous expression, E. coli BL21(DE3) from Novagen was used. ...
Streptomycetes are saprophytic bacteria that grow on complex polysaccharides, such as cellulose, starch, chitin, and chitosan. For the monomeric building blocks glucose, maltose, and N-acetylglucosamine, the metabolic pathways are well-documented, but that of glucosamine (GlcN) is largely unknown. Streptomyces nagB mutants, which lack glucosamine-6-phosphate deaminase activity, fail to grow in the presence of high concentrations of GlcN. Here, we report that mutations in the gene for the ROK-family transcriptional regulator RokL6 relieve the toxicity of GlcN in nagB mutants, as a result of elevated expression of the major facilitator superfamily (MFS) exporter SCO1448. Systems-wide analysis using RNA sequencing, ChIP-Seq, EMSAs, 5′RACE, bioinformatics, and genetics revealed that RokL6 is an autoregulator that represses the transcription of sco1448 by binding to overlapping promoters in the rokL6-sco1448 intergenic region. RokL6-independent expression of sco1448 fully relieved the toxicity of GlcN to nagB mutants. Taken together, our data show a novel system of RokL6 as a regulator that controls the expression of the MFS transporter SCO1448, which in turn protects cells against GlcN toxicity, most likely by exporting toxic metabolites out of the cell.
IMPORTANCE
Central metabolism plays a key role in the control of growth and antibiotic production in streptomycetes. Specifically, aminosugars act as signaling molecules that affect development and antibiotic production, via metabolic interference with the global repressor DasR. While aminosugar metabolism directly connects to other major metabolic routes such as glycolysis and cell wall synthesis, several important aspects of their metabolism are yet unresolved. Accumulation of N-acetylglucosamine 6-phosphate or glucosamine 6-phosphate is lethal to many bacteria, a yet unresolved phenomenon referred to as “aminosugar sensitivity.” We made use of this concept by selecting for suppressors in genes related to glucosamine toxicity in nagB mutants, which showed that the gene pair of rok-family regulatory gene rokL6 and major facilitator superfamily transporter gene sco1448 forms a cryptic rescue mechanism. Inactivation of rokL6 resulted in the expression of sco1448, which then prevents the toxicity of amino sugar-derived metabolites in Streptomyces. The systems biology of RokL6 and its transcriptional control of sco1448 shed new light on aminosugar metabolism in streptomycetes and on the response of bacteria to aminosugar toxicity.
... A total of 148 (74N, 74F) tick specimens were subjected to DNA extraction for molecular analyses. Individual ticks were crushed and their genomic DNA was extracted through a standard method of phenol-chloroform [53]. Genomic DNA was not extracted from all the morphologically identified ticks because we selected representative ticks of each tick species from their respective host in each district. ...
Simple Summary
Ticks are chelicerate arthropods that feed on blood and infest all vertebrates except fish and transmit different disease-causing agents including Rickettsia spp. to domestic and wild animals as well as humans. In the present study, we aimed to molecularly screen and genetically characterize Rickettsia spp. in various tick species infesting camels, sheep, and goats from five districts (Kohat, Dera Ismail Khan, Lower Dir, Bajaur, and Mansehra) of Khyber Pakhtunkhwa province, Pakistan. A total of 8/148 (5.4%) ticks, including four Hyalomma turanicum, two Haemaphysalis cornupunctata, one Haemaphysalis montgomeryi, and one Haemaphysalis bispinosa, were found positive for Rickettsia sp. The phylogenetic analysis of detected Rickettsia sp. based on three genetic markers (gltA, ompA, and ompB) revealed 100% identity with Rickettsia raoultii, clustered with its corresponding species reported in China, Russia, USA, Turkey, Denmark, Austria, Italy, and France. Further comprehensive studies on molecular and serosurveillance of various Rickettsia spp. in different ticks should be conducted in the region to understand the zoonotic threats due to these pathogens.
Abstract
Ticks are hematophagous ectoparasites that transmit different pathogens such as Rickettsia spp. to domestic and wild animals as well as humans. Genetic characterizations of Rickettsia spp. from different regions of Pakistan are mostly based on one or two genetic markers and are confined to small sampling areas and limited host ranges. Therefore, this study aimed to molecularly screen and genetically characterize Rickettsia spp. in various tick species infesting camels, sheep, and goats. All the collected tick specimens were morphologically identified, and randomly selected tick species (148) were screened molecularly for the detection of Rickettsia spp. by amplifying three rickettsial DNA fragments, namely, the citrate-synthase gene (gltA), outer-membrane protein A (ompA), and outer-membrane protein B (ompB). After examining 261 hosts, 161 (61.7%) hosts were found infested by 564 ticks, including 287 (50.9%) nymphs, 171 (30.3%) females, and 106 (18.8%) males in five districts (Kohat, Dera Ismail Khan, Lower Dir, Bajaur, and Mansehra). The highest occurrence was noted for Hyalomma dromedarii (number = 72, 12.8%), followed by Haemaphysalis sulcata (n = 70, 12.4%), Rhipicephalus turanicus (n = 64, 11.3%), Rhipicephalus microplus (n = 55, 9.7%), Haemaphysalis cornupunctata (n = 49, 8.7%), Hyalomma turanicum (n = 48, 8.5%), Hyalomma isaaci (n = 45, 8.0%), Haemaphysalis montgomeryi (n = 44, 7.8%), Hyalomma anatolicum (n = 42, 7.5%), Haemaphysalis bispinosa (n = 38, 6.7%), and Rhipicephalus haemaphysaloides (n = 37, 6.6%). A subset of 148 ticks were tested, in which eight (5.4%) ticks, including four Hy. turanicum, two Ha. cornupunctata, one Ha. montgomeryi, and one Ha. bispinosa, were found positive for Rickettsia sp. The gltA, ompA, and ompB sequences revealed 100% identity and were phylogenetically clustered with Rickettsia raoultii reported in China, Russia, USA, Turkey, Denmark, Austria, Italy, and France. Additionally, various reports on R. raoultii from Palearctic and Oriental regions were summarized in this study. To the best of our knowledge, this is the first report regarding genetic characterization and phylogenetic analysis of R. raoultii from Pakistan. Further studies to investigate the association between Rickettsia spp. and ticks should be encouraged to apprise effective management of zoonotic consequences.
... Th e fi nal extension was at 72 °C for 10 min. Th e PCR products with Th ermo Scientifi c MassRuler DNA Ladder Mix (Th ermo Fisher Scientifi c, Waltham, MA, USA) were separated in a 1.5% agarose gel stained with ethidium bromideand visualized under UV light [20]. ...
Th is paper describes the results of small-scale laboratory simulation of anaerobic digestion of plant biomass simultaneously contaminated with several common plant viruses to see if these pathogens can retain their biological properties (i.e., infectivity). By using a set of complementary techniques, we show that at least one of model viruses has successfully survived this simulation and was able to induce productive and symptomatic infection in susceptible host plants.
... cleotides attached to streptavidin-coated MagneSphere (Promega). Following elution in 100 µ L TE buffer, the fragments were amplified by polymerase chain reaction (PCR) using an adapter-specific primer (Adap-F) and cloned in the pGEM-T Easy Vector (Promega), transfected into Escherichia coli DH5 α cells (Invitrogen), following standard procedures (Sambrook et al . 1989). Recombinant clones were transferred to Hybond-N + membranes (GE Healthcare), which were probed with an array of 15 (CT) repeat motif, using the AlkPhos Direct Labelling and Detection System (GE Healthcare). Screening for the presence of microsatellite repeats was performed with high stringency, and positive clones were amplified in 25 ...
Here we presented 10 polymorphic microsatellite loci obtained from scarlet ibis through an enriched genomic library. The analysis of 45 individuals from three Brazilian natural populations showed allelic diversity ranged from three to 17 alleles, observed heterozygosity ranged from 0.03 to 0.92, and expected heterozygosity ranged from 0.06 to 0.92. These highly variable microsatellite loci can provide means for assessing overall genetic variation in its remnant natural populations, which may help the development of effective conservation programs.
... The new chromogenic TCBS medium consists of 10 g of peptone, 10 g of sea salts mixture, 10 g of ox bile, 10 g of sodium thiosulfate, 5 g of yeast extract, 5 g of sodium citrate, 2.2 g of sodium carbonate, 2 g of lactose, 0.5 g of sodium pyruvate and 1000ml with water and P H adjusted to 8.6 and autoclaved at 15psi/15min [34]. Genomic DNA was isolated using Proteinase-Phenol-chloroform method and used for PCR amplification according to Manufacturer's protocol [35]. The Vme drug efflux genes PCR amplification and sequencing were described previously (Matsuo T., et al. 2013) [29]. ...
... Transformation of E. coli cells with a DNA plasmid was carried out as described previously [30]. An E. coli TG1 strain was used for production of plasmids. ...
Entomopathogenic bacteria of the genus Photorhabdus secrete protease S (PrtS), which is considered a virulence factor. We found that in the Photorhabdus genomes, immediately after the prtS genes, there are genes that encode small hypothetical proteins homologous to emfourin, a recently discovered protein inhibitor of metalloproteases. The gene of emfourin-like inhibitor from Photorhabdus laumondii subsp. laumondii TT01 was cloned and expressed in Escherichia coli cells. The recombinant protein, named photorin (Phin), was purified by metal-chelate affinity and gel permeation chromatography and characterized. It has been established that Phin is a monomer and inhibits activity of protealysin and thermolysin, which, similar to PrtS, belong to the M4 peptidase family. Inhibition constants were 1.0 ± 0.3 and 10 ± 2 µM, respectively. It was also demonstrated that Phin is able to suppress proteolytic activity of P. laumondii culture fluid (half-maximal inhibition concentration 3.9 ± 0.3 nM). Polyclonal antibodies to Phin were obtained, and it was shown by immunoblotting that P. laumondii cells produce Phin. Thus, the prtS genes in entomopathogenic bacteria of the genus Photorhabdus are colocalized with the genes of emfourin-like inhibitors, which probably regulate activity of the enzyme during infection. Strict regulation of the activity of proteolytic enzymes is essential for functioning of all living systems. At the same time, the principles of regulation of protease activity by protein inhibitors remain poorly understood. Bacterial protease-inhibitor pairs, such as the PrtS and Phin pair, are promising models for in vivo studies of these principles. Bacteria of the genus Photorhabdus have a complex life cycle with multiple hosts, being both nematode symbionts and powerful insect pathogens. This provides a unique opportunity to use the PrtS and Phin pair as a model for studying the principles of protease activity regulation by proteinaceous inhibitors in the context of bacterial interactions with different types of hosts.
... Escherichia coli and B. abortus strains (Table 1) were incubated at 37°C at 200 rpm and grown on Luria Bertani Broth (LB) (Sambrook et al., 1989) or Tryptic Soy Broth (TSB) (Suárez-Esquivel et al., 2016). Additionally, culture media were supplemented with antibiotics (kanamycin 30 μg/ml, gentamicin 20 μg/ml, or ampicillin 100 μg/ml) when necessary. ...
Brucella abortus is a facultative extracellular-intracellular bacterial zoonotic pathogen worldwide. It is also a major cause of abortion in bovines, generating economic losses. The two-component regulatory system BvrR/BvrS modulates the expression of genes required to transition from extracellular to intracellular lifestyles. However, few regulatory regions of BvrR direct target genes have been studied. In this study, we characterized the regulatory region of omp25 , a gene encoding an outer membrane protein that is positively regulated by TCS BvrR/BvrS. By omp25 - lacZ reporter fusions and β-galactosidase activity assays, we found that the region between-262 and + 127 is necessary for transcriptional activity, particularly a 111-bp long fragment located from-262 to −152. In addition, we demonstrated the binding of P-BvrR to three sites within the −140 to +1 region. Two of these sites were delimited between −18 to +1 and − 99 to −76 by DNase I footprinting and called DNA regulatory boxes 1 and 2, respectively. The third binding site (box 3) was delimited from −140 to −122 by combining EMSA and fluorescence anisotropy results. A molecular docking analysis with HDOCK predicted BvrR-DNA interactions between 11, 13, and 12 amino acid residue-nucleotide pairs in boxes 1, 2, and 3, respectively. A manual sequence alignment of the three regulatory boxes revealed the presence of inverted and non-inverted repeats of five to eight nucleotides, partially matching DNA binding motifs previously described for BvrR. We propose that P-BvrR binds directly to up to three regulatory boxes and probably interacts with other transcription factors to regulate omp25 expression. This gene regulation model could apply to other BvrR target genes and to orthologs of the TCS BvrR/BvrS and Omp25 in phylogenetically closed Rhizobiales .
... All samples used in this study are listed in Table 1. DNA was extracted from muscle tissue of a male individual using the standard phenol-chloroform technique 29 . A library for low-coverage genomic sequencing was prepared using TruSeq Nano DNA Low Throughput Library Prep (Illumina), following the manufacturer's protocol. ...
The classical hypothesis proposes that the lack of recombination on sex chromosomes arises due to selection for linkage between a sex-determining locus and sexually antagonistic loci, primarily facilitated by inversions. However, cessation of recombination on sex chromosomes could be attributed also to neutral processes, connected with other chromosome rearrangements or can reflect sex-specific recombination patterns existing already before sex chromosome differentiation. Three Coleonyx gecko species share a complex X1X1X2X2/X1X2Y system of sex chromosomes evolved via a fusion of the Y chromosome with an autosome. We analyzed synaptonemal complexes and sequenced flow-sorted sex chromosomes to investigate the effect of chromosomal rearrangement on recombination and differentiation of these sex chromosomes. The gecko sex chromosomes evolved from syntenic regions that were also co-opted also for sex chromosomes in other reptiles. We showed that in male geckos, recombination is less prevalent in the proximal regions of chromosomes and is even further drastically reduced around the centromere of the neo-Y chromosome. We highlight that pre-existing recombination patterns and Robertsonian fusions can be responsible for the cessation of recombination on sex chromosomes and that such processes can be largely neutral.
... Total genomic DNA was extracted from ethanol-preserved muscle or liver tissues using standard phenol-chloroform extraction procedures (Sambrook et al. 1989) followed with isopropanol precipitation. We used the polymerase chain reaction (PCR) to amplify three mtDNA fragments: complete sequences of cytochrome b (cyt b) and NADH dehydrogenase subunit 4 (ND4) genes and a fragment of 16S rRNA gene. ...
We describe a new species of pitvipers from Trang Province of Thailand, near the Thailand–Malaysian border, based on morphological and molecular (2427 bp from cyt b, ND4, and 16S rRNA mitochondrial DNA genes) lines of evidence. Morphologically, Trimeresurus ciliaris sp. nov. is distinguished from its congeners by the following combination of morphological characters: a long papillose hemipenis; first supralabial and nasal scale fused; three to four small supraocular scales; internasals not in contact; small scale between nasal and the scale formed by the fused second supralabial and loreal present; dorsal scales in 17–17–15 rows across the body; ventral scales 172–175 in males, 171 in female; subcaudal scales 59–63 in males, 61 in female, all paired; in life an emerald-green dorsum with reddish-brown bands; creamy-white venter lacking dark dots or stripes on the lateral sides of the ventrals; white vertebral spots present in both sexes on every two or three dorsal scales; dark brown spots forming discontinuous pattern present on 1–3 lateral dorsal scale rows; males with reddish-brown postocular stripe. The new species forms a distinct clade on the phylogenetic tree of the genus Trimeresurus and differs from the morphologically similar species T. venustus by a significant divergence in cytochrome b mitochondrial DNA gene sequences (p = 12.5%). The new species is currently known from a small karstic area in the Nakawan Range spanning the border of Thailand and Malaysia, in particular in limestone forests in Trang and Satun provinces (Thailand); it likely also occurs in the adjacent parts of Perlis State (Malaysia). Our study also suggests that the taxonomy of T. kanburiensis species complex requires further studies; in particular our study suggests that the status of populations from Chumphon Province of Thailand and Pulau Langkawi Island of Malaysia should be re-assessed.
... Recombinant NS1 constructs were transformed into DH5-alpha competent Escherichia coli cells (NEB), and the sequences were verified by Sanger sequencing. HEK293T cells were transiently transfected with the recombinant NS1 constructs or empty pCAGGS using a calcium phosphate transfection protocol (71). Culture supernatant was har vested 48 hours post-transfection and cleared by filtration (0.22 µm). ...
The flavivirus non-structural protein 1 (NS1) is secreted from infected cells into the circulation and the serum levels correlate with disease severity. The effect of secreted NS1 (sNS1) on non-infected mammalian immune cells is largely unknown. Here, we expressed recombinant sNS1 proteins of tick-borne encephalitis virus (TBEV) and West Nile virus (WNV) and investigated their effects on dendritic cell (DC) effector functions. Murine bone marrow-derived DCs (BMDCs) showed reduced surface expression of co-stimulatory molecules and decreased release of pro-inflammatory cytokines when treated with sNS1 of TBEV or WNV prior to poly(I:C) stimulation. Transcriptional profiles of BMDCs that were sNS1-exposed prior to poly(I:C) stimulation showed two gene clusters that were downregulated by TBEV or WNV sNS1 and that were associated with innate and adaptive immune responses. Functionally, both sNS1 proteins modulated the capacity for BMDCs to induce specific T-cell responses as indicated by reduced IFN-γ levels in both CD4 ⁺ and CD8 ⁺ T cells after BMDC co-cultivation. In human monocyte-derived DCs, poly(I:C)-induced upregulation of co-stimulatory molecules and cytokine responses were even more strongly impaired by TBEV sNS1 or WNV sNS1 pretreatment than in the murine system. Our findings indicate that exogenous flaviviral sNS1 proteins interfere with DC-mediated stimulation of T cells, which is crucial for the initiation of cell-mediated adaptive immune responses in human flavivirus infections. Collectively, our data determine soluble flaviviral NS1 as a virulence factor responsible for a dampened immune response to flavivirus infections.
IMPORTANCE
The effective initiation of protective host immune responses controls the outcome of infection, and dysfunctional T-cell responses have previously been associated with symptomatic human flavivirus infections. We demonstrate that secreted flavivirus NS1 proteins modulate innate immune responses of uninfected bystander cells. In particular, sNS1 markedly reduced the capacity of dendritic cells to stimulate T-cell responses upon activation. Hence, by modulating cellular host responses that are required for effective antigen presentation and initiation of adaptive immunity, sNS1 proteins may contribute to severe outcomes of flavivirus disease.
... The PCR was carried out using Hercuvan GS-96 Thermal cycler (Sdn, Bhd, Malaysia) with an initial denaturation at 94°C for 3 min; followed by 34 cycles each of 94°C for 60 s, annealing at 58°C for 60 s, and extension at 72°C for 2 min; and a final elongation for 10 min at 72°C., and 4°C. PCR amplification was confirmed by amplicon was separating 5 μL of amplicon on 1% agarose gel [14] and amplicons sequenced on both strands using the above primers. Nucleotide Basic Local Alignment Search Tool (BLAST) search (http://www.ncbi.nlm.nih.gov/BLAST/) was carried out to identify the bacterium to species level and the sequences were submitted to GenBank under the accession number: MW766369. ...
Biodegradation of harmful chemicals is of paramount importance for keeping a clean environment. Anthracene is a polycyclic aromatic hydrocarbons (PAHs) with a significant pollution potential and health risk. Because of its relative toxicity it has been utilized as a model for PAHs degradation investigations. Endophytic and rhizospheric bacteria can degrade PAHs like anthracene, which is a time/cost-effective method for eco-restoration. Therefore, techniques to eradicate this contaminant from the environment are urgently needed. In this study, the possible degrading ability of anthracene by Proteus sp. Strain, BTE_BCH from Nigeria was assessed using the one-factor-at-a-time (OFAT) technique. Mineral Salt Medium was used to culture the bacterium augmented with anthracene as its only energy and carbon source, at an optimum temperature of 35°C, pH 7.5, inoculum size of 400 µL and 400 mg/L substrate concentration, after 72 h incubation time. In addition, the bacterium can tolerate 2 mg/L Cu, Hg, Fe, Cr, Zn, Cd, Ni, Pb and As. The bacterium was able to degrade 97.5% anthracene after 72 h incubation. This data could be beneficial for optimizing anthracene biodegradation environmental factors; most importantly in the cleanup of anthracene-polluted areas. Furthermore, this study revealed that Proteus sp. strain BTE_BCH might be used to biodegrade anthracene-contaminated soil since it is less expensive, easier, eco-friendly and non-pathogenic.
... As a positive control, Escherichia coli ATCC 9610 was utilised. The PCR products were amplified, then placed on a 1.5% agarose gel, stained with 1% ethidium bromide (0.5 g/mL), and seen under UV light (UV gel documentation system, Cleaver scientific Ltd, USA) [20]. ...
... The flukes were preserved in 70% ethanol and DNA was extracted through the Phenol-Chloroform extraction and Ethanol Precipitation methods (Sambrook et al., 1989). Pellets containing DNA were dried for 10 minutes followed by resuspension in 100 µl sterile TE buffer (pH 8.0). ...
The mycelium of the plant pathogenic fungus Fusarium graminearum exhibits distinct structures for vegetative growth, asexual sporulation, sexual development, virulence, and chlamydospore formation. These structures are vital for the survival and pathogenicity of the fungus, necessitating precise regulation based on environmental cues. Initially identified in Magnaporthe oryzae, the transcription factor Con7p regulates conidiation and infection-related morphogenesis, but not vegetative growth. We characterized the Con7p ortholog FgCon7, and deletion of FgCON7 resulted in severe defects in conidium production, virulence, sexual development, and vegetative growth. The mycelia of the deletion mutant transformed into chlamydospore-like structures with high chitin level accumulation. Notably, boosting FgABAA expression partially alleviated developmental issues in the FgCON7 deletion mutant. Chromatin immunoprecipitation (ChIP)–quantitative PCR (qPCR) analysis confirmed a direct genetic link between FgABAA and FgCON7. Furthermore, the chitin synthase gene Fg6550 (FGSG_06550) showed significant upregulation in the FgCON7 deletion mutant, and altering FgCON7 expression affected cell wall integrity. Further research will focus on understanding the behavior of the chitin synthase gene and its regulation by FgCon7 in F. graminearum. This study contributes significantly to our understanding of the genetic pathways that regulate hyphal differentiation and conidiation in this plant pathogenic fungus.
IMPORTANCE
The ascomycete fungus Fusarium graminearum is the primary cause of head blight disease in wheat and barley, as well as ear and stalk rot in maize. Given the importance of conidia and ascospores in the disease cycle of F. graminearum, precise spatiotemporal regulation of these biological processes is crucial. In this study, we characterized the Magnaporthe oryzae Con7p ortholog and discovered that FgCon7 significantly influences various crucial aspects of fungal development and pathogenicity. Notably, overexpression of FgABAA partially restored developmental defects in the FgCON7 deletion mutant. ChIP-qPCR analysis confirmed a direct genetic link between FgABAA and FgCON7. Furthermore, our research revealed a clear correlation between FgCon7 and chitin accumulation and the expression of chitin synthase genes. These findings offer valuable insights into the genetic mechanisms regulating conidiation and the significance of mycelial differentiation in this plant pathogenic fungus.
The Italian geographic region is characterized by complex and diversified biogeographic patterns and is represented by a high number of endemic species. Endemic species characterized by a limited distribution range should be a primary concern in conservation. This article aimed to investigate the phylogenetic and biogeographic relationships of 2 Italian endemic species of the wingless blister beetle genus Meloe Linnaeus, 1758: Meloe (Eurymeloe) apenninicus and Meloe (E.) baudii. Our inferences, based on morphological characters, 2 mitochondrial (16S and COI) and 2 nuclear (CAD and 28S) markers and the use of 3 species delimitation analyses approaches, pointed out the presence of a new Italian endemic species (M. (E.) digiuliorumsp. n.), here described, and 3 different patterns of phylogenetic and biogeographic affinities. M. digiuliorum is close to the Spanish endemic M. orobatescomb. n., revealing a possible fragmentation of the ancestor range in the Pleistocene (ca. 0.84 Mya) followed by isolation in Italy and Spain. M. apenninicus is the sister species of the European-Anatolian M. rugous and M. cfr. rugosus, and this pattern originated around the Plio-Pleistocene boundary (ca. 2.83 Mya) likely influenced by the climatic fluctuations and the presence of the Alpine barrier. Finally, 2 subspecies were referred to M. baudii: the nominal one, endemic to Italy, and the Turanian-E European M. b. glazunovistat. n., disclosing a third more recent (ca. 0.64 Mya) pattern of biogeographic disjunction.
Protein synthesis is a major energy-consuming process of the cell that requires the controlled production1–3 and turnover4,5 of ribosomes. Although the past few years have seen major advances in our understanding of ribosome biogenesis, structural insight into the degradation of ribosomes has been lacking. Here we present native structures of two distinct small ribosomal 30S subunit degradation intermediates associated with the 3′ to 5′ exonuclease ribonuclease R (RNase R). The structures reveal that RNase R binds at first to the 30S platform to facilitate the degradation of the functionally important anti-Shine–Dalgarno sequence and the decoding-site helix 44. RNase R then encounters a roadblock when it reaches the neck region of the 30S subunit, and this is overcome by a major structural rearrangement of the 30S head, involving the loss of ribosomal proteins. RNase R parallels this movement and relocates to the decoding site by using its N-terminal helix-turn-helix domain as an anchor. In vitro degradation assays suggest that head rearrangement poses a major kinetic barrier for RNase R, but also indicate that the enzyme alone is sufficient for complete degradation of 30S subunits. Collectively, our results provide a mechanistic basis for the degradation of 30S mediated by RNase R, and reveal that RNase R targets orphaned 30S subunits using a dynamic mechanism involving an anchored switching of binding sites.
Community-acquired methicillin resistant Staphylococcus aureus (CA-MRSA) strains are increasingly replacing hospital-acquired MRSA (HA-MRSA) strains in hospitalized patients leading to poor clinical outcomes. Hence, this study aimed to characterize clinical isolates of MRSA (HA-MRSA and CA-MRSA) and to understand their clonal origin. A total of 400 consecutive S. aureus clinical isolates were collected from the clinical bacteriology lab of a tertiary care hospital. All the isolates were screened for MRSA by cefoxitin disc diffusion test and mecA PCR, followed by SCCmec typing, antibiotic susceptibility testing, Panton Valentine Leukocidin (PVL) screening, and pulsed field gel electrophoresis (PFGE). Of the total 400 isolates, 134 categorized MRSA by cefoxitin, while 129 as mecA positive by PCR, of which 117 could be characterized into SCCmec types. SCCmecI and II were present in 1 isolate each, SCCmecIII in 36 (31%) representing HA-MRSA, While SCCmecIV in 51 (44%), and SCCmecV in 28 (24%) isolates representing CA-MRSA. Of all SCCmecIII isolates, 70% were multidrug resistant (MDR) while 59% of SCCmecIV and 29% of SCCmecV isolates were MDR. PVL (CA-MRSA virulence factor) positivity in mecIII, IV, V isolates was 9%, 31%, 46% respectively. PFGE typing showed MRSA clones of multiple origins. In conclusion, study showed the evolving epidemiology of HA-MRSA and CA-MRSA. CA-MRSA constituted the majority of clinical isolates amongst both community and hospital MRSA isolates. Various MDR clones of mecIV and mecV were circulating and replacing mecIII in hospital settings. SCCmecIV isolates were predominant and evolved as MDR, however, PVL was significantly associated with CA-MRSA.
The white-spotted bamboo shark (Chiloscyllium plagiosum) is of great commercial, ecological and ornamental value and has been listed as a near-threatened species in the IUCN Red List. Characterizing the mitochondrial genome (mitogenome) should be useful for biodiversity and conservation investigations. In this study, the characteristics of the mitogenome of C. plagiosum were reported. The mitogenome of C. plagiosum was 16,726 bp in length, comprising 13 protein-coding genes, 2 ribosomal RNAs, 22 transfer RNAs and one control region, which was similar to most fish species. In the control region, some conserved motifs were identified, except for motifs in the central conserved domain. In addition, phylogenetic analysis revealed C. griseum was at the basal position among investigated bamboo shark species, suggesting the third codon positions in the mitochondrial protein-coding genes should be involved in bamboo shark phylogeny. Further studies with more powerful genetic markers are needed to resolve the phylogenetic conflicts in genus Chiloscyllium. These findings may have important evolutionary and conservation implications for sound management of C. plagiosum.
Objectives:
This study aimed to investigate the genetic association between MTHFR (A1298C) SNP and preeclampsia (PE) in Punjab, Pakistan.
Methods:
A sample of 80 pregnant women (40 healthy pregnant women and 40 with PE) was pooled for genotyping MTHFR A1298C polymorphism by using the tetra-primer amplification refractory mutation system (ARMS) PCR. The Genotypic and allelic assessments were performed using various statistical techniques.
Results:
The AC genotype and C allele of MTHFR A1298C were found to be associated with decreased risk of PE (odds ratio [OR]: 0.31, risk ratio [RR]: 0.58, p = 0.01), and (odds ratio [OR]: 0.49, risk ratio [RR]: 0.61, p = 0.04), respectively.
Conclusion:
In conclusion, genetic polymorphism A1298C in MTHFR may pose a protective effect in the studied population.
The root nodule bacteria are utilized in the production of natural biological fertilizers to achieve clean agriculture by reducing chemical fertilizers. In this study, 35 samples of the root nodules associated with mung bean plants were collected from various agricultural areas of Iraq, Salah Al-Din Governorate on 4\7\2022, ten samples isolated by growing them on yeast extract mannitol agar (YEMA) and the phenotypically pure isolates were diagnosed based on the culture, microscopic and biochemical characteristics., The phenotypic diagnosis results showed that The color of the colonies was between white, creamy and yellow, Spherical, convex and smooth, gram-negative, was able to move and pink and light pink on Congo red stain medium and 9 isolates it was fast growing as it gave yellow color on YEMA-BTB bromothymol blue medium except for one isolate it was slow growing as it gave blue color and all isolates unable to grow on Hofer alkaline medium except for one isolate, The efficient isolate was selected in the production of indole acetic acid, chelating compounds and phosphate solubilization,. The results showed that isolates (M3, M4, M5, M6, M7) have a high ability for iron chelation. It was found that the (M6) isolate gave the highest phosphate solubilization and indole production, which showed a phosphorus solubilization of 39.504 mg P.L ⁻¹ and an indole production rate of 21.5 μg.ml ⁻¹ . This was followed by the isolate (M3), which showed a phosphorus solubilization of 23.723 mg P.L ⁻¹ and an indole production rate of 17.2 μ.g.ml ⁻¹ . Molecular diagnosis was performed for five competent isolates in the production of Indole acetic acid, production of chelating compounds, and phosphate solubilization. The isolates were molecularly diagnosed by PCR, the 16SrRNA gene was amplified, then the sequence of nitrogenous bases was analyzed and when matching with the global strains included in the NCBI Genetic Bank website, The results of molecular diagnosis showed that two isolates belong to Rhizobium bacteria. the results showed that (M3) isolate is %99.50 similar to the Rhizobium leguminosarum isolate OTU21_I . and the results showed that (M6) isolate is 99.48% similar to the Bradyrhizobium japonicum strain A3 and 99.96% to the Bradyrhizobium japonicum, strain:NK5 and therefore the isolates is genetically close to Rhizobium leguminosarum strain AE15 and Bradyrhizobium japonicum strain AE14 bacteria and has been recorded in the Global genome bank under accession number OP975690 and version number OP975690.1 for Rhizobium leguminosarum and accession number OP975688 and version number OP975688.1 for Bradyrhizobium japonicum and this record is the first for this bacteria which associated with mung bean in gypsiferous soils in Iraq.
The prevailing concept is that gestational alloimmune liver disease (GALD) is caused by maternal antibodies targeting a currently unknown antigen on the liver of the fetus. This leads to deposition of complement on the fetal hepatocytes and death of the fetal hepatocytes and extensive liver injury. In many cases, the newborn dies. In subsequent pregnancies early treatment of the woman with intravenous immunoglobulin can be instituted, and the prognosis for the fetus will be excellent. Without treatment the prognosis can be severe. Crucial improvements of diagnosis require identification of the target antigen. For this identification, this work was based on two hypotheses: 1. The GALD antigen is exclusively expressed in the fetal liver during normal fetal life in all pregnancies; 2. The GALD antigen is an alloantigen expressed in the fetal liver with the woman being homozygous for the minor allele and the father being, most frequently, homozygous for the major allele. We used three different experimental approaches to identify the liver target antigen of maternal antibodies from women who had given birth to a baby with the clinical GALD diagnosis: 1. Immunoprecipitation of antigens from either a human liver cell line or human fetal livers by immunoprecipitation with maternal antibodies followed by mass spectrometry analysis of captured antigens; 2. Construction of a cDNA expression library from human fetal liver mRNA and screening about 1.3 million recombinants in Escherichia coli using antibodies from mothers of babies diagnosed with GALD; 3. Exome/genome sequencing of DNA from 26 presumably unrelated women who had previously given birth to a child with GALD with husband controls and supplementary HLA typing. In conclusion, using the three experimental approaches we did not identify the GALD target antigen and the exome/genome sequencing results did not support the hypothesis that the GALD antigen is an alloantigen, but the results do not yield basis for excluding that the antigen is exclusively expressed during fetal life., which is the hypothesis we favor.
Much of our current understanding of microbiology is based on the application of genetic engineering procedures. Since their inception (more than 30 years ago), methods based largely on allelic exchange and two-step selection processes have become a cornerstone of contemporary bacterial genetics. While these tools are established for adapted laboratory strains, they have limited applicability in clinical or environmental isolates displaying a large and unknown genetic repertoire that are recalcitrant to genetic modifications. Hence, new tools allowing genetic engineering of intractable bacteria must be developed to gain a comprehensive understanding of them in the context of their biological niche. Herein, we present a method for precise, efficient and rapid engineering of the opportunistic pathogen Pseudomonas aeruginosa. This procedure relies on recombination of short single-stranded DNA facilitated by targeted double-strand DNA breaks mediated by a synthetic Cas9 coupled with the efficient Ssr recombinase. Possible applications include introducing single-nucleotide polymorphisms, short or long deletions, and short DNA insertions using synthetic single-stranded DNA templates, drastically reducing the need of PCR and cloning steps. Our toolkit is encoded on two plasmids, harboring an array of different antibiotic resistance cassettes; hence, this approach can be successfully applied to isolates displaying natural antibiotic resistances. Overall, this toolkit substantially reduces the time required to introduce a range of genetic manipulations to a minimum of five experimental days, and enables a variety of research and biotechnological applications in both laboratory strains and difficult-to-manipulate P. aeruginosa isolates.
The monothiol glutaredoxin GrxD plays an essential role in the biosynthesis of the antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG) and the biocontrol capacity of the soil bacterium Pseudomonas fluorescens 2P24. However, the detailed mechanism underlying GrxD-mediated activation of the production of 2,4-DAPG remains unclear. Here, we found that GrxD directly interacted with IbaG, a BolA protein family member. The mutation of ibaG significantly decreased 2,4-DAPG production. Furthermore, expressing ibaG restored the production of 2,4-DAPG in the grxD ibaG double mutant to wild-type levels in the presence of dithiothreitol, suggesting that IbaG was required for GrxD-mediated regulation of 2,4-DAPG production. Transcriptome sequencing analyses revealed that IbaG plays a global role in gene regulation by affecting the expression of numerous genes throughout the genome. We also demonstrated that IbaG is an important regulator of several cellular processes, including swarming motility, biofilm formation, siderophore production, and acid resistance. Altogether, our data suggest that IbaG has an essential role in 2,4-DAPG production, motility, and biofilm formation. We also propose a regulatory mechanism linking GrxD to 2,4-DAPG production via IbaG.
IMPORTANCE
The production of 2,4-diacetylphloroglucinol (2,4-DAPG) is positively influenced by the monothiol glutaredoxin GrxD in Pseudomonas fluorescens 2P24. However, the regulatory mechanism underlying GrxD-mediated regulation of 2,4-DAPG biosynthesis is mostly uncharacterized. Here, we show the function of the BolA-like protein IbaG in 2,4-DAPG biosynthesis. We also demonstrate that GrxD directly interacts with IbaG and influences the redox state of IbaG. Altogether, this work provides new insights into the role of the highly conserved IbaG protein in regulating 2,4-DAPG synthesis, biofilm formation, and other biocontrol traits of P. fluorescens .
Habitat loss and fragmentation threaten population persistence because they affect the ability of individuals to disperse between remaining patches of good-quality habitat and reduce refuge areas for populations. In cougars (Puma concolor), males are predominantly dispersers while females tend to be more philopatric. To examine the dispersal ability and philopatry of cougars in a human-dominated landscape in Brazil, we performed relatedness and spatial autocorrelation analyses based on genotyped cougars from different sampling groups: forest fragments within a human-modified matrix, continuous forest, and road-killed individuals. We found similarly high relatedness and a positive spatial autocorrelation at the shortest spatial scale (0-100 km) for both males and females from the forest fragments within a human-modified matrix. In the continuous forest and among cougars sampled as roadkills, we detected no spatial autocorrelation and observed low relatedness for both sexes. We also detected higher male:female sex ratio among road-killed individuals, likely due to the greater dispersal tendency of males. Our results confirm female philopatry in the forest fragments. However, the high relatedness and positive autocorrelation observed in the forest fragments suggest kin clustering also for males, which may be a result of unsuccessful dispersal. We reported the first evidence for a South American cougar population of unsuccessful dispersal in response to human-altered landscapes. Further research is needed to assess the specific causes of male unsuccessful dispersal and how it may affect species persistence in human-dominated landscapes.
Lead (Pb) is a non-essential metal with high toxicity, is persistent, is not biodegradable, and has no known biological function. It is responsible for severe health and environmental issues that need appropriate remediation. Therefore, microbes have thrived in a lead-contaminated environment without exhibiting any negative impacts. The present study aimed to examine the toxic effects of lead on animals and the isolation, identification, and characterization of lead-resistant bacterial strains and their biodegradation potential. After oral administration of lead for 4 weeks, mice showed an elevated level of leukocytes and a decrease in TEC, Hb, PCV, MCV, MCH, and MCHC levels. However, a decline in body weight and inflammation and oxidative stress was observed in liver tissues. To remediate toxic heavy metal, lead-resistant bacterial strains were isolated, among which Enterobacter exhibited maximum degradation potential at high lead concentrations. It was identified by molecular basis and after 16S rRNA sequencing, and 99% resemblance was observed with Enterobacter cloacae. FT-IR analysis of the bacteria illustrated the presence of functional groups, including hydroxyl, carboxyl group, sulfide, and amino groups, on the bacterial cell surface involved in the adsorption of lead. Moreover, electron microscopy (SEM) revealed the morphological and physiochemical changes in the bacterial cell after biosorption, indicating the interaction of Cu ions with functional groups. To summarize, the findings show the highly toxic effects of lead on animals and humans and its effective biodegradation by the bacterial strains in the lead-contaminated environment. This biological strategy can be an ideal alternative to remediate heavy metals from contaminated sites to clean up the environment.
Rambutan (Nephelium lappaceum L.) merupakan salah satu tanaman tropis Indonesia yang mempunyai banyak potensi ekonomi sebagai upaya diversitas buah-buahan. Pelestarian plasma nutfah rambutan perlu dilakukan lebih lanjut baik secara kualitas maupun kuantitas. Metode RAPD digunakan untuk memperoleh informasi mengenai keragaman genetik antar jenis tanaman rambutan. Penelitian dilakukan menggunakan metode RAPD (Random Amplified Polymorphic DNA) dengan 20 primer. Hasil penelitian menunjukkan bahwa seleksi 20 primer menghasilkan 5 primer terbaik yaitu (OPD 2, OPD 5, OPD 6, OPD 10, OPD 12), pada ukuran 50-2000 bp dengan total 48 pita (42 pita polimorfik dan 6 pita monomorfik). Tingkat polimorfisme keragaman antar jenis rambutan sebesar 86,24%. Hasil uji dendogram UPGMA menunjukkan adanya keragaman genetik yang tergolong tinggi pada rambutan. Terbentuk dua klaster utama dengan tingkat kesamaan antara 56-77%. Rambutan jenis Rapiah berkerabat dekat dengan Sibatuk Ganal sebesar 77%.
The ability of 7,12-dimethylbenz(a)anthracene (DMBA), a potent inducer of mammary tumors, to mimic short term effects of estradiol (17 beta-E2) on the anterior pituitary, was tested in vitro. Incubation of anterior pituitaries from ovariectomized rats with DMBA resulted in a marked depletion of membrane dopamine receptors (labeled with [3H] spiperone) and a parallel stimulation of prolactin (PRL) release. Maximal receptor depletion and PRL release were obtained after 15-30 min of incubation with 10(-8) M DMBA. These effects were reversible and already significant after a 5-min incubation. Their magnitude, dose dependency, and time course were identical to those reported for 17 beta-E2. A structurally related noncarcinogenic polycyclic aromatic hydrocarbon, phenanthrene, had no effect on [3H]spiperone binding or PRL release. When DMBA and 17 beta-E2, at suboptimal concentrations, were simultaneously added to the culture medium, no synergistic effect could be observed. When 10(-8) M of both compounds were introduced simultaneously, the decrease in dopamine receptors and the increase in PRL release were not greater than those observed in the presence of 10(-8) M of only one compound, indicating that the same mechanism(s) can be involved. These data suggest that DMBA in desensitizing lactotrophs to dopamine and in releasing PRL, by direct estrogen-like actions on anterior pituitary, may provide a hormonal state conducive to tumor development.
The effects of 17 beta-estradiol (E17 beta) on prolactin (PRL) cell proliferation and on the expression of PRL and growth hormone (GH) proteins and mRNAs were analyzed in cultured pituitary cells by immunocytochemistry, in situ hybridization, and Northern blot hybridization studies. Three different cell cultures were used: (a) normal pituitary cells; (b) GH3 tumor cell line; and (c) MtT/W15, a transplantable PRL and GH-producing pituitary tumor. E17 beta (10(-7) M) caused a significant increase in PRL cell proliferation in normal pituitary [3.9 +/- 0.4 versus 7.7 +/- 0.9% (SEM) of immunostained PRL cells with thymidine incorporation] [P less than 0.01] but produced a significant decrease in PRL cell proliferation in MtT/W15 primary cell cultures [6.7 +/- 1.0 versus 3.7 +/- 0.8%] [P less than 0.05]. PRL mRNA was significantly increased in normal pituitary and in GH3 tumor cells by E17 beta treatment. There was a significant decrease in PRL mRNA and an increase in GH mRNA expression in cultured MtT/W15 tumor cells by immunocytochemistry and in situ hybridization analyses. The percentage of cells producing both PRL and GH or mammosomatotropic cells analyzed by two different techniques declined after one week in culture in normal pituitary cells and in cultured MtT/W15 tumor cells after E17 beta treatment. These results show that E17 beta has a direct stimulatory effect on normal pituitary and GH3 cells and a direct inhibitory effect on MtT/W15 tumor cells with respect to cell proliferation and PRL hormone and mRNA expression.
Reverse hemolytic plaque assays and immunocytochemistry were used to monitor the ontogeny of individual hormone-secreting and hormone-containing cells in rats. Monodispersed anterior pituitary cells from fetal rats (sex unspecified) and neonatal rats of each sex were cultured for 24 h and then subjected to immunocytochemistry or plaque assays for PRL or GH. PRL secretors first appeared in appreciable numbers in cultures from 4-day-old animals, and by day 5, they accounted for 8-12% of all cells in culture. The percentage of GH secretors rose to a peak on day 5 (comprising approximately 40% of all cells), when the values were slightly higher than those observed previously in adults. The percentage of cultured cells from 4- to 5-day-olds that released PRL or GH was not influenced by the sex of the donor animal and was consistent with immunocytochemical estimates. Using a sequential plaque assay that enabled the detection of both GH and PRL release from the same cells, we found that of every 100 pituitary cells from 5-day-old males that released PRL and/or GH 62.5 released GH only, 1.7 released PRL alone, and the remaining 35.8 released both hormones. Almost identical proportions were found for females. These findings were confirmed using an additional variation of the plaque assay and by double staining immunocytochemistry. Taken together, these results indicate that mammosomatotropes, cells that release both GH and PRL, appear early in the neonatal development of both sexes and raise the possibility that PRL-secreting cells arise from GH-secreting cells.