Nasal Carriage Rate of Staphylococcus aureus, Its Associated Factors, and Antimicrobial Susceptibility Pattern Among Health Care Workers in Public Hospitals, Harar, Eastern Ethiopia.

Background: Staphylococcus aureus is the bacteria that colonizes the nasal nares of health-care workers and serves as a reservoir for the spread of pathogen for subsequent infections, mainly Methicillin-resistant Staphylococcus aureus. However, there is a limited study conducted regarding this topic in Harar, Eastern Ethiopia. Objective: The main objective of this study was to determine the prevalence of nasal carriage of Staphylococcus aureus, associated factors and antimicrobial susceptibility patterns among health-care workers of public hospitals in Harar, Eastern Ethiopia from May 15 to July 30, 2021. Methods: A hospital-based cross-sectional study was conducted on 295 health-care workers. A simple random sampling technique was used to select the participant. Nasal swabs were collected and cultured at 35°C for 24hrs. S. aureus was identified using the coagulase test and catalase test. Methicillin resistance S. aureus (MRSA) was screened using a cefoxitin disc on Muller Hinton agar using the Kirby-Bauer disc diffusion method. Data were entered into EPI-Info version-7 and transferred to SPSS-20 for analysis. Factors associated with nasal carriage of Staphylococcus aureus were determined by using chi-square analysis. A p-value of less than 0.05 was considered statistically significant. Results: The prevalence of Staphylococcus aureus in this study was 15.6% (95% CI: 11.7%, 20.3%) and methicillin-resistant Staphylococcus aureus was 11.2% (95% CI: 7.8%, 15.4%), respectively. Age (P < 0.001), work experience (p < 0.001), working unit (p < 0.02), antibiotic use within 3 months (p < 0.001), hand washing habit (p < 0.01), hand rub use (p < 0.001), living with smokers (p < 0.001), living with pets (p < 0.001) and having chronic diseases (p < 0.001) were found significantly associated with Staphylococcus aureus nasal carriage. Conclusion: The prevalence of Staphylococcus aureus and Methicillin-resistant Staphylococcus aureus are high in our study. The study emphasizes the need for regular surveillance among hospital staff and the environment to prevent MRSA transmission among health-care personnel.

Taurine deficiency as a driver of aging.

Aging is associated with changes in circulating levels of various molecules, some of which remain undefined. We find that concentrations of circulating taurine decline with aging in mice, monkeys, and humans. A reversal of this decline through taurine supplementation increased the health span (the period of healthy living) and life span in mice and health span in monkeys. Mechanistically, taurine reduced cellular senescence, protected against telomerase deficiency, suppressed mitochondrial dysfunction, decreased DNA damage, and attenuated inflammaging. In humans, lower taurine concentrations correlated with several age-related diseases and taurine concentrations increased after acute endurance exercise. Thus, taurine deficiency may be a driver of aging because its reversal increases health span in worms, rodents, and primates and life span in worms and rodents. Clinical trials in humans seem warranted to test whether taurine deficiency might drive aging in humans.

Vascular Endothelial Growth Factor Single Nucleotide Polymorphism +405 G/C (rs2010963) is associated with Levels, Infection Severity, and Amputation among South Indian Diabetic Foot Ulcer Patients.

Background: The regulation of vascular endothelial growth factor (VEGF) by genetic factors in T2DM and DFU still requires thorough investigation. Hence, the present study aimed to investigate the association of VEGF +405 G/C in DFU subjects and correlate it with its circulatory levels, infection severity, and amputation rate. Materials and Methods: This study registered a total of 754 participants of which group I: healthy controls (n = 297), group II: T2DM subjects (n = 242), and group III: DFU subjects (n = 215). Genotyping and levels of rs2010963 were analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and ELISA, respectively. Results: Results of the current study showed a clear decline in circulatory VEGF-A levels in DFU subjects. VEGF-A was decreased in DFU subjects with the mutant "CC" genotype. The mutant "CC" of VEGF +405G/C was also found to be more susceptible to ulcer grade (III and IV) and major amputations. Conclusion: VEGF +405G/C SNP is associated with levels, infection severity, and amputation amongst South Indian DFU patients.

Delivery of Apoplastic Extracellular Vesicles Encapsulating Green-Synthesized Silver Nanoparticles to Treat Citrus Canker.

The citrus canker pathogen Xanthomonas axonopodis has caused severe damage to citrus crops worldwide, resulting in significant economic losses for the citrus industry. To address this, a green synthesis method was used to develop silver nanoparticles with the leaf extract of Phyllanthus niruri (GS-AgNP-LEPN). This method replaces the need for toxic reagents, as the LEPN acts as a reducing and capping agent. To further enhance their effectiveness, the GS-AgNP-LEPN were encapsulated in extracellular vesicles (EVs), nanovesicles with a diameter of approximately 30-1000 nm naturally released from different sources, including plant and mammalian cells, and found in the apoplastic fluid (APF) of leaves. When compared to a regular antibiotic (ampicillin), the delivery of APF-EV-GS-AgNP-LEPN and GS-AgNP-LEPN to X. axonopodis pv. was shown to have more significant antimicrobial activity. Our analysis showed the presence of phyllanthin and nirurinetin in the LEPN and found evidence that both could be responsible for antimicrobial activity against X. axonopodis pv. Ferredoxin-NADP+ reductase (FAD-FNR) and the effector protein XopAI play a crucial role in the survival and virulence of X. axonopodis pv. Our molecular docking studies showed that nirurinetin could bind to FAD-FNR and XopAI with high binding energies (-10.32 kcal/mol and -6.13 kcal/mol, respectively) as compared to phyllanthin (-6.42 kcal/mol and -2.93 kcal/mol, respectively), which was also supported by the western blot experiment. We conclude that (a) the hybrid of APF-EV and GS-NP could be an effective treatment for citrus canker, and (b) it works via the nirurinetin-dependent inhibition of FAD-FNR and XopAI in X. axonopodis pv.

Green synthesized Se nanoparticle-mediated alleviation of salt stress in field mustard, TS-36 variety.

During this decade, selenium nanoparticles have been found to play a crucial role in helping plants endure several stress conditions, which thereby helps enhance the production of crops in such harsh environments. Globally, high salinity is considered a long-term stress in the crop fields which affects the growth and production of many crops, including mustard-one of the most important oil crops. Here, the activities of spherical-shaped selenium nanoparticles with an average particle size of 55.81 nm, synthesized and functionalized by phytochemicals of fresh grape aqueous extract, were evaluated in the salinity stress (200 mM NaCl) tolerance of mustard plants grown hydroponically in modified Hoagland's solution. These bioactive nanoparticles (30 mg L-1) have exhibited significant activity in alleviating the salt stress complications in mustard, enhancing the activities of antioxidant enzymes (SOD 41.20 %, CAT 64.10 %, APX 63.06 %, and POX 70.43 %), phenolic content (98.88 %), flavonoid content (86.90 %), and free radical scavenging activity (61.89 %). The seed germination percentage, root and shoot length, fresh and dry weight per plant, water content percentage, chlorophyll content, carbohydrate content, and protein content were significantly improved by 39.66 %, 75 %, 60.64 %, 41.2 %, 22.11 %, 1.02 %, 81.92 %, 24.65 % and 79.14 % respectively by the nano selenium application during NaCl stress compared to the control group growing under salt stress without nanoparticles. Gas chromatography-mass spectrometry chromatogram analysis inferred the interaction between the nano-selenium and mustard plants under salt stress. Besides, the in-silico analysis revealed the active molecular interactions between selenium and 20 different proteins of mustard, including glutathione peroxidase, an important antioxidant enzyme.

N6-adenomethylation of GsdmC is essential for Lgr5+ stem cell survival to maintain normal colonic epithelial morphogenesis.

Gut epithelial morphogenesis is maintained by intestinal stem cells. Here, we report that depletion of N6-adenosine methyltransferase subunit Mettl14 from gut epithelial cells in mice impaired colon mucosal morphogenesis, leading to increased mucosal permeability, severe inflammation, growth retardation, and premature death. Mettl14 ablation triggered apoptosis that depleted Lgr5+ stem cells and disrupted colonic organoid growth and differentiation, whereas the inhibition of apoptosis rescued Mettl14-deleted mice and organoids. Mettl14 depletion disrupted N6-adenomethylation on GsdmC transcripts and abolished GsdmC expression. Reconstitution of Mettl14-deleted organoids or mice with GSDMC rescued Lgr5 expression and prevented apoptosis and mouse premature death, whereas GSDMC silence eliminated LGR5 and triggered apoptosis in human colonic organoids and epithelial cells. Mechanistically, Mettl14 depletion eliminated mitochondrial GsdmC, disrupted mitochondrial membrane potential, and triggered cytochrome c release that activates the pro-apoptotic pathway. In conclusion, GsdmC N6-adenomethylation protects mitochondrial homeostasis and is essential for Lgr5+ cell survival to maintain normal colonic epithelial regeneration.

Se nanoparticles stabilized with Allamanda cathartica L. flower extract inhibited phytopathogens and promoted mustard growth under salt stress.

Selenium Nanoparticles (SeNPs) exhibit tremendous application in agriculture as antimicrobials or as nano fertilizer. Present work reports the eco-friendly synthesis of SeNPs by using Allamanda cathartica L. flower extract (aqueous) as a reducing/capping agent and selenium dioxide as a precursor. The method used here is free of any toxic reducing agents and organic solvents. The synthesis process of SeNPs took 5 h at 60 °C, confirmed by the brick red colour of the solution followed by UV-Vis spectroscopy and further characterized by XRD, FTIR, EDX and SEM. The average size (diameter) of the SeNPs were found to be 60.31 nm by DLS. It has shown strong antimicrobial activity against Pseudomonas marginalis and P. aeruginosa at 2.5, 5 and 10 mg/mL concentrations. Besides, its application improved seed germination and growth parameters of Brassica campestris (TS 36 variety) under salt stress. 25 mg/L SeNPs has improved the germination percentage by around 31%, shoot length by 92%, root length by 78% and total chlorophyll content by 49% under 200 mM NaCl stress. This SeNPs could be a potential antimicrobial agent in treating plant diseases caused by the mentioned phytopathogens, having no or minimum toxicity, in fact having positive impacts on plant growth.

Follicle-stimulating hormone-mediated decline in miR-92a-3p expression in pubertal mice Sertoli cells is crucial for germ cell differentiation and fertility.

Sertoli cells (Sc) are the sole target of follicle-stimulating hormone (FSH) in the testis and attain functional maturation post-birth to significantly augment germ cell (Gc) division and differentiation at puberty. Despite having an operational microRNA (miRNA) machinery, limited information is available on miRNA-mediated regulation of Sc maturation and male fertility. We have shown before that miR-92a-3p levels decline in pubertal rat Sc. In response to FSH treatment, the expressions of FSH Receptor, Claudin11 and Klf4 were found to be elevated in pubertal rat Sc coinciding with our finding of FSH-induced decline in miR-92a-3p levels. To investigate the association of miR-92a-3p and spermatogenesis, we generated transgenic mice where such pubertal decline of miR-92a-3p was prevented by its overexpression in pubertal Sc under proximal Rhox5 promoter, which is known to be activated specifically at puberty, in Sc. Our in vivo observations provided substantial evidence that FSH-induced decline in miR-92a-3p expression during Sc maturation acts as an essential prerequisite for the pubertal onset of spermatogenesis. Elevated expression of miR-92a-3p in post-pubertal testes results into functionally compromised Sc, leading to impairment of the blood-testis barrier formation and apoptosis of pre-meiotic Gc, ultimately culminating into infertility. Collectively, our data suggest that regulation of miR-92a-3p expression is crucial for Sc-mediated induction of active spermatogenesis at puberty and regulation of male fertility.

Enhanced lipid accumulation in microalgae through nanoparticle-mediated approach, for biodiesel production: A mini-review.

Nanoparticle application in microalgae for enhanced lipid production is an ongoing work that leads towards the contribution in biodiesel production. During this decade, metal nanoparticles are constantly being reported to have numerous applications in diverse fields, because of their unique optical, electrical, and magnetic properties. They can interact with the biomolecules of cells and thereby alters cellular metabolisms, which in turn reflects their ability to regulate some primary or secondary metabolic pathways. Nanoparticles derived from metals like Fe, Cu, and Se are taking part in redox processes and their presence in many enzymes may modulate algal metabolisms. Besides by upregulating or downregulating the expression of several genes, nanoparticle exposure can alter gene expressions in many organisms. In microalgae such as Chlorella vulgaris, C. pyrenoidosa, Scenedesmus obliquus, S. rubescens, Trachydiscus minut u s, Parachlorella kessleri, and Tetraselmis suecica; metal nanoparticle exposure in different environmental conditions have impacts on various physiological or molecular changes, thereby increasing the growth rate, biomass and lipid production. The present mini-review gives an insight into the various advantages and a future outlook on the application of nanoparticles in microalgae for biofuel production. Also, it can be proposed that nanoparticles could be useful in blocking or deactivating the AGPase enzyme (involved in the glucose to starch conversion pathway), binding to its active site, thereby increasing lipid production in microalgae that could be utilized for enhanced biodiesel production.

Declining levels of miR-382-3p at puberty trigger the onset of spermatogenesis.

A major change in the transcriptome of testicular Sertoli cells (Scs) at the onset of puberty enables them to induce robust spermatogenesis. Through comprehensive literature mining, we generated a list of genes crucial for Sc functioning and computationally predicted the microRNAs regulating them. Differential expression analysis of microRNAs in infant and pubertal rat Scs showed that miR-382-3p levels decline significantly in pubertal Scs. Interestingly, miR-382-3p was found to regulate genes like Ar and Wt1, which are crucial for functional competence of Scs. We generated a transgenic (Tg) mouse model in which pubertal decline of miR-382-3p was prevented by its overexpression in pubertal Scs. Elevated miR-382-3p restricted the functional maturation of Scs at puberty, leading to infertility. Prevention of decline in miR-382-3p expression in pubertal Scs was responsible for defective blood-testis barrier (BTB) formation, severe testicular defects, low epididymal sperm counts and loss of fertility in these mice. This provided substantial evidence that decline in levels of miR-382-3p at puberty is the essential trigger for onset of robust spermatogenesis at puberty. Hence, sustained high levels of miR-382-3p in pubertal Scs could be one of the underlying causes of idiopathic male infertility and should be considered for diagnosis and treatment of infertility.

Homeobox transcription factor Meis1 is crucial to Sertoli cell mediated regulation of male fertility.

BACKGROUND: Infertility has become a global phenomenon and constantly declining sperm count in males in modern world pose a major threat to procreation of humans. Male fertility is critically dependent on proper functioning of testicular Sertoli cells. Defective Sertoli cell proliferation and/or impaired functional maturation may be one of the underlying causes of idiopathic male infertility. Using high-throughput "omics" approach, we found binding sites for homeobox transcription factor MEIS1 on the promoters of several genes up-regulated in pubertal (mature) Sertoli cells, indicating that MEIS1 may be crucial for Sertoli cell-mediated regulation of spermatogenesis at and after puberty. OBJECTIVE: To decipher the role of transcription factor MEIS1 in Sertoli cell maturation and spermatogenesis. MATERIALS AND METHODS: Sc-specific Meis1 knockdown (KD) transgenic mice were generated using pronuclear microinjection. Morphometric and histological analysis of the testes from transgenic mice was performed to identify defects in spermatogenesis. Epididymal sperm count and litter size were analyzed to determine the effect of Meis1 knockdown on fertility. RESULTS: Sertoli cell (Sc)-specific Meis1 KD led to massive germ cell loss due to apoptosis and impaired spermatogenesis. Unlike normal pubertal Sc, the levels of SOX9 in pubertal Sc of Meis1 KD were significantly high, like immature Sc. A significant reduction in epididymal sperm count was observed in these mice. The mice were found to be infertile or sub-fertile (with reduced litter size), depending on the extent of Meis1 inhibition. DISCUSSION: The results of this study demonstrated for the first time, a role of Meis1 in Sc maturation and normal spermatogenic progression. Inhibition of Meis1 in Sc was associated with deregulated spermatogenesis and a consequent decline in fertility of the transgenic mice. CONCLUSIONS: Our results provided substantial evidence that suboptimal Meis1 expression in Sc may be one of the underlying causes of idiopathic infertility.

Pubertal down-regulation of Tetraspanin 8 in testicular Sertoli cells is crucial for male fertility.

The alarming decline in sperm count has become a global concern in the recent decades. The division and differentiation of male germ cells (Gc) into sperm are governed by Sertoli cells (Sc) upon their functional maturation during puberty. However, the roles of genes regulating pubertal maturation of Sc have not been fully determined. We have observed that Tetraspanin 8 (Tspan8) is down-regulated in Sc during puberty in rats. However, there has been no in vivo evidence for a causal link between the down-regulation of Tspan8 expression and the onset of spermatogenesis as yet. To investigate this, we generated a novel transgenic (Tg) rat, in which the natural down-regulation of Tspan8 was prevented specifically in Sc from puberty up to adulthood. Adult Tg male rats showed around 98% reduction in sperm count despite having a similar level of serum testosterone (T) as the controls. Functional maturation of Sc was impaired as indicated by elevated levels of Amh and low levels of Kitlg and Claudin11 transcripts. The integrity of the blood testis barrier was compromised due to poor expression of Gja1 and Gc apoptosis was discernible. This effect was due to a significant rise in both Mmp7 and phospho P38 MAPK in Tg rat testis. Taken together, we demonstrated that the natural down-regulation of Tspan8 in Sc during puberty is a prerequisite for establishing male fertility. This study divulges one of the aetiologies of certain forms of idiopathic male infertility where somatic cell defect, but not hormonal deficiency, is responsible for impaired spermatogenesis.

Downregulation of Sostdc1 in Testicular Sertoli Cells is Prerequisite for Onset of Robust Spermatogenesis at Puberty.

An alarming decline in sperm count of men from several countries has become a major concern for the world community. Hormones act on testicular Sertoli cells (Sc) to regulate male fertility by governing the division and differentiation of germ cells (Gc). However, there is a limited knowledge about Sc specific gene(s) regulating the spermatogenic output of the testis. Sclerostin domain-containing 1 protein (Sostdc1) is a dual BMP/Wnt regulator is predominantly expressed in the Sc of infant testes which hardly show any sign of spermatogenesis. In order to investigate the role of Sostdc1 in spermatogenic regulation, we have generated transgenic (Tg) rats which induced persistent expression of Sostdc1 in mature Sc causing reduced sperm counts. Although Sc specific Sostdc1 did not affect the function of either Sc or Leydig cells (Lc) in the adult testis of Tg rat, we observed a selective augmentation of the BMP target genes via activated phospho smad 1/5/8 signaling in Gc leading to apoptosis. Here, for the first time, we have demonstrated that Sostdc1 is a negative regulator of spermatogenesis, and provided substantial evidence that down regulation of Sostdc1 during puberty is critically essential for quantitatively and qualitatively normal spermatogenesis governing male fertility.

Syntheses, physicochemical characterization, antibacterial studies on potassium morpholine dithiocarbamate nickel (II), copper (II) metal complexes and their ligands.

Organic molecule dithiocarbamate transition metal complexes are novel and very attractive pharmaceutical targets for the management and control of antibiotic resistant bacteria. The direct reaction has synthesized new transition metal nickel (II), copper (II) complexes of potassium morpholine dithiocarbamate (K+C5H8NOS2 -) ligands and characterized by UV-visible spectroscopy, Fourier-transform infrared spectroscopy (FTIR), as well as NMR physicochemical techniques. Antibacterial bioefficacy of the ligand and its metal complexes has been investigated in vitro on the growth of Gram-positive (Staphylococcus aureus MTCC 737, Bacillus cereus MTCC 1272) and the Gram-negative (Listeria monocytogenes MTCC 657, Shigella flexeneri MTCC 1457) bacteria. The obtained electronic spectral bands are characteristic and consistent with the proposed composition of the ligand as well as its metal complexes. It also provides a further example of the bidentate coordination of dithiocarbamate ligands. Absorption peak values of FTIR are characteristic of the ligand as well as dithiocarbamate group molecules and exhibit their metal coordination. NMR 1H signal variations also correlate with the coordination mediated chemical shifts. Both the metal complexes showed significant antibacterial activity. However, enhanced antimicrobial activity of the ligands than metal complexes against Gram positive and Gram negative bacteria were observed. Thus, further study on this approach could pave a way for the development of dithiocarbamate-metal complex based antibacterial agent.

Defective Wnt3 expression by testicular Sertoli cells compromise male fertility.

Testicular Sertoli cells make a niche for the division and differentiation of germ cells. Sertoli cells respond to increased follicle-stimulating hormone (FSH) and testosterone (T) levels at the onset of puberty by producing paracrine factors which affect germ cells and trigger robust onset of spermatogenesis. Such paracrine support to germ cells is absent during infancy, despite Sertoli cells being exposed to high FSH and T within the infant testis. This situation is similar to certain cases of male idiopathic infertility where post-pubertal Sertoli cells fail to support germ cell division and differentiation in spite of endogenous or exogenous hormonal support. Defective Sertoli cells in such individuals may fail to express the full complement of their paracrine repertoire. Identification and supplementation with such factors may overcome Sertoli cells deficiencies and help trigger quantitatively and qualitatively normal differentiation of germ cells. To this end, we compared the transcriptome of FSH- and T-treated infant and pubertal monkey Sertoli cells by DNA microarray. Expression of Wnt3, a morphogen of the Wnt/ß-catenin pathway, was higher in pubertal Sertoli cells relative to infant Sertoli cells. Transgenic mice were generated by us in which Wnt3 expression was curtailed specifically in post-pubertal Sertoli cells by shRNA. Subfertility and oligozoospermia were noticed in such animals with low Wnt3 expression in post-pubertal Sertoli cells along with diminished expression of Connexin43, a gap-junctional molecule essential for germ cell development. We report that the FSH- and T-targetedf Wnt3 governs Sertoli cell-mediated regulation of spermatogenesis and hence is crucial for fertility.

Sertoli cell specific knockdown of RAR-related orphan receptor (ROR) alpha at puberty reduces sperm count in rats.

Globally, there is an alarming decline in sperm count. Very often hormonal supplementation fails to restore normal sperm count. Sertoli cells (Sc) present within seminiferous tubules provide appropriate niche and factors required for the differentiation of germ cells (Gc) into mature sperm (spermatogenesis). Functionally compromised Sc may be one of the reasons for failure of hormones to facilitate normal spermatogenesis. Although role of secretory proteins and signaling molecules of Sc has been studied well, role of transcription factors regulating sperm count has not been addressed appropriately. Retinoic acid receptor-related orphan receptor (ROR)-alpha is one of such transcription factors reported in testis but its role in testicular function is not yet known. In a separate study, we found abundant ROR-alpha binding sites on promoter regions of several genes upregulated in pubertal rat Sc as compared to infant Sc. Immunostaining studies also revealed presence of ROR alpha in nucleus of pubertal Sc. We generated a transgenic knockdown rat model expressing shRNA targeted to ROR-alpha under Sc specific promoter, which is transcriptionally active only at and after puberty. ROR-alpha knockdown animals were found to have abnormal association of Sc and Gc, including Gc sloughing and restricted release of sperm. The knockdown animals displayed compromised spermatogenesis leading to significant reduction in sperm count. This is the first report describing the Sc specific role of ROR-alpha in maintaining quantitatively normal sperm output. Identification of various such molecules can generate avenues to limit or reverse an alarmingly declining sperm count witnessed globally in men.

An integrated transcriptomics-guided genome-wide promoter analysis and next-generation proteomics approach to mine factor(s) regulating cellular differentiation.

Differential next-generation-omics approaches aid in the visualization of biological processes and pave the way for divulging important events and/or interactions leading to a functional output at cellular or systems level. To this end, we undertook an integrated Nextgen transcriptomics and proteomics approach to divulge differential gene expression of infant and pubertal rat Sertoli cells (Sc).Unlike, pubertal Sc, infant Sc are immature and fail to support spermatogenesis. We found exclusive association of 14 and 19 transcription factor binding sites to infantile and pubertal states of Sc, respectively, using differential transcriptomics-guided genome-wide computational analysis of relevant promoters employing 220 Positional Weight Matrices from the TRANSFAC database. Proteomic SWATH-MS analysis provided extensive quantification of nuclear and cytoplasmic protein fractions revealing 1,670 proteins differentially located between the nucleus and cytoplasm of infant Sc and 890 proteins differentially located within those of pubertal Sc. Based on our multi-omics approach, the transcription factor YY1 was identified as one of the lead candidates regulating differentiation of Sc.YY1 was found to have abundant binding sites on promoters of genes upregulated during puberty. To determine its significance, we generated transgenic rats with Sc specific knockdown of YY1 that led to compromised spermatogenesis.

Robust generation of transgenic mice by simple hypotonic solution mediated delivery of transgene in testicular germ cells.

Our ability to decipher gene sequences has increased enormously with the advent of modern sequencing tools, but the ability to divulge functions of new genes have not increased correspondingly. This has caused a remarkable delay in functional interpretation of several newly found genes in tissue and age specific manner, limiting the pace of biological research. This is mainly due to lack of advancements in methodological tools for transgenesis. Predominantly practiced method of transgenesis by pronuclear DNA-microinjection is time consuming, tedious, and requires highly skilled persons for embryo-manipulation. Testicular electroporation mediated transgenesis requires use of electric current to testis. To this end, we have now developed an innovative technique for making transgenic mice by giving hypotonic shock to male germ cells for the gene delivery. Desired transgene was suspended in hypotonic Tris-HCl solution (pH 7.0) and simply injected in testis. This resulted in internalization of the transgene in dividing germ-cells residing at basal compartment of tubules leading to its integration in native genome of mice. Such males generated transgenic progeny by natural mating. Several transgenic animals can be generated with minimum skill within short span of time by this easily adaptable novel technique.

An efficient method for generating a germ cell depleted animal model for studies related to spermatogonial stem cell transplantation.

BACKGROUND: Spermatogonial stem cell (SSC) transplantation (SSCT) has become important for conservation of endangered species, transgenesis and for rejuvenating testes which have lost germ cells (Gc) due to gonadotoxic chemotherapy or radiotherapy during the prepubertal phase of life. Creating a germ cell-depleted animal model for transplantation of normal or gene-transfected SSC is a prerequisite for such experimental studies. Traditionally used intraperitoneal injections of busulfan to achieve this are associated with painful hematopoietic toxicity and affects the wellbeing of the animals. Use of testicular busulfan has been reported recently to avoid this but with a very low success rate of SSCT. Therefore, it is necessary to establish a more efficient method to achieve higher SSCT without any suffering or mortality of the animals. METHODS: A solution of busulfan, ranging from 25 µg/20 µl to 100 µg/20 µl in 50 % DMSO was used for this study. Each testis received two diagonally opposite injections of 10 µl each. Only DMSO was used as control. Germ cell depletion was checked every 15 days. GFP-expressing SSC from transgenic donor mice C57BL/6-Tg (UBC-GFP) 30Scha/J were transplanted into busulfan-treated testis. Two months after SSCT, mice were analyzed for presence of colonies of donor-derived SSC and their ability to generate offspring. RESULTS: A dose of 75 µg of busulfan resulted in reduction of testis size and depletion of the majority of Gc of testis in all mice within 15 days post injection without causing mortality or a cytotoxic effect in other organs. Two months after SSCT, colonies of donor-derived Gc-expressing GFP were observed in recipient testes. When cohabitated with females, donor-derived offspring were obtained. By our method, 71 % of transplanted males sired transgenic progeny as opposed to 5.5 % by previously described procedures. About 56 % of progeny born were transgenic by our method as opposed to 1.2 % by the previously reported methods. CONCLUSIONS: We have established an efficient method of generating a germ cell-depleted animal model by using a lower dose of busulfan, injected through two diagonally opposite sites in the testis, which allows efficient colonization of transplanted SSC resulting in a remarkably higher proportion of donor-derived offspring generation.

Modern lineages of Mycobacterium tuberculosis exhibit lineage-specific patterns of growth and cytokine induction in human monocyte-derived macrophages.

BACKGROUND: Strains of Mycobacterium tuberculosis vary in virulence. Strains that have caused outbreaks in the United States and United Kingdom have been shown to subvert the innate immune response as a potential immune evasion mechanism. There is, however, little information available as to whether these patterns of immune subversion are features of individual strains or characteristic of broad clonal lineages of M. tuberculosis. METHODS: Strains from two major modern lineages (lineage 2 [East-Asian] and lineage 4 [Euro-American]) circulating in the Western Cape in South Africa as well as a comparator modern lineage (lineage 3 [CAS/Delhi]) were identified. We assessed two virulence associated characteristics: mycobacterial growth (in liquid broth and monocyte derived macrophages) and early pro-inflammatory cytokine induction. RESULTS: In liquid culture, Lineage 4 strains grew more rapidly and reached higher plateau levels than other strains (lineage 4 vs. lineage 2 p=0.0024; lineage 4 vs. lineage 3 p=0.0005). Lineage 3 strains were characterized by low and early plateau levels, while lineage 2 strains showed an intermediate growth phenotype. In monocyte-derived macrophages, lineage 2 strains grew faster than lineage 3 strains (p<0.01) with lineage 4 strains having an intermediate phenotype. Lineage 2 strains induced the lowest levels of pro-inflammatory TNF and IL-12p40 as compared to other lineages (lineage 2: median TNF 362 pg/ml, IL-12p40 91 pg/ml; lineage 3: median TNF 1818 pg/ml, IL-12p40 123 pg/ml; lineage 4: median TNF 1207 pg/ml, IL-12p40 205 pg/ml;). In contrast, lineage 4 strains induced high levels of IL-12p40 and intermediate level of TNF. Lineage 3 strains induced high levels of TNF and intermediate levels of IL-12p40. CONCLUSIONS: Strains of M. tuberculosis from the three major modern strain lineages possess distinct patterns of growth and cytokine induction. Rapid growth and immune subversion may be key characteristics to the success of these strains in different human populations.