BackgroundStrains 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.MethodsStrains 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.ResultsIn 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.ConclusionsStrains 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.

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.

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BackgroundSpermatogonial 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.MethodsA 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.ResultsA 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.ConclusionsWe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-016-0405-1) contains supplementary material, which is available to authorized users.

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/β-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.

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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.

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.