Effects of different Sertoli cell types on the maintenance of adult spermatogonial stem cells in vitro

Spermatongonial stem cells (SSCs) are unique testis cells that are able to proliferate, differentiate, and transmit genetic information to the next generation. However, the effect of different Sertoli cell types on the expression of specific SSC genes is not yet well understood. In this study, we compare the in vitro effect of adult Sertoli cells, embryonic Sertoli cells, and TM4 (a Sertoli cell line) as feeder layers on the expression of SSC genes. SSCs were isolated from the testis of adult male mice and purified by differential plating. Following enrichment, SSCs were cultivated for 1 and 2 wk in the presence of various feeders. The expression of SSC-specific genes (Mvh, ZBTB, and c-kit) was evaluated by real-time polymerase chain reaction. Our results revealed that expression of the specific SSC genes was significantly higher in the embryonic Sertoli cells after 1 and 2 wk compared to the adult Sertoli cells and the TM4 group. Our finding suggest that co-culturing of SSCs with embryonic Sertoli cells is helpful for in vitro cultivation of SSCs and might improve the self-renewal of these stem cells.     

Involvement of the cynABDS operon and the CO2-concentrating mechanism in the light-dependent transport and metabolism of cyanate by cyanobacteria

The cyanobacteria Synechococcus elongatus strain PCC7942 and Synechococcus sp. strain UTEX625 decomposed exogenously supplied cyanate (NCO-) to CO2 and NH3 through the action of a cytosolic cyanase which required HCO3- as a second substrate. The ability to metabolize NCO- relied on three essential elements: proteins encoded by the cynABDS operon, the biophysical activity of the CO2-concentrating mechanism (CCM), and light. Inactivation of cynS, encoding cyanase, and cynA yielded mutants unable to decompose cyanate. Furthermore, loss of CynA, the periplasmic binding protein of a multicomponent ABC-type transporter, resulted in loss of active cyanate transport. Competition experiments revealed that native transport systems for CO2, HCO3-, NO3-, NO2-, Cl-, PO4(2-), and SO4(2-) did not contribute to the cellular flux of NCO- and that CynABD did not contribute to the flux of these nutrients, implicating CynABD as a novel primary active NCO- transporter. In the S. elongatus strain PCC7942 DeltachpX DeltachpY mutant that is defective in the full expression of the CCM, mass spectrometry revealed that the cellular rate of cyanate decomposition depended upon the size of the internal inorganic carbon (Ci) (HCO3- + CO2) pool. Unlike wild-type cells, the rate of NCO- decomposition by the DeltachpX DeltachpY mutant was severely depressed at low external Ci concentrations, indicating that the CCM was essential in providing HCO3- for cyanase under typical growth conditions. Light was required to activate and/or energize the active transport of both NCO- and Ci. Putative cynABDS operons were identified in the genomes of diverse Proteobacteria, suggesting that CynABDS-mediated cyanate metabolism is not restricted to cyanobacteria.     

Neuroendocrine regulation of prolactin secretion in adult female rhesus monkeys during different phases of the menstrual cycle: role of neuroexcitatory amino acid (NMA)

The present study attempts to examine the role of N-methyl-D, L-aspartate (NMDA) receptors in the central regulation of prolactin (PRL) secretion, which may be involved in ovarian function and its alteration by glutamate in various phases of the menstrual cycle of female rhesus monkeys (Macaca mulatta). The results suggest that the glutaminergic component of the control system, which governs PRL secretion by utilizing NMDA receptors, may have an important role in regulating changes in PRL secretion. The response of PRL during the luteal phase of the cycle was different from that observed in follicular and menstrual phases. Steroids may influence the NMDA-dependent drive to release PRL. N-methyl-D-aspartic acid (NMA) involvement in the regulation of PRL secretion may occur through activation of the PRL-stimulating system depending on the physiological state or steroidal milieu. It is possible, therefore, that the NMA-induced release of PRL-releasing factors (PRF) and PRL are enhanced in the presence of ovarian feedback.     

Screening Currency Notes for Microbial Pathogens and Antibiotic Resistance Genes Using a Shotgun Metagenomic Approach

Fomites are a well-known source of microbial infections and previous studies have provided insights into the sojourning microbiome of fomites from various sources. Paper currency notes are one of the most commonly exchanged objects and its potential to transmit pathogenic organisms has been well recognized. Approaches to identify the microbiome associated with paper currency notes have been largely limited to culture dependent approaches. Subsequent studies portrayed the use of 16S ribosomal RNA based approaches which provided insights into the taxonomical distribution of the microbiome. However, recent techniques including shotgun sequencing provides resolution at gene level and enable estimation of their copy numbers in the metagenome. We investigated the microbiome of Indian paper currency notes using a shotgun metagenome sequencing approach. Metagenomic DNA isolated from samples of frequently circulated denominations of Indian currency notes were sequenced using Illumina Hiseq sequencer. Analysis of the data revealed presence of species belonging to both eukaryotic and prokaryotic genera. The taxonomic distribution at kingdom level revealed contigs mapping to eukaryota (70%), bacteria (9%), viruses and archae (~1%). We identified 78 pathogens including Staphylococcus aureus, Corynebacterium glutamicum, Enterococcus faecalis, and 75 cellulose degrading organisms including Acidothermus cellulolyticus, Cellulomonas flavigena and Ruminococcus albus. Additionally, 78 antibiotic resistance genes were identified and 18 of these were found in all the samples. Furthermore, six out of 78 pathogens harbored at least one of the 18 common antibiotic resistance genes. To the best of our knowledge, this is the first report of shotgun metagenome sequence dataset of paper currency notes, which can be useful for future applications including as bio-surveillance of exchangeable fomites for infectious agents.     

Selectivity profiling of novel indene H1-antihistamines for the treatment of insomnia

A series of indene analogs of the H₁-antihistamine (?)-R-dimethindene was evaluated for selectivity in the search for potentially improved sedative-hypnotics. Variation of the 6-substitutent in the indene core in combination with a pendant electron rich heterocycle led to the identification of several potent H₁-antihistamines with desirable selectivity over CYP enzymes, the M₁ muscarinic receptor and the hERG channel. These compounds were candidates for further ADME profiling and in vivo evaluation.     

A freezing technique that maintains viability of sperm from the starfish Pisaster ochraceus

Embryos and larvae of the starfish Pisaster ochraceus are excellent for studying different aspects of early development. They are clear, easily obtained, and easy to culture, making them useful for morphological studies. In addition, the adults are large, produce large numbers of gametes, and early development is synchronous so that sufficient material for biochemical work can also be obtained. One major drawback is that they have a short breeding season. Gravid adult females can be maintained in the laboratory almost year round by controlling light conditions. Unfortunately, even under carefully controlled light and temperature conditions, by late autumn, the testes of most males undergo involution making it difficult to obtain sufficient sperm to raise cultures in the autumn, winter, and early spring. In order to overcome this shortcoming, our laboratory has experimented with techniques for freezing the sperm. This has involved testing a number of different cooling rates and cryoprotectants (CPAs) in order to develop a combination that maintained viability of some of the sperms. This article presents a technique that includes a combination of cooling conditions and a cocktail of CPAs that preserves the viability of sufficient sperm such that, when combined with careful rearing techniques, it allows us to produce normal healthy larvae almost year around.     

Analysis of Proteins Expressed by an Abiotic Stress Tolerant Pseudomonas putida (NBAII-RPF9) Isolate Under Saline and High Temperature Conditions

Pseudomonas putida (NBAII-RPF9) was identified as an abiotic stress tolerant bacterium capable of growing at 45 °C as well as in 1 M NaCl. The proteins expressed by this bacterium when subjected to these two stresses were analyzed by 2D gel and MALDI-TOF/MS. Two parameters viz., heat/saline shock (20 min at 45 °C/1 M solid NaCl added at mid log phase and incubated for 1 h) and heat/saline tolerance (24 h growth at 45 °C/in 1 M NaCl) were studied. Under heat shock 13 upregulated proteins and 1 downregulated protein were identified and under tolerance 6 upregulated proteins were identified. GroES and GroEL proteins were expressed under both tolerance and shock. Under saline shock 11 upregulated proteins were identified whereas under saline tolerance 6 upregulated proteins were identified and all these proteins had pI between 3 and 10 with molecular weights ranging from 14.3 to 97 kDa. Aspartate carbamoyltransferase was common under both the saline conditions studied. The analysis revealed involvement of heat stress responsive molecular chaperones and membrane proteins during heat stress. During salt stress, proteins involved in metabolic processes were found to be upregulated to favor growth and adaptation of the bacterium. Heat shock chaperones viz., DnaK and DnaJ were expressed under both saline and heat stress. This is the first report of protein profile obtained from a single bacterium under saline and heat stress and the studies reveal the complex mechanisms adapted by the organism to survive under high temperature or saline conditions.