Optimization of DNA extraction from human urinary samples for mycobiome community profiling.

IntroductionRecent data suggest the urinary tract hosts a microbial community of varying composition, even in the absence of infection. Culture-independent methodologies, such as next-generation sequencing of conserved ribosomal DNA sequences, provide an expansive look at these communities, identifying both common commensals and fastidious organisms. A fundamental challenge has been the isolation of DNA representative of the entire resident microbial community, including fungi.Materials and methodsWe evaluated multiple modifications of commonly-used DNA extraction procedures using standardized male and female urine samples, comparing resulting overall, fungal and bacterial DNA yields by quantitative PCR. After identifying protocol modifications that increased DNA yields (lyticase/lysozyme digestion, bead beating, boil/freeze cycles, proteinase K treatment, and carrier DNA use), all modifications were combined for systematic confirmation of optimal protocol conditions. This optimized protocol was tested against commercially available methodologies to compare overall and microbial DNA yields, community representation and diversity by next-generation sequencing (NGS).ResultsOverall and fungal-specific DNA yields from standardized urine samples demonstrated that microbial abundances differed significantly among the eight methods used. Methodologies that included multiple disruption steps, including enzymatic, mechanical, and thermal disruption and proteinase digestion, particularly in combination with small volume processing and pooling steps, provided more comprehensive representation of the range of bacterial and fungal species. Concentration of larger volume urine specimens at low speed centrifugation proved highly effective, increasing resulting DNA levels and providing greater microbial representation and diversity.ConclusionsAlterations in the methodology of urine storage, preparation, and DNA processing improve microbial community profiling using culture-independent sequencing methods. Our optimized protocol for DNA extraction from urine samples provided improved fungal community representation. Use of this technique resulted in equivalent representation of the bacterial populations as well, making this a useful technique for the concurrent evaluation of bacterial and fungal populations by NGS

Estrous Cyclicity in Mice During Simulated Weightlessness

Hindlimb unloading (HU) is a rodent model system used to simulate weightlessness experienced in space. However, some effects of this approach on rodent physiology are under-studied, specifically the effects on ovarian estrogen production which drives the estrous cycle. To resolve this deficiency, we conducted a ground-based validation study using the HU model, while monitoring estrous cycles in 16-weeks-old female C57BL6 mice. Animals were exposed to HU for 12 days following a 3 day HU cage acclimation period, and estrous cycling was analyzed in HU animals (n=22), normally loaded HU Cage Pair-Fed controls (CPF; n=22), and Vivarium controls fed ad libitum (VIV; n=10). Pair feeding was used to control for potential nutritional deficits on ovarian function. Vaginal cells were sampled daily in all mice via saline lavage. Cells were dried and stained with crystal violet, and the smears evaluated using established vaginal cytology techniques by two individuals blinded to the animal treatment group. Estrous cyclicity was disrupted in nearly all HU and CPF mice, while those maintained in VIV had an average normal cycle length of 4.8+/- 0.5 days, with all stages in the cycle visibly observed. CPF and HU animals arrested in the diestrous phase, which precedes the pre-ovulatory estrogen surge. Additionally, infection-like symptoms characterized by vaginal discharge and swelling arose in several HU animals, which we suspect was due to an inability of these mice to properly groom themselves, and/or due to the change in the gravity vector relative to the vaginal opening, which prevented drainage of the lavage solution. Pair-feeding resulted in similar weight gains of HU and CPF (1.5% vs 3.0%, respectively). The current results indicate that pair-feeding controlled weight gain and that the HU cage alone influenced estrous cyclicity. Thus, longer acclimation needs to be tested to determine if and when normal estrous cycling resumes in non-loaded mice in HU cages prior to HU testing. Future studies might also examine whether modifications to the vaginal lavage procedure might prevent the onset of the infection-like symptoms, and allow estrous cyclicity to be measured in this model system. Research supported by NNX15AB48G to JST

A novel method for the analysis of particle coating behaviour via contact spreading in a tumbling drum: Effect of coating liquid viscosity

Spray coating is a common method of distributing liquids over powders, especially in the pharmaceutical, detergent and food industries. During this process, liquid drops are deposited on the surface of particles. Liquid is then transferred between particles via particle collisions, in a process called liquid contact spreading. This contact spreading process facilitates inter-particle coating, in which wetting, de-wetting, mixing and drying are occurring simultaneously. This work presents the first experimental study of the mechanism of liquid contact spreading. In this work, a novel experimental method has been developed to investigate the mechanism of contact spreading, incorporating a newly developed image analysis technique, based on colourimetric measurements, to quantitatively determine coating behaviour via contact spreading. Here, experiments designed to isolate the contact spreading coating mechanism were performed in a tumbling drum using a model material system; alumina particles and dyed polyethylene glycol solutions of varying viscosities. The coating uniformity was quantified by the variation in inter-particle coating; the coefficient of variation (CoV). For all systems, the uniformity of the coating increased with time until the CoV decreased to an asymptotic value. The rate of the decrease in the CoV was successfully fitted using an exponential decay function. The viscosity of the coating solution had a significant effect on the rate of liquid transfer; the lower the viscosity the faster the contact spreading process. This effect is attributed to differences in the formation and stability of liquid bridges between the particles, influencing the extent of liquid transfer. The results also show that in most cases examined here, viscous forces play a main role in the contact spreading process, and the contribution of capillary forces are minimal. This understanding could assist the design and scale up for the wet coating processes

Bioaccumulation and physiological responses of the turtle Chelydra serpentina exposed to polychlorinated biphenyls during early life stages.

Despite the North American production ban of polychlorinated biphenyls (PCBs), PCBs are ubiquitous in the environment and in wildlife tissues. Chelydra serpentina serpentina (common snapping turtle) have been used as environmental indicators of PCB pollution upwards of 40 years given their high site fidelity and high trophic position. Despite their long use as indicators of PCB contamination, the effects of PCBs in reptiles remain largely unknown. In this study, we performed two experiments to assess i) bioaccumulation and ii) toxicity of PCBs to 1-month-old C. s. serpentina, to aid in interpretation of PCB burdens. Food pellets were spiked at an environmentally relevant concentration (0.45 μg/g) of the PCB mixture Aroclor 1254 to model hepatic bioaccumulation and depuration, through feeding, for 31 days and clean food for 50 days, respectively. No significant differences in PCB concentrations were observed in liver tissue over the course of the experiment, suggesting that juvenile turtles can likely metabolize low environmentally occurring concentrations of PCBs. Additionally, a dose-response experiment, performed to determine hepatic toxicity and bioaccumulation in juvenile C. s. serpentina, showed a 1.8-fold increase in hepatic expression of cyp1a when fed A1254-spiked pellets (12.7 μg/g; range 0–12.7 μg/g). This gene induction correlates with the significant increase of group 3 PCB congeners measured in the turtle liver, which are known to be metabolized by CYP1A. This study indicates that C. s. serpentina may be a good environmental indicator for PCBs, while more research is needed to assess the effects of body burdens in wild C. s. serpentina

N-phenyl-1-naphthylamine (PNA) Accumulates in Snapping Turtle (Chelydra serpentina) Liver Activating the Detoxification Pathway.

Substituted phenylamine antioxidants (SPAs) are used in Canadian industrial processes. SPAs, specifically N-phenyl-1-naphthylamine (PNA), have received very little attention despite their current use in Canada and their expected aquatic and environmental releases. There is a research gap regarding the effects of PNA in wildlife; therefore, Chelydra serpentina (common snapping turtle) was studied due to its importance as an environmental indicator species. A chronic experiment was performed using PNA spiked food (0 to 3446 ng/g) to determine its toxicity to juvenile C. serpentina. A significant increase in cyp1a mRNA level was observed in the liver of turtles exposed to 3446 ng/g PNA, suggesting that phase I detoxification is activated in the exposed animals. Additionally, a significant decrease in cyp2b transcript level was observed at the two lowest PNA doses, likely indicating another metabolic alteration for PNA. This study helped determine the molecular effects associated with a PNA exposure in reptiles

Vitamin D and SARS-CoV-2 virus/COVID-19 disease

The spread of novel SARS-CoV-2 virus, and the disease COVID-19 that is caused by SARS-CoV-2, continues apace. Saving lives and slowing the worldwide pandemic remain of utmost importance to everyone: the public, healthcare professionals, scientists, industry and governments

Vitamin D and SARS-Co V-2 virus/COVID-19 disease

Summary for social mediaVitamin D is essential for good health, especially bone and muscle health. Many people have low blood levels of vitamin D, especially in winter or if confined indoors, because summer sunshine is the main source of vitamin D for most people. Government vitamin D intake recommendations for the general population are 400 IU (10 µg) per day for the UK7 and 600 IU (15 µg) per day for the USA (800 IU (20 µg) per day for >70 years) and the EU.9 Taking a daily supplement (400 IU /day (10 µg/day) in the UK) and eating foods that provide vitamin D is particularly important for those self-isolating with limited exposure to sunlight. Vitamin D intakes greater than the upper limit of 4000 IU (100 µg) per day may be harmful and should be avoided unless under personal medical/clinical advice by a qualified health professional

The Impact of Long-Term Exposure to Space Environment on Adult Mammalian Organisms: A Study on Mouse Thyroid and Testis

Hormonal changes in humans during spaceflight have been demonstrated but the underlying mechanisms are still unknown. To clarify this point thyroid and testis/epididymis, both regulated by anterior pituitary gland, have been analyzed on long-term space-exposed male C57BL/10 mice, either wild type or pleiotrophin transgenic, overexpressing osteoblast stimulating factor-1. Glands were submitted to morphological and functional analysis

The Molecular Chaperone Hsp90α Is Required for Meiotic Progression of Spermatocytes beyond Pachytene in the Mouse

The molecular chaperone Hsp90 has been found to be essential for viability in all tested eukaryotes, from the budding yeast to Drosophila. In mammals, two genes encode the two highly similar and functionally largely redundant isoforms Hsp90α and Hsp90β. Although they are co-expressed in most if not all cells, their relative levels vary between tissues and during development. Since mouse embryos lacking Hsp90β die at implantation, and despite the fact that Hsp90 inhibitors being tested as anti-cancer agents are relatively well tolerated, the organismic functions of Hsp90 in mammals remain largely unknown. We have generated mouse lines carrying gene trap insertions in the Hsp90α gene to investigate the global functions of this isoform. Surprisingly, mice without Hsp90α are apparently normal, with one major exception. Mutant male mice, whose Hsp90β levels are unchanged, are sterile because of a complete failure to produce sperm. While the development of the male reproductive system appears to be normal, spermatogenesis arrests specifically at the pachytene stage of meiosis I. Over time, the number of spermatocytes and the levels of the meiotic regulators and Hsp90 interactors Hsp70-2, NASP and Cdc2 are reduced. We speculate that Hsp90α may be required to maintain and to activate these regulators and/or to disassemble the synaptonemal complex that holds homologous chromosomes together. The link between fertility and Hsp90 is further supported by our finding that an Hsp90 inhibitor that can cross the blood-testis barrier can partially phenocopy the genetic defects