Raman microspectroscopy was used to quantify freezing response of cells to various cooling rates and solution compositions. The distribution pattern of cytochrome c in individual cells was used as a measure of cell viability in the frozen state and this metric agreed well with the population-averaged viability and trypan blue staining experiments. Raman imaging of cells demonstrated that intracellular ice formation (IIF) was common and did not necessarily result in cell death. The amount of intracellular ice as well as ice crystal size played a role in determining whether or not ice inside the cell was a lethal event. Intracellular ice crystals were colocated to the sections of cell membrane in close proximity to extracellular ice. Increasing the distance between extracellular ice and cell membrane decreased the incidence of IIF. Reducing the effective stiffness of the cell membrane by disrupting the actin cytoskeleton using cytochalasin D increased the amount of IIF. Strong intracellular osmotic gradients were observed when IIF was present. These observations support the hypothesis that interactions between the cell membrane and extracellular ice result in IIF. Raman spectromicroscopy provides a powerful tool for observing IIF and understanding its role in cell death during freezing, and enables the development, to our knowledge, of new and improved cell preservation protocols.

The microbiome inherited at birth exerts marked effects on immune programming with long-term health consequences. Here, we demonstrated that the gut, vaginal, and oral microbial diversity of pregnant women with SARS-CoV-2 infection is reduced, and women with early infections exhibit a different vaginal microbiota composition compared to healthy controls at the time of delivery. Accordingly, infants born to pregnant women with early SARS-CoV-2 infection exhibit a unique oral microbiota dominated by Streptococcus species. Together, we demonstrated that SARS-CoV-2 infections during pregnancy, particularly early infections, are associated with lasting changes in the microbiome of pregnant women compromising the initial microbial seed of their infant. Our results highlight the importance of further exploring the impact of SARS-CoV-2 on the infant's microbiome-dependent immune programming.

Background Infants receive their first bacteria from their birthing parent. This newly acquired microbiome plays a pivotal role in developing a robust immune system, the cornerstone of long-term health. Results We demonstrated that the gut, vaginal, and oral microbial diversity of pregnant women with SARS-CoV-2 infection is reduced, and women with early infections exhibit a different vaginal microbiota composition at the time of delivery compared to their healthy control counterparts. Accordingly, a low relative abundance of two Streptococcus sequence variants (SV) was predictive of infants born to pregnant women with SARS-CoV-2 infection. Conclusions Our data suggest that SARS-CoV-2 infections during pregnancy, particularly early infections, are associated with lasting changes in the microbiome of pregnant women, compromising the initial microbial seed of their infant. Our results highlight the importance of further exploring the impact of SARS-CoV-2 on the infant’s microbiome-dependent immune programming.

Introduction:Oxidation-reduction (redox) reactions, and the redox potential (RP) that must be maintained for proper cell function, lie at the heart of physiologic processes in critical illness. Imbalance in RP reflects systemic oxidative stress, and whole blood RP measures have been shown to correlate with oxygen debt level over time in swine traumatic shock. We hypothesize that RP measures reflect changing concentrations of metabolites involved in oxidative stress. To test this hypothesis, we compared blood and urine RP with concentrations of multiple metabolites in a swine traumatic shock model to identify meaningful RP-metabolite relationships.Methods:Seven swine were subjected to traumatic shock. Mixed venous (MV) RP, urine RP, and concurrent MV and urine metabolite concentrations were assessed at baseline, max O2 Debt (80 mL/kg), end resuscitation, and 2 h post-resuscitation. RP was measured at collection via open circuit potential using nanoporous gold electrodes with Ag/AgCl reference and a ParstatMC potentiostat. Metabolite concentrations were measured by quantitative 1H-NMR spectroscopy. MV and urine RP were compared with time-matched metabolites across all swine. LASSO regression with leave-one-out cross validation was used to determine meaningful RP/metabolite relationships. Metabolites had to maintain magnitude and direction of coefficients across 6 or more swine to be considered as having a meaningful relationship. KEGG IDs of these metabolites were uploaded into Metscape for pathway identification and evaluation for physiologic function.Results:Meaningful metabolite relationships (and mean coefficients across cross-validation folds) with MV RP included: choline (−6.27), ATP (−4.39), glycine (5.93), ADP (1.84), glucose (15.96), formate (−13.09), pyruvate (6.18), and taurine (−7.18). Relationships with urine RP were: betaine (4.81), urea (4.14), glycine (−2.97), taurine (10.32), 3-hydroxyisobutyrate (−7.67), N-phenylacetylglycine, PAG (−14.52), hippurate (12.89), and formate (−5.89). These meaningful metabolites were found to scavenge extracellular peroxide (pyruvate), inhibit ROS and activate cellular antioxidant defense (taurine), act as indicators of antioxidant mobilization against oxidative stress (glycine + PAG), and reflect renal hydroxyl radical trapping (hippurate), among other activities.Conclusions:Real-time RP measures demonstrate significant relationships with metabolites attributable to metabolic pathways involved in systemic responses to oxidative stress, as well as those involved in these processes. These data support RP measures as a feasible, biologically relevant marker of oxidative stress. As a direct measure of redox state, RP may be a useful biomarker and clinical tool in guiding diagnosis and therapy in states of increased oxidative stress and may offer value as a marker for organ injury in these states as well.