Bridging the Gap in Cryopreservation Mechanism: Unraveling the Interplay between Structure, Dynamics, and Thermodynamics in Cryoprotectant Aqueous Solutions.

Cryoprotectants play a crucial role in preserving biological material, ensuring their viability during storage and facilitating crucial applications such as the conservation of medical compounds, tissues, and organs for transplantation. However, the precise mechanism by which cryoprotectants modulate the thermodynamic properties of water to impede the formation and growth of ice crystals, thus preventing long-term damage, remains elusive. This is evident in the use of empirically optimized recipes for mixtures that typically contain DMSO, glycerol, and various sugar constituents. Here, we use terahertz calorimetry, Overhauser nuclear polarization, and molecular dynamics simulations to show that DMSO exhibits a robust structuring effect on water around its methyl groups, reaching a maximum at a DMSO mole fraction of XDMSO = 0.33. In contrast, glycerol exerts a smaller water-structuring effect, even at higher concentrations (Scheme 1). These results potentially suggest that the wrapped water around DMSO's methyl group, which can be evicted upon ligand binding, may render DMSO a more surface-active cryoprotectant than glycerol, while glycerol may participate more as a viscogen that acts on the entire sample. These findings shed light on the molecular intricacies of cryoprotectant solvation behavior and have potentially significant implications for optimizing cryopreservation protocols.

Nanoscale water-polymer interactions tune macroscopic diffusivity of water in aqueous poly(ethylene oxide) solutions.

The separation and anti-fouling performance of water purification membranes is governed by both macroscopic and molecular-scale water properties near polymer surfaces. However, even for poly(ethylene oxide) (PEO) - ubiquitously used in membrane materials - there is little understanding of whether or how the molecular structure of water near PEO surfaces affects macroscopic water diffusion. Here, we probe both time-averaged bulk and local water dynamics in dilute and concentrated PEO solutions using a unique combination of experimental and simulation tools. Pulsed-Field Gradient NMR and Overhauser Dynamic Nuclear Polarization (ODNP) capture water dynamics across micrometer length scales in sub-seconds to sub-nanometers in tens of picoseconds, respectively. We find that classical models, such as the Stokes-Einstein and Mackie-Meares relations, cannot capture water diffusion across a wide range of PEO concentrations, but that free volume theory can. Our study shows that PEO concentration affects macroscopic water diffusion by enhancing the water structure and altering free volume. ODNP experiments reveal that water diffusivity near PEO is slower than in the bulk in dilute solutions, previously not recognized by macroscopic transport measurements, but the two populations converge above the polymer overlap concentration. Molecular dynamics simulations reveal that the reduction in water diffusivity occurs with enhanced tetrahedral structuring near PEO. Broadly, we find that PEO does not simply behave like a physical obstruction but directly modifies water's structural and dynamic properties. Thus, even in simple PEO solutions, molecular scale structuring and the impact of polymer interfaces is essential to capturing water diffusion, an observation with important implications for water transport through structurally complex membrane materials.

Angiotensin II acts through Rac1 to upregulate pendrin: role of NADPH oxidase.

Angiotensin II increases apical plasma membrane pendrin abundance and function. This study explored the role of the small GTPase Rac1 in the regulation of pendrin by angiotensin II. To do this, we generated intercalated cell (IC) Rac1 knockout mice and observed that IC Rac1 gene ablation reduced the relative abundance of pendrin in the apical region of intercalated cells in angiotensin II-treated mice but not vehicle-treated mice. Similarly, the Rac1 inhibitor EHT 1864 reduced apical pendrin abundance in angiotensin II-treated mice, through a mechanism that does not require aldosterone. This IC angiotensin II-Rac1 signaling cascade modulates pendrin subcellular distribution without significantly changing actin organization. However, NADPH oxidase inhibition with APX 115 reduced apical pendrin abundance in vivo in angiotensin II-treated mice. Moreover, superoxide dismutase mimetics reduced Cl- absorption in angiotensin II-treated cortical collecting ducts perfused in vitro. Since Rac1 is an NADPH subunit, Rac1 may modulate pendrin through NADPH oxidase-mediated reactive oxygen species production. Because pendrin gene ablation blunts the pressor response to angiotensin II, we asked if pendrin blunts the angiotensin II-induced increase in kidney superoxide. Although kidney superoxide was similar in vehicle-treated wild-type and pendrin knockout mice, it was lower in angiotensin II-treated pendrin-null kidneys than in wild-type kidneys. We conclude that angiotensin II acts through Rac1, independently of aldosterone, to increase apical pendrin abundance. Rac1 may stimulate pendrin, at least partly, through NADPH oxidase. This increase in pendrin abundance contributes to the increment in blood pressure and kidney superoxide content seen in angiotensin II-treated mice.NEW & NOTEWORTHY This study defines a new signaling mechanism by which angiotensin II modulates oxidative stress and blood pressure.

Relationships between Water's Structure and Solute Affinity at Polypeptoid Brush Surfaces.

Excellent antifouling surfaces are generally thought to create a tightly bound layer of water that resists solute adsorption, and highly hydrophilic surfaces such as those with zwitterionic functionalities are of significant current interest as antifoulant strategies. However, despite significant proofs-of-concept, we still lack a fundamental understanding of how the nanoscopic structure of this hydration layer translates to reduced fouling, how surface chemistry can be tuned to achieve antifouling through hydration water, and why, in particular, zwitterionic surfaces seem so promising. Here, we use molecular dynamics simulations and free energy calculations to investigate the molecular relationships among surface chemistry, hydration water structure, and surface-solute affinity across a variety of surface-decorated chemistries. Specifically, we consider polypeptoid-decorated surfaces that display well-known experimental antifouling capabilities and that can be synthesized sequence specifically, with precise backbone positioning of, e.g., charged groups. Through simulations, we calculate the affinities of a range of small solutes to polypeptoid brush surfaces of varied side-chain chemistries. We then demonstrate that measures of the structure of surface hydration water in response to a particular surface chemistry signal solute-surface affinity; specifically, we find that zwitterionic chemistries produce solute-surface repulsion through highly coordinated hydration water while suppressing tetrahedral structuring around the solute, in contrast to uncharged surfaces that show solute-surface affinity. Based on the relationship of this structural perturbation to the affinity of small-molecule solutes, we propose a molecular mechanism by which zwitterionic surface chemistries enhance solute repulsion, with broader implications for the design of antifouling surfaces.

Regulatory T cells play a crucial role in maintaining sperm tolerance and male fertility.

Regulatory T cells (Tregs) modulate tissue homeostatic processes and immune responses. Understanding tissue-Treg biology will contribute to developing precision-targeting treatment strategies. Here, we show that Tregs maintain the tolerogenic state of the testis and epididymis, where sperm are produced and mature. We found that Treg depletion induces severe autoimmune orchitis and epididymitis, manifested by an exacerbated immune cell infiltration [CD4 T cells, monocytes, and mononuclear phagocytes (MPs)] and the development of antisperm antibodies (ASA). In Treg-depleted mice, MPs increased projections toward the epididymal lumen as well as invading the lumen. ASA-bound sperm enhance sperm agglutination and might facilitate sperm phagocytosis. Tolerance breakdown impaired epididymal epithelial function and altered extracellular vesicle cargo, both of which play crucial roles in the acquisition of sperm fertilizing ability and subsequent embryo development. The affected mice had reduced sperm number and motility and severe fertility defects. Deciphering these immunoregulatory mechanisms may help to design new strategies to treat male infertility, as well as to identify potential targets for immunocontraception.

Local solvation structures govern the mixing thermodynamics of glycerol-water solutions.

Glycerol is a major cryoprotective agent and is widely used to promote protein stabilization. By a combined experimental and theoretical study, we show that global thermodynamic mixing properties of glycerol and water are dictated by local solvation motifs. We identify three hydration water populations, i.e., bulk water, bound water (water hydrogen bonded to the hydrophilic groups of glycerol) and cavity wrap water (water hydrating the hydrophobic moieties). Here, we show that for glycerol experimental observables in the THz regime allow quantification of the abundance of bound water and its partial contribution to the mixing thermodynamics. Specifically, we uncover a 1 : 1 connection between the population of bound waters and the mixing enthalpy, which is further corroborated by the simulation results. Therefore, the changes in global thermodynamic quantity - mixing enthalpy - are rationalized at the molecular level in terms of changes in the local hydrophilic hydration population as a function of glycerol mole fraction in the full miscibility range. This offers opportunities to rationally design polyol water, as well as other aqueous mixtures to optimize technological applications by tuning mixing enthalpy and entropy based on spectroscopic screening.

A Bifunctional PARP-HDAC Inhibitor with Activity in Ewing Sarcoma.

PURPOSE: Histone deacetylase (HDAC) inhibition has been shown to induce pharmacologic "BRCAness" in cancer cells with proficient DNA repair activity. This provides a rationale for exploring combination treatments with HDAC and PARP inhibition in cancer types that are insensitive to single-agent PARP inhibitors (PARPi). Here, we report the concept and characterization of a novel bifunctional PARPi (kt-3283) with dual activity toward PARP1/2 and HDAC enzymes in Ewing sarcoma cells. EXPERIMENTAL DESIGN: Inhibition of PARP1/2 and HDAC was measured using PARP1/2, HDAC activity, and PAR formation assays. Cytotoxicity was assessed by IncuCyte live cell imaging, CellTiter-Glo, and spheroid assays. Cell-cycle profiles were determined using propidium iodide staining and flow cytometry. DNA damage was examined by γH2AX expression and comet assay. Inhibition of metastatic potential by kt-3283 was evaluated via ex vivo pulmonary metastasis assay (PuMA). RESULTS: Compared with FDA-approved PARP (olaparib) and HDAC (vorinostat) inhibitors, kt-3283 displayed enhanced cytotoxicity in Ewing sarcoma models. The kt-3283-induced cytotoxicity was associated with strong S and G2-M cell-cycle arrest in nanomolar concentration range and elevated DNA damage as assessed by γH2AX tracking and comet assays. In three-dimensional spheroid models of Ewing sarcoma, kt-3283 showed efficacy in lower concentrations than olaparib and vorinostat, and kt-3283 inhibited colonization of Ewing sarcoma cells in the ex vivo PuMA model. CONCLUSIONS: Our data demonstrate the preclinical justification for studying the benefit of dual PARP and HDAC inhibition in the treatment of Ewing sarcoma in a clinical trial and provides proof-of-concept for a bifunctional single-molecule therapeutic strategy.

Relationships between Molecular Structural Order Parameters and Equilibrium Water Dynamics in Aqueous Mixtures.

Water's unique thermophysical properties and how it mediates aqueous interactions between solutes have long been interpreted in terms of its collective molecular structure. The seminal work of Errington and Debenedetti [Nature 2001, 409, 318-321] revealed a striking hierarchy of relationships among the thermodynamic, dynamic, and structural properties of water, motivating many efforts to understand (1) what measures of water structure are connected to different experimentally accessible macroscopic responses and (2) how many such structural metrics are adequate to describe the collective structural behavior of water. Diffusivity constitutes a particularly interesting experimentally accessible equilibrium property to investigate such relationships because advanced NMR techniques allow the measurement of bulk and local water dynamics in nanometer proximity to molecules and interfaces, suggesting the enticing possibility of measuring local diffusivities that report on water structure. Here, we apply statistical learning methods to discover persistent structure-dynamic correlations across a variety of simulated aqueous mixtures, from alcohol-water to polypeptoid-water systems. We investigate a variety of molecular water structure metrics and find that an unsupervised statistical learning algorithm (namely, sequential feature selection) identifies only two or three independent structural metrics that are sufficient to predict water self-diffusivity accurately. Surprisingly, the translational diffusivity of water across all mixed systems studied here is strongly correlated with a measure of tetrahedral order given by water's triplet angle distribution. We also identify a separate small number of structural metrics that well predict an important thermodynamic property, the excess chemical potential of an idealized methane-sized hydrophobe in water. Ultimately, we offer a Bayesian method of inferring water structure by using only structure-dynamics linear regression models with experimental Overhauser dynamic nuclear polarization (ODNP) measurements of water self-diffusivity. This study thus quantifies the relationships among several distinct structural order parameters in water and, through statistical learning, reveals the potential to leverage molecular structure to predict fundamental thermophysical properties. In turn, these findings suggest a framework for solving the inverse problem of inferring water's molecular structure using experimental measurements such as ODNP studies that probe local water properties.

Tizoxanide Antiviral Activity on Dengue Virus Replication.

Dengue virus is an important circulating arbovirus in Brazil responsible for high morbidity and mortality worldwide, representing a huge economic and social burden, in addition to affecting public health. In this study, the biological activity, toxicity, and antiviral activity against dengue virus type 2 (DENV-2) of tizoxanide (TIZ) was evaluated in Vero cell culture. TIZ has a broad spectrum of action in inhibiting different pathogens, including bacteria, protozoa, and viruses. Cells were infected for 1 h with DENV-2 and then treated for 24 h with different concentrations of the drug. The quantification of viral production indicated the antiviral activity of TIZ. The protein profiles in infected Vero cells treated and not treated with TIZ were analyzed using the label-free quantitative proteomic approach. TIZ was able to inhibit virus replication mainly intracellularly after DENV-2 penetration and before the complete replication of the viral genome. Additionally, the study of the protein profile of infected not-treated and infected-treated Vero cells showed that TIZ interferes with cellular processes such as intracellular trafficking and vesicle-mediated transport and post-translational modifications when added after infection. Our results also point to the activation of immune response genes that would eventually lead to a decrease of DENV-2 production. TIZ is a promising therapeutic molecule for the treatment of DENV-2 infections.

Targeting Nrf2 with 3 H-1,2-dithiole-3-thione to moderate OXPHOS-driven oxidative stress attenuates IL-17A-induced psoriasis.

Psoriasis, a chronic autoimmune disease characterized by the hyperproliferation of keratinocytes in the epidermis and parakeratosis, significantly impacts quality of life. Interleukin (IL)- 17A dominates the pathogenesis of psoriasis and facilitates reactive oxygen species (ROS) accumulation, which exacerbates local psoriatic lesions. Biologic treatment provides remarkable clinical efficacy, but its high cost and unignorable side effects limit its applications. 3 H-1,2-Dithiole-3-thione (D3T) possesses compelling antioxidative capacities against several diseases through the nuclear factor erythroid 2-related factor 2 (Nrf2) cascade. Hence, we aimed to evaluate the effect and mechanism of D3T in psoriasis. We found that D3T attenuates skin thickening and scaling by inhibiting IL-17A-secreting γδT cells in imiquimod (IMQ)-induced psoriatic mice. Interleukin-17A markedly enhanced IL-6 and IL-8 expression, lipid peroxidation, the contents of nitric oxide and hydrogen peroxide, oxidative phosphorylation and the MAPK/NF-κB pathways in keratinocytes. IL-17A also inhibited the Nrf2-NQO1-HO-1 axis and the activities of superoxide dismutase and glutathione peroxidase. D3T significantly reversed these parameters in IL-17A-treated keratinocytes. ML-385, a Nrf2 neutralizer, failed to improve D3T-induced anti-inflammatory and antioxidative effects in IL-17A-treated keratinocytes. We conclude that targeting Nrf2 with D3T to diminish oxidative and inflammatory damage in keratinocytes may attenuate psoriasis.

Intracellular sites of AQP2 S256 phosphorylation identified using inhibitors of the AQP2 recycling itinerary.

Vasopressin (VP)-regulated aquaporin-2 (AQP2) trafficking between cytoplasmic vesicles and the plasma membrane of kidney principal cells is essential for water homeostasis. VP affects AQP2 phosphorylation at several serine residues in the COOH-terminus; among them, serine 256 (S256) appears to be a major regulator of AQP2 trafficking. Mutation of this serine to aspartic acid, which mimics phosphorylation, induces constitutive membrane expression of AQP2. However, the intracellular location(s) at which S256 phosphorylation occurs remains elusive. Here, we used strategies to block AQP2 trafficking at different cellular locations in LLC-PK1 cells and monitored VP-stimulated phosphorylation of S256 at these sites by immunofluorescence and Western blot analysis with phospho-specific antibodies. Using methyl-ß-cyclodextrin, cold block or bafilomycin, and taxol, we blocked AQP2 at the plasma membrane, in the perinuclear trans-Golgi network, and in scattered cytoplasmic vesicles, respectively. Regardless of its cellular location, VP induced a significant increase in S256 phosphorylation, and this effect was not dependent on a functional microtubule cytoskeleton. To further investigate whether protein kinase A (PKA) was responsible for S256 phosphorylation in these cellular compartments, we created PKA-null cells and blocked AQP2 trafficking using the same procedures. We found that S256 phosphorylation was no longer increased compared with baseline, regardless of AQP2 localization. Taken together, our data indicate that AQP2 S256 phosphorylation can occur at the plasma membrane, in the trans-Golgi network, or in cytoplasmic vesicles and that this event is dependent on the expression of PKA in these cells.NEW & NOTEWORTHY Phosphorylation of aquaporin-2 by PKA at serine 256 (S256) occurs in various subcellular locations during its recycling itinerary, suggesting that the protein complex necessary for AQP2 S256 phosphorylation is present in these different recycling stations. Furthermore, we showed, using PKA-null cells, that PKA activity is required for vasopressin-induced AQP2 phosphorylation. Our data reveal a complex spatial pattern of intracellular AQP2 phosphorylation at S256, shedding new light on the role of phosphorylation in AQP2 membrane accumulation.

Neutrophils incite and macrophages avert electrical storm after myocardial infarction.

Sudden cardiac death, arising from abnormal electrical conduction, occurs frequently in patients with coronary heart disease. Myocardial ischemia simultaneously induces arrhythmia and massive myocardial leukocyte changes. In this study, we optimized a mouse model in which hypokalemia combined with myocardial infarction triggered spontaneous ventricular tachycardia in ambulatory mice, and we showed that major leukocyte subsets have opposing effects on cardiac conduction. Neutrophils increased ventricular tachycardia via lipocalin-2 in mice, whereas neutrophilia associated with ventricular tachycardia in patients. In contrast, macrophages protected against arrhythmia. Depleting recruited macrophages in Ccr2 -/- mice or all macrophage subsets with Csf1 receptor inhibition increased both ventricular tachycardia and fibrillation. Higher arrhythmia burden and mortality in Cd36 -/- and Mertk -/- mice, viewed together with reduced mitochondrial integrity and accelerated cardiomyocyte death in the absence of macrophages, indicated that receptor-mediated phagocytosis protects against lethal electrical storm. Thus, modulation of leukocyte function provides a potential therapeutic pathway for reducing the risk of sudden cardiac death.

4-Ethylguaiacol Modulates Neuroinflammation and Promotes Heme Oxygenase-1 Expression to Ameliorate Brain Injury in Ischemic Stroke.

Ischemic stroke is caused by a sudden reduction in cerebral blood flow that subsequently induces a complex cascade of pathophysiological responses, leading to brain inflammation and irreversible infarction. 4-ethylguaiacol (4-EG) is reported to suppress inflammatory immune responses. However, whether 4-EG exerts anti-inflammatory effects in ischemic stroke remains unexplored. We evaluated the therapeutic potential of 4-EG and examined the cellular and molecular mechanisms underlying the protective effects of 4-EG in ischemic stroke. The effect of 4-EG in ischemic stroke was determined by using a transient middle cerebral artery occlusion (MCAO) animal model followed by exploring the infarct size, neurological deficits, microglia activation, inflammatory cytokine production, blood-brain barrier (BBB) disruption, brain endothelial cell adhesion molecule expression, and microglial heme oxygenase-1 (HO-1) expression. Nrf2-/- and HO-1 inhibitor ZnPP-treated mice were also subjected to MCAO to evaluate the role of the Nrf2/HO-1 pathway in 4-EG-mediated protection in ischemic stroke. We found that 4-EG attenuated infarct size and neurological deficits, and lessened BBB disruption in ischemic stroke. Further investigation revealed that 4-EG suppressed microglial activation, peripheral inflammatory immune cell infiltration, and brain endothelial cell adhesion molecule upregulation in the ischemic brain. Finally, we identified that the protective effect of 4-EG in ischemic stroke was abolished in Nrf2-/- and ZnPP-treated MCAO mice. Our results identified that 4-EG confers protection against ischemic stroke and reveal that the protective effect of 4-EG in ischemic stroke is mediated through the induction of the Nrf2/HO1 pathway. Thus, our findings suggest that 4-EG could be developed as a novel therapeutic agent for the treatment of ischemic stroke.

High-throughput nuclear resonance time domain interferometry using annular slits.

Nuclear resonance time domain interferometry (NR-TDI) is used to study the slow dynamics of liquids (that do not require Mössbauer isotopes) at atomic and molecular length scales. Here the TDI method of using a stationary two-line magnetized 57Fe foil as a source and a stationary single-line stainless steel foil analyzer is employed. The new technique of adding an annular slit in front of a single silicon avalanche photodiode detector enables a wide range of momentum transfers (1 to 100 nm-1 by varying the distance between the annular slits and sample) with a high count rate of up to 160 Hz with a Δq resolution of ±1.7 nm-1 at q = 14 nm-1. The sensitivity of this method in determining relaxation times is quantified and discussed. The Kohlrausch-Williams-Watts (KWW) model was used to extract relaxation times for glycerol. These relaxation times give insight into the dynamics of the electron density fluctuations of glycerol as a function of temperature and momentum transfers.

Cerebrospinal fluid can exit into the skull bone marrow and instruct cranial hematopoiesis in mice with bacterial meningitis.

Interactions between the immune and central nervous systems strongly influence brain health. Although the blood-brain barrier restricts this crosstalk, we now know that meningeal gateways through brain border tissues facilitate intersystem communication. Cerebrospinal fluid (CSF), which interfaces with the glymphatic system and thereby drains the brain's interstitial and perivascular spaces, facilitates outward signaling beyond the blood-brain barrier. In the present study, we report that CSF can exit into the skull bone marrow. Fluorescent tracers injected into the cisterna magna of mice migrate along perivascular spaces of dural blood vessels and then travel through hundreds of sub-millimeter skull channels into the calvarial marrow. During meningitis, bacteria hijack this route to invade the skull's hematopoietic niches and initiate cranial hematopoiesis ahead of remote tibial sites. As skull channels also directly provide leukocytes to meninges, the privileged sampling of brain-derived danger signals in CSF by regional marrow may have broad implications for inflammatory neurological disorders.

Simultaneous stabilization of actin cytoskeleton in multiple nephron-specific cells protects the kidney from diverse injury.

Chronic kidney diseases and acute kidney injury are mechanistically distinct kidney diseases. While chronic kidney diseases are associated with podocyte injury, acute kidney injury affects renal tubular epithelial cells. Despite these differences, a cardinal feature of both acute and chronic kidney diseases is dysregulated actin cytoskeleton. We have shown that pharmacological activation of GTPase dynamin ameliorates podocyte injury in murine models of chronic kidney diseases by promoting actin polymerization. Here we establish dynamin's role in modulating stiffness and polarity of renal tubular epithelial cells by crosslinking actin filaments into branched networks. Activation of dynamin's crosslinking capability by a small molecule agonist stabilizes the actomyosin cortex of the apical membrane against injury, which in turn preserves renal function in various murine models of acute kidney injury. Notably, a dynamin agonist simultaneously attenuates podocyte and tubular injury in the genetic murine model of Alport syndrome. Our study provides evidence for the feasibility and highlights the benefits of novel holistic nephron-protective therapies.

A non-dividing cell population with high pyruvate dehydrogenase kinase activity regulates metabolic heterogeneity and tumorigenesis in the intestine.

Although reprogramming of cellular metabolism is a hallmark of cancer, little is known about how metabolic reprogramming contributes to early stages of transformation. Here, we show that the histone deacetylase SIRT6 regulates tumor initiation during intestinal cancer by controlling glucose metabolism. Loss of SIRT6 results in an increase in the number of intestinal stem cells (ISCs), which translates into enhanced tumor initiating potential in APCmin mice. By tracking down the connection between glucose metabolism and tumor initiation, we find a metabolic compartmentalization within the intestinal epithelium and adenomas, where a rare population of cells exhibit features of Warburg-like metabolism characterized by high pyruvate dehydrogenase kinase (PDK) activity. Our results show that these cells are quiescent cells expressing +4 ISCs and enteroendocrine markers. Active glycolysis in these cells suppresses ROS accumulation and enhances their stem cell and tumorigenic potential. Our studies reveal that aerobic glycolysis represents a heterogeneous feature of cancer, and indicate that this metabolic adaptation can occur in non-dividing cells, suggesting a role for the Warburg effect beyond biomass production in tumors.

B lymphocyte-derived acetylcholine limits steady-state and emergency hematopoiesis.

Autonomic nerves control organ function through the sympathetic and parasympathetic branches, which have opposite effects. In the bone marrow, sympathetic (adrenergic) nerves promote hematopoiesis; however, how parasympathetic (cholinergic) signals modulate hematopoiesis is unclear. Here, we show that B lymphocytes are an important source of acetylcholine, a neurotransmitter of the parasympathetic nervous system, which reduced hematopoiesis. Single-cell RNA sequencing identified nine clusters of cells that expressed the cholinergic α7 nicotinic receptor (Chrna7) in the bone marrow stem cell niche, including endothelial and mesenchymal stromal cells (MSCs). Deletion of B cell-derived acetylcholine resulted in the differential expression of various genes, including Cxcl12 in leptin receptor+ (LepR+) stromal cells. Pharmacologic inhibition of acetylcholine signaling increased the systemic supply of inflammatory myeloid cells in mice and humans with cardiovascular disease.

Stress, Anxiety, Depression, and Perfectionism Among Graduate Students in Health Sciences Programs.

Graduate students in occupational therapy, physical therapy, physician assistant, and speech-language pathology programs in the United States were surveyed using an online questionnaire in spring 2020. The 1,916 students reported on mental health diagnoses and completed the Depression, Anxiety, and Stress Scale and three Hewitt-Flett Perfectionism subscales. Students in all four programs scored significantly higher than national norms on depression, anxiety, and stress; and females scored higher than males on anxiety and stress. There were small differences between students in the four programs, with speech-language pathology students demonstrating higher levels of depression, anxiety, and stress. First-year students had higher levels of anxiety than third-year students, and third-year students had lower levels of stress than both first-year and second-year students. All three perfectionism subscales were positively, although weakly, correlated to depression, anxiety, and stress. Finally, students reported significantly higher rates of anxiety after the onset of the COVID-19 pandemic than prior to the pandemic. These findings can be used to inform health science graduate programs of concerns related to student stress, anxiety, and depression as they re-evaluate program or curricular design.