Thermochemical Studies of Small Carbohydrates.

Despite their prevalence in biomass and importance in biochemistry, there is still much to be learned about simple carbohydrates. Gas-phase calculations are reported here on two trioses and three tetroses. For aldotetroses, both the open-chain and furanose forms are considered. Enthalpies of reduction to polyols are calculated at the CBS-APNO level of theory, and comparisons to simple aldehydes and ketones are made. Heats of formation are calculated in two ways with overall good agreement. The heat of formation of glyceraldehyde obtained from modified HEAT calculations is also reported. Finally, calculated bond energies are presented, and the influence of the structure on the bond energies is discussed.

Calponin 1 inhibits agonist-induced ERK activation and decreases calcium sensitization in vascular smooth muscle.

Smooth muscle cell (SMC) contraction and vascular tone are modulated by phosphorylation and multiple modifications of the thick filament, and thin filament regulation of SMC contraction has been reported to involve extracellular regulated kinase (ERK). Previous studies in ferrets suggest that the actin-binding protein, calponin 1 (CNN1), acts as a scaffold linking protein kinase C (PKC), Raf, MEK and ERK, promoting PKC-dependent ERK activation. To gain further insight into this function of CNN1 in ERK activation and the regulation of SMC contractility in mice, we generated a novel Calponin 1 knockout mouse (Cnn1 KO) by a single base substitution in an intronic CArG box that preferentially abolishes expression of CNN1 in vascular SMCs. Using this new Cnn1 KO mouse, we show that ablation of CNN1 has two effects, depending on the cytosolic free calcium level: (1) in the presence of elevated intracellular calcium caused by agonist stimulation, Cnn1 KO mice display a reduced amplitude of stress and stiffness but an increase in agonist-induced ERK activation; and (2) during intracellular calcium depletion, in the presence of an agonist, Cnn1 KO mice exhibit increased duration of SM tone maintenance. Together, these results suggest that CNN1 plays an important and complex modulatory role in SMC contractile tone amplitude and maintenance.

Single-cell analysis of shared signatures and transcriptional diversity during zebrafish development.

During development, animals generate distinct cell populations with specific identities, functions, and morphologies. We mapped transcriptionally distinct populations across 489,686 cells from 62 stages during wild-type zebrafish embryogenesis and early larval development (3-120 h post-fertilization). Using these data, we identified the limited catalog of gene expression programs reused across multiple tissues and their cell-type-specific adaptations. We also determined the duration each transcriptional state is present during development and identify unexpected long-term cycling populations. Focused clustering and transcriptional trajectory analyses of non-skeletal muscle and endoderm identified transcriptional profiles and candidate transcriptional regulators of understudied cell types and subpopulations, including the pneumatic duct, individual intestinal smooth muscle layers, spatially distinct pericyte subpopulations, and recently discovered best4+ cells. To enable additional discoveries, we make this comprehensive transcriptional atlas of early zebrafish development available through our website, Daniocell.

Recycling of modified H2A-H2B provides short-term memory of chromatin states.

Chromatin landscapes are disrupted during DNA replication and must be restored faithfully to maintain genome regulation and cell identity. The histone H3-H4 modification landscape is restored by parental histone recycling and modification of new histones. How DNA replication impacts on histone H2A-H2B is currently unknown. Here, we measure H2A-H2B modifications and H2A.Z during DNA replication and across the cell cycle using quantitative genomics. We show that H2AK119ub1, H2BK120ub1, and H2A.Z are recycled accurately during DNA replication. Modified H2A-H2B are segregated symmetrically to daughter strands via POLA1 on the lagging strand, but independent of H3-H4 recycling. Post-replication, H2A-H2B modification and variant landscapes are quickly restored, and H2AK119ub1 guides accurate restoration of H3K27me3. This work reveals epigenetic transmission of parental H2A-H2B during DNA replication and identifies cross talk between H3-H4 and H2A-H2B modifications in epigenome propagation. We propose that rapid short-term memory of recycled H2A-H2B modifications facilitates restoration of stable H3-H4 chromatin states.

Profiling Chromatin Accessibility on Replicated DNA with repli-ATAC-Seq.

Open or accessible chromatin typifies euchromatic regions and helps define cell type-specific transcription programs. DNA replication massively disorders chromatin composition and structure, and how accessible regions are affected by and recover from this disruption has been unclear. Here, we present repli-ATAC-seq, a protocol to profile accessible chromatin genome-wide on replicated DNA starting from 100,000 cells. In this method, replicated DNA is labeled with a short 5-ethynyl-2'-deoxyuridine (EdU) pulse in cultured cells and isolated from a population of tagmented fragments for amplification and next-generation sequencing. Repli-ATAC-seq provides high-resolution information on chromatin dynamics after DNA replication and reveals new insights into the interplay between DNA replication, transcription, and the chromatin landscape.

Quantifying propagation of DNA methylation and hydroxymethylation with iDEMS.

DNA methylation is a critical epigenetic mark in mammalian cells. Many aspects of DNA methylation maintenance have been characterized; however, the exact kinetics of post-replicative methylation maintenance remain a subject of debate. Here we develop isolation of DNA by 5-ethynyl-deoxyuridine labelling for mass spectrometry (iDEMS), a highly sensitive, quantitative mass spectrometry-based method for measuring DNA modifications on metabolically labelled DNA. iDEMS reveals an unexpectedly hemi-methylated landscape on nascent DNA. Combining iDEMS with metabolic labelling reveals that methylation maintenance is outpaced by cell division in mouse embryonic stem cells. Our approach shows that hydroxymethylation is perpetually asymmetric between sister strands in favour of the parental, template strand. iDEMS can be coupled with immunoprecipitation of chromatin proteins, revealing features of DNA methylation-histone modification crosstalk and suggesting a model for interplay between methylation and nucleosome assembly. iDEMS therefore elucidates long-standing questions about DNA modification propagation and provides an important orthogonal technology to understanding this process in dynamic cellular contexts.

The importance of dystrophin and the dystrophin associated proteins in vascular smooth muscle.

This review details the role of dystrophin and the dystrophin associated proteins (DAPs) in the vascular smooth muscle. Dystrophin is most comprehensively studied in the skeletal muscle due to serious symptoms found related to the skeletal muscle of patients with muscular dystrophy. Mutations in the dystrophin gene, or DAPs genes, result in a wide range of muscular dystrophies. In skeletal muscle, dystrophin is known to act to as a cytoskeletal stabilization protein and protects cells against contraction-induced damage. In skeletal muscle, dystrophin stabilizes the plasma membrane by transmitting forces generated by sarcomeric contraction to the extracellular matrix (ECM). Dystrophin is a scaffold that binds the dystroglycan complex (DGC) and has many associated proteins (DAPs). These DAPs include sarcoglycans, syntrophins, dystroglycans, dystrobrevin, neuronal nitric oxide synthase, and caveolins. The DAPs provide biomechanical support to the skeletal or cardiac plasma membrane during contraction, and loss of one or several of these DAPs leads to plasma membrane fragility. Dystrophin is expressed near the plasma membrane of all muscles, including cardiac and vascular smooth muscle, and some neurons. Dystrophic mice have noted biomechanical irregularities in the carotid arteries and spontaneous motor activity in portal vein altered when compared to wild type mice. Additionally, some studies suggest the vasculature of patients and animal models with muscular dystrophy is abnormal. Although the function of dystrophin and the DAPs in vascular smooth muscle is not thoroughly established in the field, this review makes the point that these proteins are expressed, and important and further study is warranted.

Enhancing the Mentoring Experience for Underrepresented Students.

Undergraduate research is a valuable experience that increases the likelihood of a STEM major to continue on to postgraduate training in their field. For students from groups underrepresented in the biomedical sciences, a strong mentoring relationship during this undergraduate period is a key component in preparing them for the next stage of their education and can have a significant influence on their ability to persist in the pipeline. Although the ideal scenario to increase the diversity of the biomedical workforce is to provide more BIPOC (Black, Indigenous, People of Color) faculty mentors for our undergraduates, we also need to develop strategies to provide strong mentoring experiences for our BIPOC students when those mentors are not in great number. At Xavier University of Louisiana, we have used our NIH BUILD Project Pathways program to look more closely at the mentor matching process. Throughout the past seven years, we have moved from the traditional mentor, research-focused matching process to a student-centered process. The lessons learned here can be used by any University looking to craft an inclusive undergraduate research program to meet the needs of all students, but in particular a diverse student population.

Functional Remodeling of the Contractile Smooth Muscle Cell Cortex, a Provocative Concept, Supported by Direct Visualization of Cortical Remodeling.

Considerable controversy has surrounded the functional anatomy of the cytoskeleton of the contractile vascular smooth muscle cell. Recent studies have suggested a dynamic nature of the cortical cytoskeleton of these cells, but direct proof has been lacking. Here, we review past studies in this area suggesting a plasticity of smooth muscle cells. We also present images testing these suggestions by using the technique of immunoelectron microscopy of metal replicas to directly visualize the cortical actin cytoskeleton of the contractile smooth muscle cell along with interactions by representative cytoskeletal binding proteins. We find the cortical cytoskeletal matrix to be a branched, interconnected network of linear actin bundles. Here, the focal adhesion proteins talin and zyxin were localized with nanometer accuracy. Talin is reported in past studies to span the integrin-cytoplasm distance in fibroblasts and zyxin is known to be an adaptor protein between alpha-actinin and VASP. In response to activation of signal transduction with the alpha-agonist phenylephrine, we found that no movement of talin was detectable but that the zyxin-zyxin spacing was statistically significantly decreased in the smooth muscle cells examined. Contractile smooth muscle is often assumed to have a fixed cytoskeletal structure. Thus, the results included here are important in that they directly support the concept at the electron microscopic level that the focal adhesion of the contractile smooth muscle cell has a dynamic nature and that the protein-protein interfaces showing plasticity are protein-specific.

Ageing causes an aortic contractile dysfunction phenotype by targeting the expression of members of the extracellular signal-regulated kinase pathway.

The extracellular signal-regulated kinase (ERK) pathway is a well-known regulator of vascular smooth muscle cell proliferation, but it also serves as a regulator of caldesmon, which negatively regulates vascular contractility. This study examined whether aortic contractile function requires ERK activation and if this activation is regulated by ageing. Biomechanical experiments revealed that contractile responses to the alpha1-adrenergic agonist phenylephrine are attenuated specifically in aged mice, which is associated with downregulation of ERK phosphorylation. ERK inhibition attenuates phenylephrine-induced contractility, indicating that the contractile tone is at least partially ERK-dependent. To explore the mechanisms of this age-related downregulation of ERK phosphorylation, we transfected microRNAs, miR-34a and miR-137 we have previously shown to increase with ageing and demonstrated that in A7r5 cells, both miRs downregulate the expression of Src and paxillin, known regulators of ERK signalling, as well as ERK phosphorylation. Further studies in aortic tissues transfected with miRs show that miR-34a but not miR-137 has a negative effect on mRNA levels of Src and paxillin. Furthermore, ERK phosphorylation is decreased in aortic tissue treated with the Src inhibitor PP2. Increases in miR-34a and miR-137 with ageing downregulate the expression of Src and paxillin, leading to impaired ERK signalling and aortic contractile dysfunction.

Animal Transmission of SARS-CoV-2 and the Welfare of Animals during the COVID-19 Pandemic.

The accelerated pace of research into Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) necessitates periodic summaries of current research. The present paper reviews virus susceptibilities in species with frequent human contact, and factors that are best predictors of virus susceptibility. Species reviewed were those in contact with humans through entertainment, pet, or agricultural trades, and for whom reports (either anecdotal or published) exist regarding the SARS-CoV-2 virus and/or the resulting disease state COVID-19. Available literature was searched using an artificial intelligence (AI)-assisted engine, as well as via common databases, such as Web of Science and Medline. The present review focuses on susceptibility and transmissibility of SARS-CoV-2, and polymorphisms in transmembrane protease serine 2 (TMPRSS2) and angiotensin-converting enzyme 2 (ACE2) that contribute to species differences. Dogs and pigs appear to have low susceptibility, while ferrets, mink, some hamster species, cats, and nonhuman primates (particularly Old World species) have high susceptibility. Precautions may therefore be warranted in interactions with such species, and more selectivity practiced when choosing appropriate species to serve as models for research.

Vascular aging, the vascular cytoskeleton and aortic stiffness.

Vascular aging, aortic stiffness and hypertension are mechanistically interrelated. The perspective presented here will focus mainly on the molecular mechanisms of age-associated increases in the stiffness of the vascular smooth muscle cell (VSMC). This review will highlight the mechanisms by which the VSMC contributes to disorders of vascular aging. Distinct functional sub-components of the vascular cell and the molecular mechanisms of the protein-protein interactions, signaling mechanisms and intracellular trafficking processes in the setting of the aging aorta will be detailed.

BCL11B Regulates Arterial Stiffness and Related Target Organ Damage.

[Figure: see text].

Non-muscle myosin II regulates aortic stiffness through effects on specific focal adhesion proteins and the non-muscle cortical cytoskeleton.

Non-muscle myosin II (NMII) plays a role in many fundamental cellular processes including cell adhesion, migration, and cytokinesis. However, its role in mammalian vascular function is not well understood. Here, we investigated the function of NMII in the biomechanical and signalling properties of mouse aorta. We found that blebbistatin, an inhibitor of NMII, decreases agonist-induced aortic stress and stiffness in a dose-dependent manner. We also specifically demonstrate that in freshly isolated, contractile, aortic smooth muscle cells, the non-muscle myosin IIA (NMIIA) isoform is associated with contractile filaments in the core of the cell as well as those in the non-muscle cell cortex. However, the non-muscle myosin IIB (NMIIB) isoform is excluded from the cell cortex and colocalizes only with contractile filaments. Furthermore, both siRNA knockdown of NMIIA and NMIIB isoforms in the differentiated A7r5 smooth muscle cell line and blebbistatin-mediated inhibition of NM myosin II suppress agonist-activated increases in phosphorylation of the focal adhesion proteins FAK Y925 and paxillin Y118. Thus, we show in the present study, for the first time that NMII regulates aortic stiffness and stress and that this regulation is mediated through the tension-dependent phosphorylation of the focal adhesion proteins FAK and paxillin.

Aging-induced microbleeds of the mouse thalamus compared to sensorimotor and memory defects.

The aim of this study is to investigate the relationship between aging and brain vasculature health. Three groups of mice, 3, 17-18, and 24 months, comparable to young adult, middle age, and old human were studied. Prussian blue histology and fast imaging with steady precession T2∗-weighted magnetic resonance imaging were used to quantify structural changes in the brain across age groups. The novel object recognition test was used to assess behavioral changes associated with anatomical changes. This study is the first to show that the thalamus is the most vulnerable brain region in the mouse model for aging-induced vascular damage. Magnetic resonance imaging data document the timeline of accumulation of thalamic damage. Histological data reveal that the majority of vascular damage accumulates in the ventroposterior nucleus and mediodorsal thalamic nucleus. Functional studies indicate that aging-induced vascular damage in the thalamus is associated with memory and sensorimotor deficits. This study points to the possibility that aging-associated vascular disease is a factor in irreversible brain damage as early as middle age.

The brains of aged mice are characterized by altered tissue diffusion properties and cerebral microbleeds.

BACKGROUND: Brain aging is a major risk factor in the progression of cognitive diseases including Alzheimer's disease (AD) and vascular dementia. We investigated a mouse model of brain aging up to 24 months old (mo). METHODS: A high field (11.7T) MRI protocol was developed to characterize specific features of brain aging including the presence of cerebral microbleeds (CMBs), morphology of grey and white matter, and tissue diffusion properties. Mice were selected from age categories of either young (3 mo), middle-aged (18 mo), or old (24 mo) and fed normal chow over the duration of the study. Mice were imaged in vivo with multimodal MRI, including conventional T2-weighted (T2W) and T2*-weighted (T2*W) imaging, followed by ex vivo diffusion-weighted imaging (DWI) and T2*W MR-microscopy to enhance the detection of microstructural features. RESULTS: Structural changes observed in the mouse brain with aging included reduced cortical grey matter volume and enlargement of the brain ventricles. A remarkable age-related change in the brains was the development of CMBs found starting at 18 mo and increasing in total volume at 24 mo, primarily in the thalamus. CMBs presence was confirmed with high resolution ex vivo MRI and histology. DWI detected further brain tissue changes in the aged mice including reduced fractional anisotropy, increased radial diffusion, increased mean diffusion, and changes in the white matter fibers visualized by color-coded tractography, including around a large cortical CMB. CONCLUSIONS: The mouse is a valuable model of age-related vascular contributions to cognitive impairment and dementia (VCID). In composite, these methods and results reveal brain aging in older mice as a multifactorial process including CMBs and tissue diffusion alterations that can be well characterized by high field MRI.

Chromatin replication and epigenetic cell memory.

Propagation of the chromatin landscape across cell divisions is central to epigenetic cell memory. Mechanistic analysis of the interplay between DNA replication, the cell cycle, and the epigenome has provided insights into replication-coupled chromatin assembly and post-replicative chromatin maintenance. These breakthroughs are critical for defining how proliferation impacts the epigenome during cell identity changes in development and disease. Here we review these findings in the broader context of epigenetic inheritance across mitotic cell division.

BUILDING INTEGRATED PATHWAYS TO INDEPENDENCE FOR DIVERSE BIOMEDICAL RESEARCHERS: PROJECT PATHWAYS.

Diversity of backgrounds and life experiences on scientific teams is known to lead to more innovative ideas and better scientific products. However, in the United States, the percentages of individuals from underrepresented racial and ethnic groups who obtain doctoral degrees in the Sciences continue to be significantly lower than their percentages in the population. This has resulted in the need for nation-wide initiatives to remedy this inequality, and consequently produce more productive teams of scientific minds. Xavier University of Louisiana is a historically Black and Catholic university that is widely recognized in the US for the success of its undergraduate Science, Technology, Engineering, and Mathematics (STEM) programs. Project Pathways at Xavier is one of ten federally-funded Building Infrastructure Leading to Diversity (BUILD) programs with the overarching goal of diversifying the Biomedical research workforce. Project Pathways is designed as a holistic, integrated, and coordinated program across Biomedical academic departments, student academic and career support offices, and the University's faculty development center. The overall hypothesis of Project Pathways is that if individuals from groups underrepresented in scientific research careers are provided with a) early awareness and deepening exposure to Biomedical careers, b) supportive relationships as they move through the pathway, c) suitable infrastructure, and d) meaningful engagement in Biomedical research experiences and adequate research resources, then a higher number will succeed in entering and successfully completing graduate programs, leading to increased diversity in the Biomedical research workforce. Here, the significant strides of this program during its first five-year funding cycle are presented.

Transcription Restart Establishes Chromatin Accessibility after DNA Replication.

DNA replication is highly disruptive to chromatin, leading to eviction of nucleosomes, RNA polymerase, and regulatory factors. When and how transcription resumes on DNA following DNA replication is unknown. Here we develop a replication-coupled assay for transposase-accessible chromatin (repli-ATAC-seq) to investigate active chromatin restoration post-replication in mouse embryonic stem cells. We find that nascent chromatin is inaccessible and transcriptionally silenced, with accessibility and RNA polymerase occupancy re-appearing within 30 minutes. Chromatin accessibility restores differentially genome wide, with super enhancers regaining transcription factor occupancy faster than other genomic features. We also identify opportunistic and transiently accessible chromatin within gene bodies after replication. Systematic inhibition of transcription shows that transcription restart is required to re-establish active chromatin states genome wide and resolve opportunistic binding events resulting from DNA replication. Collectively, this establishes a central role for transcription in overcoming the genome-wide chromatin inaccessibility imposed by DNA replication every cell division.