Characterization of Multicellular Niches Supporting Hematopoietic Stem Cells Within Distinct Zones.

Previous studies of hematopoietic stem cells (HSCs) primarily focused on single cell-based niche models, yielding fruitful but conflicting findings 1-5 . Here we report our investigation on the fetal liver (FL) as the primary fetal hematopoietic site using spatial transcriptomics. Our study reveals two distinct niches: the portal-vessel (PV) niche and the sinusoidal niche. The PV niche, composing N-cadherin (N-cad) Hi Pdgfrα + mesenchymal stromal cells (MSCs), endothelial cells (ECs), and N-cad Lo Albumin + hepatoblasts, maintains quiescent and multipotential FL-HSCs. Conversely, the sinusoidal niche, comprising ECs, hepatoblasts and hepatocytes, as well as potential macrophages and megakaryocytes, supports proliferative FL-HSCs biased towards myeloid lineages. Unlike prior reports on the role of Cxcl12, with its depletion from vessel-associated stromal cells leading to 80% of HSCs' reduction in the adult bone marrow (BM) 6,7 , depletion of Cxcl12 via Cdh2 CreERT (encoding N-cad) induces altered localization of HSCs from the PV to the sinusoidal niches, resulting in an increase of HSC number but with myeloid-bias. Similarly, we discovered that adult BM encompasses two niches within different zones, each composed of multi-cellular components: trabecular bone area (TBA, or metaphysis) supporting deep-quiescent HSCs, and central marrow (CM, or diaphysis) fostering heterogenous proliferative HSCs. This study transforms our understanding of niches by shifting from single cell-based to multicellular components within distinct zones, illuminating the intricate regulation of HSCs tailored to their different cycling states.

Plasticity of the mitotic spindle in response to karyotype variation.

Karyotypes, composed of chromosomes, must be accurately partitioned by the mitotic spindle for optimal cell health. However, it is unknown how underlying characteristics of karyotypes, such as chromosome number and size, govern the scaling of the mitotic spindle to ensure accurate chromosome segregation and cell proliferation. We utilize budding yeast strains engineered with fewer chromosomes, including just two "mega chromosomes," to study how spindle size and function are responsive to, and scaled by, karyotype. We determined that deletion and overexpression of spindle-related genes are detrimental to the growth of strains with two chromosomes, suggesting that mega chromosomes exert altered demands on the spindle. Using confocal microscopy, we demonstrate that cells with fewer but longer chromosomes have smaller spindle pole bodies, fewer microtubules, and longer spindles. Moreover, using electron tomography and confocal imaging, we observe elongated, bent anaphase spindles with fewer core microtubules in strains with mega chromosomes. Cells harboring mega chromosomes grow more slowly, are delayed in mitosis, and a subset struggle to complete chromosome segregation. We propose that the karyotype of the cell dictates the microtubule number, type, spindle pole body size, and spindle length, subsequently influencing the dynamics of mitosis, such as the rate of spindle elongation and the velocity of pole separation. Taken together, our results suggest that mitotic spindles are highly plastic ultrastructures that can accommodate and adjust to a variety of karyotypes, even within a species.

Pilar Cyst of the Dorsal Hand: A Rare Presentation of a Common Cyst.

Pilar cysts are common benign cysts of follicular origin that typically arise in areas of skin containing dense hair follicles such as the scalp. Here we describe a unique case of a young woman who was found to have a pilar cyst on the dorsum of her hand, a rather atypical location given the relative lack of pilosebaceous units. This case illustrates the variability in pilar cyst presentation and the importance of considering a pilar cyst in the differential diagnosis of a patient presenting with a tumor of the dorsal hand.

Intranasal Oxytocin for Obesity.

BACKGROUND: Accumulating preclinical and preliminary translational evidence shows that the hypothalamic peptide oxytocin reduces food intake, increases energy expenditure, and promotes weight loss. It is currently unknown whether oxytocin administration is effective in treating human obesity. METHODS: In this randomized, double-blind, placebo-controlled trial, we randomly assigned adults with obesity 1:1 (stratified by sex and obesity class) to receive intranasal oxytocin (24 IU) or placebo four times daily for 8 weeks. The primary end point was change in body weight (kg) from baseline to week 8. Key secondary end points included change in body composition (total fat mass [g], abdominal visceral adipose tissue [cm2], and liver fat fraction [proportion; range, 0 to 1; higher values indicate a higher proportion of fat]), and resting energy expenditure (kcal/day; adjusted for lean mass) from baseline to week 8 and caloric intake (kcal) at an experimental test meal from baseline to week 6. RESULTS: Sixty-one participants (54% women; mean age ± standard deviation, 33.6 ± 6.2 years; body-mass index [the weight in kilograms divided by the square of the height in meters], 36.9 ± 4.9) were randomly assigned. There was no difference in body weight change from baseline to week 8 between oxytocin and placebo groups (0.20 vs. 0.26 kg; P=0.934). Oxytocin (vs. placebo) was not associated with beneficial effects on body composition or resting energy expenditure from baseline to week 8 (total fat: difference [95% confidence interval], 196.0 g [-1036 to 1428]; visceral fat: 3.1 cm2 [-11.0 to 17.2]; liver fat: -0.01 [-0.03 to 0.01]; resting energy expenditure: -64.0 kcal/day [-129.3 to 1.4]). Oxytocin compared with placebo was associated with reduced caloric intake at the test meal (-31.4 vs. 120.6 kcal; difference [95% confidence interval], -152.0 kcal [-302.3 to -1.7]). There were no serious adverse events. Incidence and severity of adverse events did not differ between groups. CONCLUSIONS: In this randomized, placebo-controlled trial in adults with obesity, intranasal oxytocin administered four times daily for 8 weeks did not reduce body weight. (Funded by the National Institute of Diabetes and Digestive and Kidney Diseases and others; ClinicalTrials.gov number, NCT03043053.).

Defining a core configuration for human centromeres during mitosis.

The centromere components cohesin, CENP-A, and centromeric DNA are essential for biorientation of sister chromatids on the mitotic spindle and accurate sister chromatid segregation. Insight into the 3D organization of centromere components would help resolve how centromeres function on the mitotic spindle. We use ChIP-seq and super-resolution microscopy with single particle averaging to examine the geometry of essential centromeric components on human chromosomes. Both modalities suggest cohesin is enriched at pericentromeric DNA. CENP-A localizes to a subset of the α-satellite DNA, with clusters separated by ~562 nm and a perpendicular intervening ~190 nM wide axis of cohesin in metaphase chromosomes. Differently sized α-satellite arrays achieve a similar core structure. Here we present a working model for a common core configuration of essential centromeric components that includes CENP-A nucleosomes, α-satellite DNA and pericentromeric cohesion. This configuration helps reconcile how centromeres function and serves as a foundation to add components of the chromosome segregation machinery.

Bacterial Vaginosis in Postmenopausal Women.

Purpose of Review: Bacterial vaginosis (BV) is the most common vaginal infection worldwide, but most research has been conducted in premenopausal women. After menopause, endogenous estrogen production decreases, often leading to the genitourinary syndrome of menopause (GSM), characterized by vulvovaginal dryness and irritation. The estrogen-deficient postmenopausal state results in an elevated vaginal pH and depletion of vaginal lactobacilli. Use of traditional BV diagnostics (Amsel criteria, Nugent score) is difficult in post-menopausal women, especially those not on estrogen replacement therapy, as these methods were originally developed in premenopausal women. In this review, we discuss recent clinical data on BV in postmenopausal women, difficulties in diagnosis using traditional methods, the role of BV molecular diagnostics, and our current expert opinion for managing BV in this population. Recent Findings: BV prevalence has been found to range between 2%-57% among postmenopausal women per Amsel and Nugent criteria. This is likely an over-estimate of the true prevalence due to limitations in these criteria which were only validated in pre-menopausal women. Despite increasing diagnostic options for BV in recent years, including highly sensitive and specific BV nucleic acid amplification tests (NAATs), the physiologic changes of menopause and limited inclusion of postmenopausal women in clinical studies, diagnosis is difficult in this population. Recent studies utilizing 16s rRNA gene sequencing suggest that the vaginal microbiota of premenopausal and postmenopausal women is quite different, even if BV is not present. Data also suggest that obese postmenopausal women have significantly lower rates of BV compared to non-obese postmenopausal women, although further research is needed in this area. Multiple treatment options exist for vaginal atrophy and BV in this population. Summary: Data are limited regarding optimal diagnostic approaches for BV in postmenopausal women; BV NAATs and 16s rRNA gene sequencing may have a role for diagnosing BV in symptomatic women although further studies are needed. Menopausal women with characteristic vaginal symptoms and an elevated vaginal pH should be initially treated for estrogen deficiency prior to considering a diagnosis of BV; subsequent treatment for BV should be driven by symptoms.

Expression of Envelope Protein Encoded by Endogenous Retrovirus K102 in Rheumatoid Arthritis Neutrophils.

Many patients suffering from autoimmune diseases have autoantibodies against proteins encoded by genomic retroelements, suggesting that normal epigenetic silencing is insufficient to prevent the production of the encoded proteins for which immune tolerance appears to be limited. One such protein is the transmembrane envelope (Env) protein encoded by human endogenous retrovirus K (HERV-K). We reported recently that patients with rheumatoid arthritis (RA) have IgG autoantibodies that recognize Env. Here, we use RNA sequencing of RA neutrophils to analyze HERV-K expression and find that only two loci with an intact open-reading frame for Env, HERV-K102, and K108 are expressed, but only the former is increased in RA. In contrast, other immune cells express more K108 than K102. Patient autoantibodies recognized endogenously expressed Env in breast cancer cells and in RA neutrophils but not healthy controls. A monoclonal anti-Env antibody also detected Env on the surface of RA neutrophils but very little on the surface of other immune cells. We conclude that HERV-K102 is the locus that produces Env detectable on the surface of neutrophils in RA. The low levels of HERV-K108 transcripts may contribute only marginally to cell surface Env on neutrophils or other immune cells in some patients.

Morphological changes and two Nodal paralogs drive left-right asymmetry in the squamate veiled chameleon (C. calyptratus).

The ancestral mode of left-right (L-R) patterning involves cilia in the L-R organizer. However, the mechanisms regulating L-R patterning in non-avian reptiles remains an enigma, since most squamate embryos are undergoing organogenesis at oviposition. In contrast, veiled chameleon (Chamaeleo calyptratus) embryos are pre-gastrula at oviposition, making them an excellent organism for studying L-R patterning evolution. Here we show that veiled chameleon embryos lack motile cilia at the time of L-R asymmetry establishment. Thus, the loss of motile cilia in the L-R organizers is a synapomorphy of all reptiles. Furthermore, in contrast to avians, geckos and turtles, which have one Nodal gene, veiled chameleon exhibits expression of two paralogs of Nodal in the left lateral plate mesoderm, albeit in non-identical patterns. Using live imaging, we observed asymmetric morphological changes that precede, and likely trigger, asymmetric expression of the Nodal cascade. Thus, veiled chameleons are a new and unique model for studying the evolution of L-R patterning.

Split-GFP Complementation to Study the Nuclear Membrane Proteome Using Microscopy.

Defining the proteome of any given subcellular compartment provides insight into the activities and functions within that organelle. Understanding the composition of the nuclear envelope (NE) using traditional methods such as biochemical subcellular fractionation has been challenging due to the continuity of the NE and the endoplasmic reticulum. Here, we describe how split green fluorescent protein (split-GFP) was adapted to determine and define the NE proteome. This system is able to resolve protein topology and distinguish localization to the inner or outer nuclear membranes (INM or ONM).

A multifaceted stewardship intervention helps curb steroid overprescribing in hospitalized patients with acute exacerbations of COPD.

PURPOSE: Corticosteroid overprescribing is well documented in real-world practice. There is currently no evidence to guide best practices for steroid stewardship. The aim of this study was to assess the effects of a 3-part stewardship intervention strategy on inpatient steroid prescribing in patients with acute exacerbations of COPD (AECOPD). SUMMARY: Investigators implemented a 3-part stewardship initiative consisting of (1) an anonymous survey for providers on steroid prescribing in a simplified case of AECOPD, (2) face-to-face education and review of survey results, and (3) prospective audit and feedback from a clinical pharmacist. This was a quasi-experimental before-and-after study evaluating hospitalized adults diagnosed with AECOPD in two 12-month study periods before (April 2019-March 2020) and after (May 2020-April 2021) implementation. The primary outcome was mean inpatient steroid dosing. Secondary outcomes were duration of therapy, length of stay (LOS), 30-day readmissions, 30-day mortality, and incidence of hyperglycemia. Per power analysis, there were 27 patients per cohort. The interventions resulted in a significant reduction in prednisone equivalents during hospitalization: 118 mg vs 53 mg (P = 0.0003). This decrease was similar in ICU (160 mg vs 61 mg, P = 0.008) and non-ICU (102 mg vs 49 mg, P = 0.004) locations. There was no significant difference in duration of therapy (8 days vs 7 days, P = 0.44), length of stay (3.3 days vs 3.9 days, P = 0.21), 30-day mortality (4% vs 7%, P = 0.55), 30-day readmissions (15% vs 7%, P = 0.39), or rate of hyperglycemia (48% vs 44%, P = 0.78). CONCLUSION: A multifaceted stewardship intervention significantly reduced steroid dosing in hospitalized AECOPD patients. This reduction was not associated with known deleterious effects.

A distinct inner nuclear membrane proteome in Saccharomyces cerevisiae gametes.

The inner nuclear membrane (INM) proteome regulates gene expression, chromatin organization, and nuclear transport; however, it is poorly understood how changes in INM protein composition contribute to developmentally regulated processes, such as gametogenesis. We conducted a screen to determine how the INM proteome differs between mitotic cells and gametes. In addition, we used a strategy that allowed us to determine if spores synthesize their INM proteins de novo, rather than inheriting their INM proteins from the parental cell. This screen used a split-GFP complementation system, where we were able to compare the distribution of all C-terminally tagged transmembrane proteins in Saccharomyces cerevisiae in gametes to that of mitotic cells. Gametes contain a distinct INM proteome needed to complete gamete formation, including expression of genes linked to cell wall biosynthesis, lipid biosynthetic and metabolic pathways, protein degradation, and unknown functions. Based on the inheritance pattern, INM components are made de novo in the gametes. Whereas mitotic cells show a strong preference for proteins with small extraluminal domains, gametes do not exhibit this size preference likely due to the changes in the nuclear permeability barrier during gametogenesis. Taken together, our data provide evidence for INM changes during gametogenesis and shed light on mechanisms used to shape the INM proteome of spores.

Tumor-initiating stem cell shapes its microenvironment into an immunosuppressive barrier and pro-tumorigenic niche.

Tumor-initiating stem cells (TSCs) are critical for drug resistance and immune escape. However, the mutual regulations between TSC and tumor microenvironment (TME) remain unclear. Using DNA-label retaining, single-cell RNA sequencing (scRNA-seq), and other approaches, we investigated intestinal adenoma in response to chemoradiotherapy (CRT), thus identifying therapy-resistant TSCs (TrTSCs). We find bidirectional crosstalk between TSCs and TME using CellPhoneDB analysis. An intriguing finding is that TSCs shape TME into a landscape that favors TSCs for immunosuppression and propagation. Using adenoma-organoid co-cultures, niche-cell depletion, and lineaging tracing, we characterize a functional role of cyclooxygenase-2 (Cox-2)-dependent signaling, predominantly occurring between tumor-associated monocytes and macrophages (TAMMs) and TrTSCs. We show that TAMMs promote TrTSC proliferation through prostaglandin E2 (PGE2)-PTGER4(EP4) signaling, which enhances ß-catenin activity via AKT phosphorylation. Thus, our study shows that the bidirectional crosstalk between TrTSC and TME results in a pro-tumorigenic and immunosuppressive contexture.

Repeated evolution of circadian clock dysregulation in cavefish populations.

Circadian rhythms are nearly ubiquitous throughout nature, suggesting they are critical for survival in diverse environments. Organisms inhabiting largely arrhythmic environments, such as caves, offer a unique opportunity to study the evolution of circadian rhythms in response to changing ecological pressures. Populations of the Mexican tetra, Astyanax mexicanus, have repeatedly invaded caves from surface rivers, where individuals must contend with perpetual darkness, reduced food availability, and limited fluctuations in daily environmental cues. To investigate the molecular basis for evolved changes in circadian rhythms, we investigated rhythmic transcription across multiple independently-evolved cavefish populations. Our findings reveal that evolution in a cave environment has led to the repeated disruption of the endogenous biological clock, and its entrainment by light. The circadian transcriptome shows widespread reductions and losses of rhythmic transcription and changes to the timing of the activation/repression of core-transcriptional clock. In addition to dysregulation of the core clock, we find that rhythmic transcription of the melatonin regulator aanat2 and melatonin rhythms are disrupted in cavefish under darkness. Mutants of aanat2 and core clock gene rorca disrupt diurnal regulation of sleep in A. mexicanus, phenocopying circadian modulation of sleep and activity phenotypes of cave populations. Together, these findings reveal multiple independent mechanisms for loss of circadian rhythms in cavefish populations and provide a platform for studying how evolved changes in the biological clock can contribute to variation in sleep and circadian behavior.

Adults with Cerebral Palsy Require Ongoing Neurologic Care: A Systematic Review.

Cerebral palsy (CP) neurologic care and research efforts typically focus on children. However, most people with CP are adults. Adults with CP are at increased risk of new neurologic conditions, such as stroke and myelopathy, that require ongoing neurologic surveillance to distinguish them from baseline motor impairments. Neurologic factors could also contribute to the motor function decline, chronic pain, and chronic fatigue that are commonly experienced by adults with CP. Based on a systematic literature review, we suggest (1) guidelines for neurologic surveillance and neurologist referral and (2) clinical research questions regarding the evolving neurologic risks for adults with CP. ANN NEUROL 2021;89:860-871.

Gait features of dystonia in cerebral palsy.

AIM: To determine the features cited by motor phenotyping experts when identifying dystonia in people with cerebral palsy (CP). METHOD: Dystonia identification in CP, particularly when comorbid with spasticity, can be difficult. The dystonia diagnostic criterion standard remains subjective visual identification by expert consensus. For this qualitative study, we conducted an inductive thematic analysis of consensus-building discussions between three pediatric movement disorder physicians as they identified the presence or absence of dystonia in gait videos of 40 participants with spastic CP and periventricular leukomalacia. RESULTS: Unanimous consensus about the presence or absence of dystonia was achieved for 34 out of 40 videos. Two main themes were present during consensus-building discussions as videos were evaluated for dystonia: (1) unilateral leg or foot adduction that was variable over time, and (2) difficulty in identifying dystonia. Codes contributing to the first theme were more likely to be cited by a discussant when they felt dystonia was present (as opposed to absent) in a video (χ2 test, p=0.004). DISCUSSION: These results describe the gait features cited by experts during consensus-building discussion as they identify dystonia in ambulatory people with CP. Qualitative thematic analysis of these discussions could help codify the subjective process of dystonia diagnosis.

A computational method for immune repertoire mining that identifies novel binders from different clonotypes, demonstrated by identifying anti-pertussis toxoid antibodies.

Due to their shared genetic history, antibodies from the same clonotype often bind to the same epitope. This knowledge is used in immune repertoire mining, where known binders are used to search bulk sequencing repertoires to identify new binders. However, current computational methods cannot identify epitope convergence between antibodies from different clonotypes, limiting the sequence diversity of antigen-specific antibodies that can be identified. We describe how the antibody binding site, the paratope, can be used to cluster antibodies with common antigen reactivity from different clonotypes. Our method, paratyping, uses the predicted paratope to identify these novel cross clonotype matches. We experimentally validated our predictions on a pertussis toxoid dataset. Our results show that even the simplest abstraction of the antibody binding site, using only the length of the loops involved and predicted binding residues, is sufficient to group antigen-specific antibodies and provide additional information to conventional clonotype analysis. Abbreviations: BCR: B-cell receptor; CDR: complementarity-determining region; PTx: pertussis toxoid.

In Situ Hematopoietic Stem Cell Imaging.

Although immunohistochemistry of tissue sections has been the gold standard for analyzing tissue structure and cellular localization, this approach has significant shortcomings when it comes to analyzing complex and heterogeneous tissues such as the bone marrow with rare cells like hematopoietic stem cells (HSCs). Hence, studying rare cells and their relationship with the surrounding heterogenous microenvironment requires visualization of specifically labeled cells within large intact tissues in three dimensions. Here, we describe a whole mount sternal bone marrow imaging method which has enabled detailed quantitative and qualitative analysis of rare HSCs within the sternal tissue. The methodology is broadly applicable for examining the 3D architecture of niche cells in relation to HSCs.

Astrocyte deletion of α2-Na/K ATPase triggers episodic motor paralysis in mice via a metabolic pathway.

Familial hemiplegic migraine is an episodic neurological disorder characterized by transient sensory and motor symptoms and signs. Mutations of the ion pump α2-Na/K ATPase cause familial hemiplegic migraine, but the mechanisms by which α2-Na/K ATPase mutations lead to the migraine phenotype remain incompletely understood. Here, we show that mice in which α2-Na/K ATPase is conditionally deleted in astrocytes display episodic paralysis. Functional neuroimaging reveals that conditional α2-Na/K ATPase knockout triggers spontaneous cortical spreading depression events that are associated with EEG low voltage activity events, which correlate with transient motor impairment in these mice. Transcriptomic and metabolomic analyses show that α2-Na/K ATPase loss alters metabolic gene expression with consequent serine and glycine elevation in the brain. A serine- and glycine-free diet rescues the transient motor impairment in conditional α2-Na/K ATPase knockout mice. Together, our findings define a metabolic mechanism regulated by astrocytic α2-Na/K ATPase that triggers episodic motor paralysis in mice.