T cell-derived protein S engages TAM receptor signaling in dendritic cells to control the magnitude of the immune response.

Dendritic cell (DC) activation is essential for the induction of immune defense against pathogens, yet needs to be tightly controlled to avoid chronic inflammation and exaggerated immune responses. Here, we identify a mechanism of immune homeostasis by which adaptive immunity, once triggered, tempers DC activation and prevents overreactive immune responses. T cells, once activated, produced Protein S (Pros1) that signaled through TAM receptor tyrosine kinases in DCs to limit the magnitude of DC activation. Genetic ablation of Pros1 in mouse T cells led to increased expression of costimulatory molecules and cytokines in DCs and enhanced immune responses to T cell-dependent antigens, as well as increased colitis. Additionally, PROS1 was expressed in activated human T cells, and its ability to regulate DC activation was conserved. Our results identify a heretofore unrecognized, homeostatic negative feedback mechanism at the interface of adaptive and innate immunity that maintains the physiological magnitude of the immune response.

Tracking intracellular forces and mechanical property changes in mouse one-cell embryo development.

Cells comprise mechanically active matter that governs their functionality, but intracellular mechanics are difficult to study directly and are poorly understood. However, injected nanodevices open up opportunities to analyse intracellular mechanobiology. Here, we identify a programme of forces and changes to the cytoplasmic mechanical properties required for mouse embryo development from fertilization to the first cell division. Injected, fully internalized nanodevices responded to sperm decondensation and recondensation, and subsequent device behaviour suggested a model for pronuclear convergence based on a gradient of effective cytoplasmic stiffness. The nanodevices reported reduced cytoplasmic mechanical activity during chromosome alignment and indicated that cytoplasmic stiffening occurred during embryo elongation, followed by rapid cytoplasmic softening during cytokinesis (cell division). Forces greater than those inside muscle cells were detected within embryos. These results suggest that intracellular forces are part of a concerted programme that is necessary for development at the origin of a new embryonic life.

Transcriptome asymmetry within mouse zygotes but not between early embryonic sister blastomeres.

Transcriptome regionalization is an essential polarity determinant among metazoans, directing embryonic axis formation during normal development. Although conservation of this principle in mammals is assumed, recent evidence is conflicting and it is not known whether transcriptome asymmetries exist within unfertilized mammalian eggs or between the respective cleavage products of early embryonic divisions. We here address this by comparing transcriptome profiles of paired single cells and sub-cellular structures obtained microsurgically from mouse oocytes and totipotent embryos. Paired microsurgical spindle and remnant samples from unfertilized metaphase II oocytes possessed distinguishable profiles. Fertilization produces a totipotent 1-cell embryo (zygote) and associated spindle-enriched second polar body whose paired profiles also differed, reflecting spindle transcript enrichment. However, there was no programmed transcriptome asymmetry between sister cells within 2- or 3-cell embryos. Accordingly, there is transcriptome asymmetry within mouse oocytes, but not between the sister blastomeres of early embryos. This work places constraints on pre-patterning in mammals and provides documentation correlating potency changes and transcriptome partitioning at the single-cell level.

Full-term mouse development by abolishing Zn2+-dependent metaphase II arrest without Ca2+ release.

In vertebrates, a rise in intracellular free Ca(2+) (Ca(2+)(i)) levels during fertilization initiates second metaphase (mII) exit and the developmental programme. The Ca(2+) rise has long been considered to be crucial for development, but verifying this contribution would benefit from defining its role during fertilization. Here, we delineate the role of Ca(2+) release during mII exit in wild-type mouse eggs and show that it is dispensable for full-term development. Exit from mII can be induced by Zn(2+)-specific sequestration without Ca(2+) release, eliciting Cyclin B degradation in a manner dependent upon the proteasome pathway and intact microtubules, but not accompanied by degradation of the meiotic regulator Emi2. Parthenogenotes generated by Zn(2+) sequestration developed in vitro with normal expression of Ca(2+)-sensitive genes. Meiotic exit induced by either Ca(2+) oscillations or a single Ca(2+) rise in oocytes containing a signaling-deficient sperm resulted in comparable developmental rates. In the absence of Ca(2+) release, full-term development occurred approximately 50% less efficiently, but at readily detectable rates, with the birth of 27 offspring. These results show in intact mouse oocytes that Zn(2+) is essential for mII arrest and suggest that triggering meiotic exit is the sole indispensable developmental role of Ca(2+) signaling in mammalian fertilization.

Mouse Emi2 as a distinctive regulatory hub in second meiotic metaphase.

The oocytes of vertebrates are typically arrested at metaphase II (mII) by the cytostatic factor Emi2 until fertilization. Regulatory mechanisms in Xenopus Emi2 (xEmi2) are understood in detail but contrastingly little is known about the corresponding mechanisms in mammals. Here, we analyze Emi2 and its regulatory neighbours at the molecular level in intact mouse oocytes. Emi2, but not xEmi2, exhibited nuclear targeting. Unlike xEmi2, separable N- and C-terminal domains of mouse Emi2 modulated metaphase establishment and maintenance, respectively, through indirect and direct mechanisms. The C-terminal activity was mapped to the potential phosphorylation target Tx(5)SxS, a destruction box (D-box), a lattice of Zn(2+)-coordinating residues and an RL domain. The minimal region of Emi2 required for its cytostatic activity was mapped to a region containing these motifs, from residue 491 to the C terminus. The cytostatic factor Mos-MAPK promoted Emi2-dependent metaphase establishment, but Mos autonomously disappeared from meiotically competent mII oocytes. The N-terminal Plx1-interacting phosphodegron of xEmi2 was apparently shifted to within a minimal fragment (residues 51-300) of mouse Emi2 that also contained a calmodulin kinase II (CaMKII) phosphorylation motif and which was efficiently degraded during mII exit. Two equimolar CaMKII gamma isoform variants were present in mII oocytes, neither of which phosphorylated Emi2 in vitro, consistent with the involvement of additional factors. No evidence was found that calcineurin is required for mouse mII exit. These data support a model in which mammalian meiotic establishment, maintenance and exit converge upon a modular Emi2 hub via evolutionarily conserved and divergent mechanisms.

Meat-eating justification and relationship closeness with veg*n family, friends, and romantic partners.

A study of 190 omnivores examined their meat-eating justification (MEJ) beliefs and relationship closeness with veg*n friends, family members, and romantic partners; and how relationship closeness changes after veg*n diets are adopted. Denial and dissociation MEJs predicted lower closeness, whereas the hierarchical MEJ predicted higher closeness. Results also showed that relationship closeness significantly decreased for frequency and diversity of activities after adoption of veg*n diets. Closeness in terms of strength significantly increased after adoption of veg*n diets. A significant interaction was found between relationship type and time in which frequency of interactions decreased for friends and family after adoption of veg*n diets but did not change for romantic partners. These results suggest that only MEJs with moral considerations that elicit meat-related cognitive dissonance reduce relationship closeness after the adoption of veg*n diets; and that relationship closeness decreases after the adoption of veg*n diets only with friends or family members.

The initiation of mammalian embryonic transcription: to begin at the beginning.

Gamete (sperm and oocyte) genomes are transcriptionally silent until embryonic genome activation (EGA) following fertilization. EGA in humans had been thought to occur around the eight-cell stage, but recent findings suggest that it is triggered in one-cell embryos, by fertilization. Phosphorylation and other post-translational modifications during fertilization may instate transcriptionally favorable chromatin and activate oocyte-derived transcription factors (TFs) to initiate EGA. Expressed genes lay on cancer-associated pathways and their identities predict upregulation by MYC and other cancer-associated TFs. One interpretation of this is that the onset of EGA, and the somatic cell trajectory to cancer, are mechanistically related: cancer initiates epigenetically. We describe how fertilization might be linked to the initiation of EGA and involve distinctive processes recapitulated in cancer.

A program of successive gene expression in mouse one-cell embryos.

At the moment of union in fertilization, sperm and oocyte are transcriptionally silent. The ensuing onset of embryonic transcription (embryonic genome activation [EGA]) is critical for development, yet its timing and profile remain elusive in any vertebrate species. We here dissect transcription during EGA by high-resolution single-cell RNA sequencing of precisely synchronized mouse one-cell embryos. This reveals a program of embryonic gene expression (immediate EGA [iEGA]) initiating within 4 h of fertilization. Expression during iEGA produces canonically spliced transcripts, occurs substantially from the maternal genome, and is mostly downregulated at the two-cell stage. Transcribed genes predict regulation by transcription factors (TFs) associated with cancer, including c-Myc. Blocking c-Myc or other predicted regulatory TF activities disrupts iEGA and induces acute developmental arrest. These findings illuminate intracellular mechanisms that regulate the onset of mammalian development and hold promise for the study of cancer.

A Retrospective Study on the Impact of COVID-19 on Emergency General Surgery.

Background The coronavirus pandemic has caused global disruption to all aspects of life. This disturbance has been most notable in the medical world. Political, societal, medical, and behavioral alterations have forced emergency surgical practices to adapt. This study investigated the impact of coronavirus 2019 (COVID-19) at a busy surgical center. Methodology This is a retrospective observational study. Three study periods were analyzed: pre-COVID, first wave, and second wave. Data were collected on referrals, diagnoses, investigations, management pathways, outcomes, patient behavior, and consultant practice. A one-way analysis of variance (ANOVA test) was used for the analysis of parametric data and the Mann-Whitney U test for non-parametric data. Results Declining numbers of patients presented across the three periods. There was a severe disruption in performing emergency general surgeries during the first wave, propagated by alterations in clinical decision-making, as well as fluctuations in societal and patient behavior. Despite the effects of the second wave being significantly more profound in terms of hospitalization and COVID-related mortality, a paradoxical, gradual return to the norm was noted, which was seen in referral pathways, imaging decisions, and management strategies. Conclusion Our data is suggestive of society, both within and outside the medical sphere, adjusting to life with COVID-19.

Human embryonic genome activation initiates at the one-cell stage.

In human embryos, the initiation of transcription (embryonic genome activation [EGA]) occurs by the eight-cell stage, but its exact timing and profile are unclear. To address this, we profiled gene expression at depth in human metaphase II oocytes and bipronuclear (2PN) one-cell embryos. High-resolution single-cell RNA sequencing revealed previously inaccessible oocyte-to-embryo gene expression changes. This confirmed transcript depletion following fertilization (maternal RNA degradation) but also uncovered low-magnitude upregulation of hundreds of spliced transcripts. Gene expression analysis predicted embryonic processes including cell-cycle progression and chromosome maintenance as well as transcriptional activators that included cancer-associated gene regulators. Transcription was disrupted in abnormal monopronuclear (1PN) and tripronuclear (3PN) one-cell embryos. These findings indicate that human embryonic transcription initiates at the one-cell stage, sooner than previously thought. The pattern of gene upregulation promises to illuminate processes involved at the onset of human development, with implications for epigenetic inheritance, stem-cell-derived embryos, and cancer.

The impact of the COVID-19 pandemic on elective laparoscopic cholecystectomy: A retrospective Cohort study.

The coronavirus disease 2019 (COVID-19) pandemic had a significant impact on elective surgery for benign disease. We examined the effects of COVID-19 related delays on the outcomes of patients undergoing elective laparoscopic cholecystectomy (LC) in an upper gastrointestinal surgery unit in the UK. We have analysed data retrospectively of patients undergoing elective LC between 01/03/2019 to 01/05/2019 and 01/04/2021 to 11/06/2021. Demographics, waiting time to surgery, intra-operative details and outcome data were compared between the two cohorts. Indications for surgery were grouped as inflammatory (acute cholecystitis, gallstone pancreatitis, CBD stone with cholangitis) or non-inflammatory (biliary colic, gallbladder polyps, CBD stone without cholangitis). A p value of <0.05 was used for statistical significance. Out of the 159 patients included, 106 were operated pre-pandemic and 53 during the pandemic recovery phase. Both groups had similar age, gender, ASA-grades and BMI. In the pre-pandemic group, 68 (64.2%) were operated for a non-inflammatory pathology compared to 19 (35.8%) from the recovery phase cohort (p < 0.001). The waiting time to surgery was significantly higher amongst patients operated during the recovery phase (p = 0000.1). Less patients had complete cholecystectomy during the pandemic recovery phase (p = 0.04). There were no differences in intraoperative times and patient outcomes. These results demonstrate the impact of COVID-19 related delays to our cohort, however due to the retrospective nature of this study, the current results need to be backed up by higher evidence in order for strong recommendations to be made.

Bariatric Surgery as a Viable Treatment for Idiopathic Intracranial Hypertension: a Case Series and Review of Literature.

PURPOSE: Idiopathic intracranial hypertension is a significant cause of preventable blindness. Patients suffer from debilitating headaches, pulsatile tinnitus, nausea, vomiting, photophobia and radicular pain. At this rate, treatment cost will increase to 462.7 million pounds sterling annually by 2030. Weight loss is the only proven disease-modifying therapy for reversal of idiopathic intracranial hypertension. Bariatric surgery leads to superlative weight loss and reversal of related comorbidities. The case series and literature review aim to raise awareness of bariatric surgery as a safe and effective treatment modality for idiopathic intracranial hypertension. MATERIAL AND METHODS: The literature review comprises three systematic analysis and one randomised control trial which were identified after a PubMed search. In the case series, we have included four patients with a preoperative diagnosis of long-standing idiopathic intracranial hypertension. They were referred to our department for bariatric surgery by the neuro-ophthalmologist between January and December 2018. They were followed up for 2 years after bariatric surgery. RESULTS: All four patients were women with a mean age of 34 years. Mean body mass index reduced from 47.3 kg/m2 before surgery to 30 kg/m2 at the end of 2 years after surgery. They showed significant improvement or resolution in their symptoms related to idiopathic intracranial hypertension, and none of them required further cerebrospinal fluid pressure reducing procedures. CONCLUSION: Bariatric surgery is a safe and effective method of treating idiopathic intracranial hypertension. It is superior compared to medical management and cerebrospinal fluid pressure reducing procedures which have high rates of recurrence.

Use of Telemedicine to Expedite and Expand Care During COVID-19.

INTRODUCTION: The novel coronavirus disease 2019 (COVID-19) created challenges with access to care including increased burden on healthcare systems and potential exposure risks for vulnerable patients. To address these needs, Rush University Medical Center created a virtual, urgent care program specifically designed to address these challenges during the COVID-19 pandemic. METHODS: This was a retrospective study analyzing adult patients with COVID-19-related telemedicine visits performed between March 1-June 30, 2020. COVID-19-related telemedicine visits refer to those who used the "Concern for Coronavirus" module. We assessed the total number of telemedicine visits using this module, percentage with a subsequent emergency department (ED) visit within seven days, and outcomes (ie, hospitalization status, intubation, and death) of patients who presented to the ED for evaluation. Data are presented using descriptive statistics. RESULTS: A total of 2,974 adult patients accessed the program via the COVID-19 module over the four-month period. Of those, 142 patients (4.8%) had an ED visit within seven days. Only 14 patients (0.5%) required admission. One patient was intubated, and there were no deaths among the telemedicine population. CONCLUSION: The data suggests that telemedicine may be a safe and effective way to screen and treat patients with possible COVID-19, while reducing potential burdens on EDs.

Migalastat Tissue Distribution: Extrapolation From Mice to Humans Using Pharmacokinetic Modeling and Comparison With Agalsidase Beta Tissue Distribution in Mice.

Approved therapies for Fabry disease (FD) include migalastat, an oral pharmacological chaperone, and agalsidase beta and agalsidase alfa, 2 forms of enzyme replacement therapy. Broad tissue distribution may be beneficial for clinical efficacy in FD, which has severe manifestations in multiple organs. Here, migalastat and agalsidase beta biodistribution were assessed in mice and modeled using physiologically based pharmacokinetic (PBPK) analysis, and migalastat biodistribution was subsequently extrapolated to humans. In mice, migalastat concentration was highest in kidneys and the small intestine, 2 FD-relevant organs. Agalsidase beta was predominantly sequestered in the liver and spleen (organs unaffected in FD). PBPK modeling predicted that migalastat 123 mg every other day resulted in concentrations exceeding the in vitro half-maximal effective concentration in kidneys, small intestine, skin, heart, and liver in human subjects. However, extrapolation of mouse agalsidase beta concentrations to humans was unsuccessful. In conclusion, migalastat may distribute to tissues that are inaccessible to intravenous agalsidase beta in mice, and extrapolation of mouse migalastat concentrations to humans showed adequate tissue penetration, particularly in FD-relevant organs.

Genomic imprinting in mouse blastocysts is predominantly associated with H3K27me3.

In mammalian genomes, differentially methylated regions (DMRs) and histone marks including trimethylation of histone 3 lysine 27 (H3K27me3) at imprinted genes are asymmetrically inherited to control parentally-biased gene expression. However, neither parent-of-origin-specific transcription nor imprints have been comprehensively mapped at the blastocyst stage of preimplantation development. Here, we address this by integrating transcriptomic and epigenomic approaches in mouse preimplantation embryos. We find that seventy-one genes exhibit previously unreported parent-of-origin-specific expression in blastocysts (nBiX: novel blastocyst-imprinted expressed). Uniparental expression of nBiX genes disappears soon after implantation. Micro-whole-genome bisulfite sequencing (µWGBS) of individual uniparental blastocysts detects 859 DMRs. We further find that 16% of nBiX genes are associated with a DMR, whereas most are associated with parentally-biased H3K27me3, suggesting a role for Polycomb-mediated imprinting in blastocysts. nBiX genes are clustered: five clusters contained at least one published imprinted gene, and five clusters exclusively contained nBiX genes. These data suggest that early development undergoes a complex program of stage-specific imprinting involving different tiers of regulation.

CD133 identifies a human bone marrow stem/progenitor cell sub-population with a repertoire of secreted factors that protect against stroke.

The reparative properties of bone marrow stromal cells (BMSCs) have been attributed in part to the paracrine action of secreted factors. We isolated typical human BMSCs by plastic adherence and compared them with BMSC sub-populations isolated by magnetic-activated cell sorting against CD133 (CD133-derived BMSCs, CD133BMSCs) or CD271 [p75 low-affinity nerve growth factor receptor (p75LNGFR), p75BMSCs]. Microarray assays of expressed genes, and enzyme-linked immunosorbent assays (ELISAs) of selected growth factors and cytokines secreted under normoxic and hypoxic conditions demonstrated that the three transit-amplifying progenitor cell populations were distinct from one another. CD133BMSC-conditioned medium (CdM) was superior to p75BMSC CdM in protecting neural progenitor cells against cell death during growth factor/nutrient withdrawal. Intracardiac (arterial) administration of concentrated CD133BMSC CdM provided neuroprotection and significantly reduced cortical infarct volumes in mice following cerebral ischemia. In support of the paracrine hypothesis for BMSC action, intra-arterial infusion of CD133BMSC CdM provided significantly greater protection against stroke compared with the effects of CD133BMSC (cell) administration. CdM from CD133BMSCs also provided superior protection against stroke compared with that conferred by CdM from p75BMSCs or typically isolated BMSCs. CD133 identifies a sub-population of nonhematopoietic stem/progenitor cells from adult human bone marrow, and CD133BMSC CdM may provide neuroprotection for patients with stroke.

Transient Cell-to-Cell Signaling Before Mitosis in Cultures of Human Bone Marrow-Derived Mesenchymal Stem/Stromal Cells.

Some types of cells, if not all, that undergo signal exchanges in culture need to contact other cells for various reasons, such as cell-to-cell contact for growth inhibition. However, signal exchanges by cell-to-cell contact before proliferation have never been reported. Using time-lapse recording, we discovered the emergence of several astonishing cell-to-cell contact modes in bone marrow-derived mesenchymal stem/stromal cells (MSCs) before the cells divided. When the cells contacted with another, a huge temporary synapse-like structure formed for molecule exchanges; a cell-tissue particle was taken in by a recipient cell; two cell membranes formed infusion-like structure for a short time; and even a 20-µm long and 5-µm wide cell tail was grafted to another cell. A total of 87% of cells underwent cell-to-cell contact before dividing. After epidermal growth factor-green fluorescent protein (EGF-GFP) vectors were transfected into MSCs and the cells were cocultured with unmanipulated MSCs, the unmanipulated MSCs took in EGF-GFP particles from EGF-GFP expressed MSCs, immediately increased in mitogen genes, and then divided. These results suggest that cells which may lack signal molecules may need to obtain these molecules from other cells through various types of cell-to-cell contact, as mentioned above. Our study provided valuable information to better understand the behaviors of cell-to-cell contact and communication before mitosis.

Points to Consider: Best Practices to Identify Particle Entry Routes along the Manufacturing Process for Parenteral Formulations.

During the processes involved in pharmaceutical manufacturing, particulate matter may be introduced into a product from a variety of sources and at different points in the manufacturing process. Companies design quality at the beginning of the process to ensure against defects and strive to manufacture products that meet the pharmacopeial standard of being "practically/essentially free" of particles, which can be challenging, though necessary. As particulate matter recalls are predominantly associated with parenteral products, most companies employ a quality risk management program to identify critical parameters or conditions that could affect product quality or patient safety and incorporate systemic and procedural controls to mitigate or reduce the probability of their occurrence. Yet, determining where particulates are most likely to enter the process, what types of materials are most vulnerable, and how the size and number of particles might affect product quality can be very complex. Visual inspection and sampling of the manufactured drug product are designed to control the risk of particulate contamination; building prevention controls will ensure sustainability. This concept paper highlights the necessity of a more thorough understanding of the failure mechanisms that result in particle contamination across a range of products, such as elastomeric components and glass, and processes, such as the formulation and filling of injectables. The goal is to identify process steps within the end-to-end manufacturing process that are most critical to particle generation and entering of visible particles into the final drug product.LAY ABSTRACT: This concept paper highlights the necessity of a more thorough understanding of the failure mechanisms that result in particle contamination across a range of products, such as elastomeric components and glass, and processes, such as the formulation and filling of injectables. The goal is to identify process steps within the end-to-end manufacturing process that are most critical to particle generation and entering of visible particles into the final drug product.

Practice Innovation, Health Care Utilization and Costs in a Network of Federally Qualified Health Centers and Hospitals for Medicaid Enrollees.

State Medicaid programs need cost-effective strategies to provide high-quality care that is accessible to individuals with low incomes and limited resources. Integrated delivery systems have been formed to provide care across the continuum, but creating a shared vision for improving community health can be challenging. Medical Home Network was created as a network of primary care providers and hospital systems providing care to Medicaid enrollees, guided by the principles of egalitarian governance, practice-level care coordination, real-time electronic alerts, and pay-for-performance incentives. This analysis of health care utilization and costs included 1,189,195 Medicaid enrollees. After implementation of Medical Home Network, a risk-adjusted increase of $9.07 or 4.3% per member per month was found over the 2 years of implementation compared with an increase of $17.25 or 9.3% per member per month, before accounting for the cost of care management fees and other financial incentives, for Medicaid enrollees within the same geographic area with a primary care provider outside of Medical Home Network. After accounting for care coordination fees paid to providers, the net risk-adjusted cost reduction was $11.0 million.