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The ability to identify T cells that recognize specific peptide antigens bound to major histocompatibility complex (MHC) molecules has enabled enumeration and molecular characterization of the lymphocytes responsible for cell-mediated immunity. Fluorophore-labeled peptide:MHC class I (p:MHCI) tetramers are well-established reagents for identifying antigen-specific CD8 + T cells by flow cytometry, but efforts to extend the approach to CD4 + T cells have been less successful, perhaps owing to lower binding strength between CD4 and MHC class II (MHCII) molecules. Here we show that p:MHCII tetramers engineered by directed evolution for enhanced CD4 binding outperform conventional tetramers for the detection of cognate T cells. Using the engineered tetramers, we identified about twice as many antigen-specific CD4 + T cells in mice immunized against multiple peptides than when using traditional tetramers. CD4 affinity-enhanced p:MHCII tetramers, therefore, allow direct sampling of antigen-specific CD4 + T cells that cannot be accessed with conventional p:MHCII tetramer technology. These new reagents could provide a deeper understanding of the T cell repertoire., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Humans have been dealing with health problems for millions of years. Normal health services need well-trained personnel and high-cost diagnostic tests, which forces patients to go to hospitals if medical treatment is required. To address this, prototype testing has been carried out into the wearable drug delivery health care perspectives. Researchers have devised a wide variety of formulations for the treatment of various diseases at home by performing real-time monitoring of different routes of drug administration such as ocular, transdermal, intraoral, intracochlear, and several more. A comprehensive review of the different types of wearable drug delivery systems with respect to their manufacturing, mechanism of action and specifications has been done. In the pharmaceutical context, these devices are technologically well-equipped interfaces for diverse physicochemical signals. Above mentioned information with a broader perspective has also been discussed in this article. Several wearable drug delivery systems have been introduced in the market in recent years. But a lot of testing needs to be conducted to address the numerous obstacles before the wearable devices are successfully launched in the market.

Purpose: Osteoarthritis (OA) is one of the most common causes of chronic pain and a leading cause of disability in the US. The objective of this study was to examine the clinical and economic burden of OA by pain severity., Patients and Methods: We used nationally representative survey data. Adults ≥18 years with self-reported physician-diagnosed OA and experiencing OA pain were included in the study. OA pain severity was measured using the Short Form McGill Pain Questionnaire Visual Analog Scale (SF-MPQ-VAS). Data were collected for demographics, clinical characteristics, health-related quality of life (HRQoL), productivity, OA treatment, adherence to pain medication, and healthcare resource utilization. Univariate analysis was performed to examine differences between respondents with moderate-to-severe OA pain vs those with mild OA pain., Results: Higher proportions of respondents with moderate-to-severe OA pain (n=3798) compared with mild OA pain (n=2038) were female (69.4% vs 57.3%), <65 years of age (54.8% vs 43.4%), and not employed (70.6% vs 64.5%). Respondents with moderate-to-severe OA pain experienced OA pain daily (80.8% vs 48.8%), were obese (53.0% vs 40.5%), had more comorbidities (sleep disturbance, insomnia, depression, and anxiety), and reported significantly poorer health status and HRQoL, and greater productivity and activity impairment (all P <0.05). Moderate-to-severe OA pain respondents were prescribed significantly more pain medications than mild OA pain respondents (41.0% vs 17.0%) and had higher adherence (75.9% vs 64.1%) yet were less satisfied with their pain medications (all P <0.001). Outpatient and emergency room visits, and hospitalizations in the 6 months prior to the survey were significantly higher in moderate-to-severe OA pain respondents vs those with mild OA pain (all P <0.05)., Conclusion: Patient and clinical burden was significantly greater in moderate-to-severe OA pain respondents vs mild OA pain respondents and may inform decision-making for appropriate resource allocation and effective management strategies that target specific subgroups., Competing Interests: The findings of this manuscript have been presented at the ISPOR 2020 Virtual Annual Meeting; May 18–20, 2020 and at PAINWeek 2020 Virtual Meeting; September 11–13, 2020. The following authors are employees of Pfizer with stock and/or stock options: PS, ST, CB, DM, and BE. RR is an employee and stockholder of Eli Lilly and Company. AS was financially compensated for providing editorial support in the drafting of this manuscript. The authors report no other conflicts of interest in this work., (© 2021 Schepman et al.)

Vascular calcification (VC) is one of the major causes of cardiovascular morbidity and mortality in patients with chronic kidney disease (CKD). VC is a complex process expressing similarity to bone metabolism in onset and progression. VC in CKD is promoted by various factors not limited to hyperphosphatemia, Ca/Pi imbalance, uremic toxins, chronic inflammation, oxidative stress, and activation of multiple signaling pathways in different cell types, including vascular smooth muscle cells (VSMCs), macrophages, and endothelial cells. In the current review, we provide an in-depth analysis of the various kinds of VC, the clinical significance and available therapies, significant contributions from multiple cell types, and the associated cellular and molecular mechanisms for the VC process in the setting of CKD. Thus, we seek to highlight the key factors and cell types driving the pathology of VC in CKD in order to assist in the identification of preventative, diagnostic, and therapeutic strategies for patients burdened with this disease.

A key challenge in single-cell RNA-sequencing (scRNA-seq) data analysis is batch effects that can obscure the biological signal of interest. Although there are various tools and methods to correct for batch effects, their performance can vary. Therefore, it is important to understand how batch effects manifest to adjust for them. Here, we systematically explore batch effects across various scRNA-seq datasets according to magnitude, cell type specificity, and complexity. We developed a cell-specific mixing score (cms) that quantifies mixing of cells from multiple batches. By considering distance distributions, the score is able to detect local batch bias as well as differentiate between unbalanced batches and systematic differences between cells of the same cell type. We compare metrics in scRNA-seq data using real and synthetic datasets and whereas these metrics target the same question and are used interchangeably, we find differences in scalability, sensitivity, and ability to handle differentially abundant cell types. We find that cell-specific metrics outperform cell type-specific and global metrics and recommend them for both method benchmarks and batch exploration., (© 2021 Lütge et al.)

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can infect any human host, but kidney transplant recipients (KTR) are considered more susceptible on the basis of previous experience with other viral infections. We evaluated rates of hospital complications between SARS-CoV-2-positive KTR and comparator groups., Methods: We extracted data from the electronic health record on patients who were hospitalized with SARS-CoV-2, testing at six hospitals from March 4 through September 9, 2020. We compared outcomes between SARS-CoV-2-positive KTR and controls: SARS-CoV-2-positive non-KTR, SARS-CoV-2-negative KTR, and SARS-CoV-2-negative non-KTR., Results: Of 31,540 inpatients, 3213 tested positive for SARS-CoV-2. There were 32 SARS-CoV-2-positive and 224 SARS-CoV-2-negative KTR. SARS-CoV-2-positive KTR had higher ferritin levels (1412; interquartile range, 748-1749 versus 553; interquartile range, 256-1035; P <0.01) compared with SARS-CoV-2-positive non-KTR. SARS-CoV-2-positive KTR had higher rates of ventilation (34% versus 14%, P <0.01; versus 9%, P <0.01; versus 5%, P <0.01), vasopressor use (41% versus 16%, P <0.01; versus 17%, P <0.01; versus 12%, P <0.01), and AKI (47% versus 15%, P <0.01; versus 23%, P <0.01; versus 10%, P <0.01) compared with SARS-CoV-2-positive non-KTR, SARS-CoV-2-negative KTR, and SARS-CoV-2-negative non-KTR, respectively. SARS-CoV-2-positive KTR continued to have increased odds of ventilation, vasopressor use, and AKI compared with SARS-CoV-2-positive non-KTR independent of Elixhauser score, Black race, and baseline eGFR. Mortality was not significantly different between SARS-CoV-2-positive KTR and non-KTR, but there was a notable trend toward higher mortality in SARS-CoV-2-positive KTR (25% versus 16%, P =0.15, respectively)., Conclusions: Hospitalized SARS-CoV-2-positive KTR had a high rate of mortality and hospital complications, such as requiring ventilation, vasopressor use, and AKI. Additionally, they had higher odds of hospital complications compared with SARS-CoV-2-positive non-KTR after adjusting for Elixhauser score, Black race, and baseline eGFR. Future studies with larger sample size of KTR are needed to validate our findings., Podcast: This article contains a podcast at https://dts.podtrac.com/redirect.mp3/www.asn-online.org/media/podcast/K360/2021&#95;03&#95;25&#95;KID0005652020.mp3., Competing Interests: E. Marin reports having consultancy agreements with Natera and Veloxis; reports being a scientific advisor or member of the Kidney360 Review Board; and reports having other interests/relationships as a member of the American Society of Nephrology and American Society of Transplantation. D. Moledina reports a patent to Systems and Methods to Diagnose Acute Interstitial Nephritis pending. F. Wilson reports having consultancy agreements with Translational Catalyst, LLC; reports having an ownership interest in Efference, LLC; reports being a scientific advisor or member of the Editorial Board of the American Journal of Kidney Disease and the Editorial Board of CJASN; and reports other interests/relationships as Board of Directors of Gaylord Health Care and Medical Commentator of Medscape. All remaining authors have nothing to disclose., (Copyright © 2021 by the American Society of Nephrology.)

Objective: To evaluate COVID-19 vaccine breakthrough infections among immunocompromised (IC) individuals., Methods: Individuals vaccinated with BNT162b2 were selected from the US HealthVerity database (10 December 2020  to  8   July 2021). COVID-19 vaccine breakthrough infections were examined in fully vaccinated (≥14 days after 2nd dose) IC individuals (IC cohort), 12 mutually exclusive IC condition groups, and a non-IC cohort. IC conditions were identified using an algorithm based on diagnosis codes and immunosuppressive (IS) medication usage., Results: Of 1,277,747 individuals ≥16 years of age who received 2 BNT162b2 doses, 225,796 (17.7%) were identified as IC (median age: 58 years; 56.3% female). The most prevalent IC conditions were solid malignancy (32.0%), kidney disease (19.5%), and rheumatologic/inflammatory conditions (16.7%). Among the fully vaccinated IC and non-IC cohorts, a total of 978 breakthrough infections were observed during the study period; 124 (12.7%) resulted in hospitalization and 2 (0.2%) were inpatient deaths. IC individuals accounted for 38.2% ( N  = 374) of all breakthrough infections, 59.7% ( N  = 74) of all hospitalizations, and 100% ( N  = 2) of inpatient deaths. The proportion with breakthrough infections was 3 times higher in the IC cohort compared to the non-IC cohort ( N  = 374 [0.18%] vs. N  = 604 [0.06%]; unadjusted incidence rates were 0.89 and 0.34 per 100 person-years, respectively. Organ transplant recipients had the highest incidence rate; those with >1 IC condition, antimetabolite usage, primary immunodeficiencies, and hematologic malignancies also had higher incidence rates compared to the overall IC cohort. Incidence rates in older (≥65 years old) IC individuals were generally higher versus younger IC individuals (<65)., Limitations: This retrospective analysis relied on coding accuracy and had limited capture of COVID-19 vaccine receipt., Conclusions: COVID-19 vaccine breakthrough infections are rare but are more common and severe in IC individuals. The findings from this large study support the FDA authorization and CDC recommendations to offer a 3rd vaccine dose to increase protection among IC individuals.

Objective: The aims of this study were to evaluate health outcomes and the economic burden of hospitalized COVID-19 patients in the United States., Methods: Hospitalized patients with a primary or secondary discharge diagnosis code for COVID-19 (ICD-10 code U07.1) from 1 April to 31 October 2020 were identified in the Premier Healthcare COVID-19 Database. Patient demographics, hospitalization characteristics, and concomitant medical conditions were assessed. Hospital length of stay (LOS), in-hospital mortality, hospital charges, and hospital costs were evaluated overall and stratified by age groups, insurance types, and 4 COVID-19 disease progression states based on intensive care unit (ICU) and invasive mechanical ventilation (IMV) usage., Results: Of the 173,942 hospitalized COVID-19 patients, the median age was 63 years, 51.0% were male, and 48.5% were covered by Medicare. The most prevalent concomitant medical conditions were cardiovascular disease (73.5%), hypertension (64.8%), diabetes (40.7%), obesity (27.0%), and chronic kidney disease (24.2%). Approximately one-fifth (21.9%) of the hospitalized COVID-19 patients were admitted to the ICU and 16.9% received IMV; most patients (73.6%) did not require ICU admission or IMV, and 12.4% required both. The median hospital LOS was 5 days, in-hospital mortality was 13.6%, median hospital charges were $43,986, and median hospital costs were $12,046. Hospital LOS and in-hospital mortality increased with ICU and/or IMV usage and age; hospital charges and costs increased with ICU and/or IMV usage. Patients with both ICU and IMV usage had the longest median hospital LOS (15 days), highest in-hospital mortality (53.8%), and highest hospital charges ($198,394) and hospital costs ($54,402)., Limitations: This retrospective administrative database analysis relied on coding accuracy and a subset of admissions with validated/reconciled hospital costs., Conclusions: This study summarizes the severe health outcomes and substantial hospital costs of hospitalized COVID-19 patients in the US. The findings support the urgent need for rapid implementation of effective interventions, including safe and efficacious vaccines.

Single-cell RNA sequencing (scRNA-seq) has become an empowering technology to profile the transcriptomes of individual cells on a large scale. Early analyses of differential expression have aimed at identifying differences between subpopulations to identify subpopulation markers. More generally, such methods compare expression levels across sets of cells, thus leading to cross-condition analyses. Given the emergence of replicated multi-condition scRNA-seq datasets, an area of increasing focus is making sample-level inferences, termed here as differential state analysis; however, it is not clear which statistical framework best handles this situation. Here, we surveyed methods to perform cross-condition differential state analyses, including cell-level mixed models and methods based on aggregated pseudobulk data. To evaluate method performance, we developed a flexible simulation that mimics multi-sample scRNA-seq data. We analyzed scRNA-seq data from mouse cortex cells to uncover subpopulation-specific responses to lipopolysaccharide treatment, and provide robust tools for multi-condition analysis within the muscat R package.

To understand how neural-immune-associated genes and pathways contribute to neurodegenerative disease pathophysiology, we performed a systematic functional genomic analysis in purified microglia and bulk tissue from mouse and human AD, FTD, and PSP. We uncover a complex temporal trajectory of microglial-immune pathways involving the type 1 interferon response associated with tau pathology in the early stages, followed by later signatures of partial immune suppression and, subsequently, the type 2 interferon response. We find that genetic risk for dementias shows disease-specific patterns of pathway enrichment. We identify drivers of two gene co-expression modules conserved from mouse to human, representing competing arms of microglial-immune activation (NAct) and suppression (NSupp) in neurodegeneration. We validate our findings by using chemogenetics, experimental perturbation data, and single-cell sequencing in post-mortem brains. Our results refine the understanding of stage- and disease-specific microglial responses, implicate microglial viral defense pathways in dementia pathophysiology, and highlight therapeutic windows., Competing Interests: Declaration of Interests D.H.G. has received research funding from Takeda Pharmaceuticals and Hoffman-LaRoche. D.M. is a full-time employee of F. Hoffmann-La Roche, Basel, Switzerland., (Published by Elsevier Inc.)

The high morbidity and mortality of COVID-19 in immunocompetent patients raises significant concern for immunosuppressed kidney transplant recipients (KTRs). This level of concern, both on the part of the KTRs and transplant professionals, is heightened by a lack of prior knowledge on how Severe Acute Respiratory Syndrome 2 virus (SARS-CoV-2) may manifest differently in immunosuppressed patients. Characterizing how KTRs may present differently than the general population would allow for more targeted and timely evaluation and treatment of KTRs with COVID-19 infection. METHODS: Without prior knowledge of how this virus would affect our transplant center's delivery of care to KTRs who are SARS-CoV-2 positive or patients under investigation, and in the setting of limited testing availability, we initiated a quality assurance and improvement project (QAPI) to track KTRs followed at our transplant center through the SARS-CoV-2 testing process. RESULTS: Of the 53 symptomatic patients, 20 (38%) tested positive for SARS-CoV-2 either on presentation to the emergency department or referral to a designated outpatient testing center. In addition, 16 (80%) of the 20 patients who tested positive required inpatient treatment. Intriguingly, patients with a history of polyoma BK viremia (BKV) had a higher incidence of testing positive for SARS-CoV-2 compared to patients without a history of BKV (80% and 28%, respectively; P = .002). The Positive Predictive Value and Likelihood ratio was 80% and 6.6 for this association, respectively. Among our KTRs tested, those receiving belatacept had a lower likelihood of testing positive for SARS-CoV-2. This finding approached, but did not achieve, statistical significance (P = .06)., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Microcystin-leucine arginine (MC-LR) is a potent liver toxin produced by freshwater cyanobacteria, also known as blue-green algae. While harmful algal blooms are increasing in frequency and severity worldwide, there is still no established method for the diagnosis and assessment of MC-LR induced liver damage. The guidelines for MC-LR safe exposure limits have been previously established based on healthy animal studies, however we have previously demonstrated that pre-existing non-alcoholic fatty liver disease (NAFLD) increases susceptiblity to the hepatotoxic effects of MC-LR. In this study, we sought to investigate the suitability of clinically used biomarkers of liver injury, specifically alanine aminotransferase (ALT) and alkaline phosphatase (ALP), as potential diagnostic tools for liver damage induced by chronic low dose administration of MC-LR in the setting of pre-existing NAFLD. In our Leprdb/J mouse model of NAFLD, we found that while MC-LR induced significant histopathologic damage in the setting of NAFLD, gene expression of ALT and ALP failed to increase with MC-LR exposure. Serum ALT and ALP also failed to increase with MC-LR exposure, except for a moderate increase in ALP with the highest dose of MC-LR used (100 μg/kg). In HepG2 human liver epithelial cells, we observed that increasing MC-LR exposure levels do not lead to an increase in ALT or ALP gene expression, intracellular enzyme activity, or extracellular activity, despite a significant increase in MC-LR induced cytotoxicity. These findings demonstrate that ALT and ALP may be unsuitable as diagnostic biomarkers for MC-LR induced liver damage., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Viral entry mechanisms for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are an important aspect of virulence. Proposed mechanisms involve host cell membrane-bound angiotensin-converting enzyme 2 (ACE2), type II transmembrane serine proteases (TTSPs), such as transmembrane serine protease isoform 2 (TMPRSS2), lysosomal endopeptidase Cathepsin L (CTSL), subtilisin-like proprotein peptidase furin (FURIN), and even potentially membrane bound heparan sulfate proteoglycans. The distribution and expression of many of these genes across cell types representing multiple organ systems in healthy individuals has recently been demonstrated. However, comorbidities such as diabetes and cardiovascular disease are highly prevalent in patients with Coronavirus Disease 2019 (COVID-19) and are associated with worse outcomes. Whether these conditions contribute directly to SARS-CoV-2 virulence remains unclear. Here, we show that the expression levels of ACE2, TMPRSS2 and other viral entry-related genes, as well as potential downstream effector genes such as bradykinin receptors, are modulated in the target organs of select disease states. In tissues, such as the heart, which normally express ACE2 but minimal TMPRSS2, we found that TMPRSS2 as well as other TTSPs are elevated in individuals with comorbidities compared to healthy individuals. Additionally, we found the increased expression of viral entry-related genes in the settings of hypertension, cancer, or smoking across target organ systems. Our results demonstrate that common comorbidities may contribute directly to SARS-CoV-2 virulence and we suggest new therapeutic targets to improve outcomes in vulnerable patient populations.

Water-lean CO 2 capture solvents show promise for more efficient and cost-effective CO 2 capture, although their long-term behavior in operation has yet to be well studied. New observations of extended structure solvent behavior show that some solvent formulations transform into a glass-like phase upon aging at operating temperatures after contact with CO 2 . The glassification of a solvent would be detrimental to a carbon-capture process due to plugging of infrastructure, introducing a critical need to decipher the underlying principles of this phenomenon to prevent it from happening. We present the first integrated theoretical and experimental study to characterize the nano-structure of metastable and glassy states of an archetypal single-component alkanolguanidine carbon-capture solvent and assess how minute changes in atomic-level interactions convert the solvent between metastable and glass-like states. Small-angle neutron scattering and neutron diffraction coupled with small- and wide-angle X-ray scattering analysis demonstrate that minute structural changes in solution precipitae reversible aggregation of zwitterionic alkylcarbonate clusters in solution. Our findings indicate that our test system, an alkanolguanidine, exhibits a first-order phase transition, similar to a glass transition, at approximately 40 °C-close to the operating absorption temperature for post-combustion CO 2 capture processes. We anticipate that these phenomena are not specific to this system, but are present in other classes of colvents as well. We discuss how molecular-level interactions can have vast implications for solvent-based carbon-capture technologies, concluding that fortunately in this case, glassification of water-lean solvents can be avoided as long as the solvent is run above its glass transition temperature.

Although CD4 + T cell memory is a critical component of adaptive immunity, antigen-specific CD4 + T cell recall responses to secondary infection have been inadequately studied. Here we examine the kinetics of the secondary response in an important immunological model, infection with attenuated Listeria monocytogenes (Lm). We identify CD4 + T cell subsets that preferentially expand during a secondary response and highlight the importance of prime-boost strategies in expanding and maintaining antigen-specific, tissue-resident memory CD4 + T cells. Following intravenous infection with an attenuated strain of Lm, we found that total antigen-specific CD4 + T cells responded more robustly in secondary compared with primary infection, reaching near-peak levels in secondary lymphoid organs (SLOs) and the liver by three days post-infection. During the secondary response, CD4 + T cells also contracted more quickly. Primary Lm infection generated two main classes of effector cells: Th1 cells that assist macrophages and T follicular helper (Tfh) cells that aid B cells in antibody production. We found that during the secondary response, a population of Ly6C + Tfh cells emerged in SLOs and was the basis for the skewing of this response to a Tfh phenotype. Deletion of T-bet in T cells precluded development of Ly6C + Tfh cells, but did not alter anti-Lm antibody responses. Moreover, during recall responses, CD49a + Th1 cells preferentially expanded and accumulated in the liver, achieving a new set point. Parabiosis experiments indicated that, in contrast to Tfh cells and most splenic Th1 cells, the majority of CD49a + Th1 cells in the liver were tissue resident. Overall, these data demonstrate a robust secondary CD4 + T cell response that differs in kinetics and composition from the primary response and provide insight into targets to enhance both peripheral and tissue-resident CD4 + T cell responses., (Copyright © 2020 Malhotra, Burrack, Jenkins and Frosch.)

Capturing carbon dioxide from post-combustion gas streams is an energy-intensive process that is required prior to either converting or sequestering CO 2 . Although a few commercial 1st and 2nd generation aqueous amine technologies have been proposed, the cost of capturing CO 2 with these technologies remains high. One approach to decrease costs of capture has been the development of water-lean solvents that aim to increase efficiency by reducing the water content in solution. Water-lean solvents, such as γ-aminopropyl aminosilicone/triethylene glycol (GAP/TEG), are promising technologies, with the potential to halve the parasitic load to a coal-fired power plant, albeit only if high solution viscosities and hydrolysis of the siloxane moieties can be mitigated. This study concerns an integrated multidisciplinary approach to overhaul the GAP/TEG solvent system at the molecular level to mitigate hydrolysis while also reducing viscosity. Cosolvents and diluents are found to have negligible effects on viscosity and are not needed. This finding allows for the design of single-component siloxane-free diamine derivatives with site-specific incorporation of selective chemical moieties for direct placement and orientation of hydrogen bonding to reduce viscosity. Ultimately, these new formulations are less susceptible to hydrolysis and exhibit up to a 98 % reduction in viscosity compared to the initial GAP/TEG formulation., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The thalamic reticular nucleus (TRN), the major source of thalamic inhibition, regulates thalamocortical interactions that are critical for sensory processing, attention and cognition 1-5 . TRN dysfunction has been linked to sensory abnormality, attention deficit and sleep disturbance across multiple neurodevelopmental disorders 6-9 . However, little is known about the organizational principles that underlie its divergent functions. Here we performed an integrative study linking single-cell molecular and electrophysiological features of the mouse TRN to connectivity and systems-level function. We found that cellular heterogeneity in the TRN is characterized by a transcriptomic gradient of two negatively correlated gene-expression profiles, each containing hundreds of genes. Neurons in the extremes of this transcriptomic gradient express mutually exclusive markers, exhibit core or shell-like anatomical structure and have distinct electrophysiological properties. The two TRN subpopulations make differential connections with the functionally distinct first-order and higher-order thalamic nuclei to form molecularly defined TRN-thalamus subnetworks. Selective perturbation of the two subnetworks in vivo revealed their differential role in regulating sleep. In sum, our study provides a comprehensive atlas of TRN neurons at single-cell resolution and links molecularly defined subnetworks to the functional organization of thalamocortical circuits.

Harmful algal blooms (HAB) have become a major health concern worldwide, not just to humans that consume and recreate on contaminated waters, but also to the fauna that inhabit the environments surrounding affected areas. HABs contain heterotrophic bacteria, cyanobacterial lipopolysaccharide, and cyanobacterial toxins such as microcystins, that can cause severe toxicity in many aquatic species as well as bioaccumulation within various organs. Thus, the possibility of trophic transference of this toxin through the food chain has potentially important health implications for other organisms in the related food web. While some species have developed adaptions to attenuate the toxic effects of HAB toxins, there are still numerous species that remain vulnerable, including Lithobates catesbeiana (American bullfrog) tadpoles. In the current study we demonstrate that acute, short-term exposure of tadpoles to HAB toxins containing 1 µg/L (1 nmol/L) of total microcystins for only 7 days results in significant liver and intestinal toxicity within tadpoles. Exposed tadpoles had increased intestinal diameter, decreased intestinal fold heights, and a constant number of intestinal folds, indicating pathological intestinal distension, similar to what is seen in various disease processes, such as toxic megacolon. HAB-toxin-exposed tadpoles also demonstrated hepatocyte hypertrophy with increased hepatocyte binucleation consistent with carcinogenic and oxidative processes within the liver. Both livers and intestines of HAB-toxin-exposed tadpoles demonstrated significant increases in protein carbonylation consistent with oxidative stress and damage. These findings demonstrate that short-term exposure to HAB toxins, including microcystins, can have significant adverse effects in amphibian populations. This acute, short-term toxicity highlights the need to evaluate the influence HAB toxins may have on other vulnerable species within the food web and how those may ultimately also impact human health.

Inflammatory Bowel Disease (IBD) is one of the most common gastrointestinal (GI) disorders around the world, and includes diagnoses such as Crohn's disease and ulcerative colitis. The etiology of IBD is influenced by genetic and environmental factors. One environmental perturbagen that is not well studied within the intestines is microcystin-leucine arginine (MC-LR), which is a toxin produced by cyanobacteria in freshwater environments around the world. We recently reported that MC-LR has limited effects within the intestines of healthy mice, yet interestingly has significant toxicity within the intestines of mice with pre-existing colitis induced by dextran sulfate sodium (DSS). MC-LR was found to prolong DSS-induced weight loss, prolong DSS-induced bloody stools, exacerbate DSS-induced colonic shortening, exacerbate DSS-induced colonic ulceration, and exacerbate DSS-induced inflammatory cytokine upregulation. In addition, we previously reported a significant increase in expression of the pro-inflammatory receptor CD40 in the colons of these mice, along with downstream products of CD40 activation, including plasminogen activator inhibitor-1 (PAI-1) and monocyte chemoattractant protein-1 (MCP-1). In the current study, we demonstrate that knocking out CD40 attenuates the effects of MC-LR in mice with pre-existing colitis by decreasing the severity of weight loss, allowing a full recovery in bloody stools, preventing the exacerbation of colonic shortening, preventing the exacerbation of colonic ulceration, and preventing the upregulation of the pro-inflammatory and pro-fibrotic cytokines IL-1β, MCP-1, and PAI-1. We also demonstrate the promising efficacy of a CD40 receptor blocking peptide to ameliorate the effects of MC-LR exposure in a proof-of-concept study. Our findings suggest for the first time that MC-LR acts through a CD40-dependent mechanism to exacerbate colitis.

Background Renal artery stenosis is a common cause of renal ischemia, contributing to the development of chronic kidney disease. To investigate the role of local CD40 expression in renal artery stenosis, Goldblatt 2-kidney 1-clip surgery was performed on hypertensive Dahl salt-sensitive rats (S rats) and genetically modified S rats in which CD40 function is abolished ( Cd40 mutant ). Methods and Results Four weeks following the 2-kidney 1-clip procedure, Cd40 mutant rats demonstrated significantly reduced blood pressure and renal fibrosis in the ischemic kidneys compared with S rat controls. Similarly, disruption of Cd40 resulted in reduced 24-hour urinary protein excretion in Cd40 mutant rats versus S rat controls (46.2±1.9 versus 118.4±5.3 mg/24 h; P <0.01), as well as protection from oxidative stress, as indicated by increased paraoxonase activity in Cd40 mutant rats versus S rat controls ( P <0.01). Ischemic kidneys from Cd40 mutant rats demonstrated a significant decrease in gene expression of the profibrotic mediator, plasminogen activator inhibitor-1 ( P <0.05), and the proinflammatory mediators, C-C motif chemokine ligand 19 ( P <0.01), C-X-C Motif Chemokine Ligand 9 ( P <0.01), and interleukin-6 receptor ( P <0.001), compared with S rat ischemic kidneys, as assessed by quantitative PCR assay. Reciprocal renal transplantation documented that CD40 exclusively expressed in the kidney contributes to ischemia-induced renal fibrosis. Furthermore, human CD40-knockout proximal tubule epithelial cells suggested that suppression of CD40 signaling significantly inhibited expression of proinflammatory and -fibrotic genes. Conclusions Taken together, our data suggest that activation of CD40 induces a significant proinflammatory and -fibrotic response and represents an attractive therapeutic target for treatment of ischemic renal disease.

Background Recent studies have highlighted a critical role for a group of natriuretic hormones, cardiotonic steroid (CTS), in mediating renal inflammation and fibrosis associated with volume expanded settings, such as chronic kidney disease. Immune cell adhesion is a critical step in the inflammatory response; however, little is currently understood about the potential regulatory role of CTS signaling in this setting. Herein, we tested the hypothesis that CTS signaling through Na + /K + -ATPase α-1 (NKA α-1) enhances immune cell recruitment and adhesion to renal epithelium that ultimately advance renal inflammation. Methods and Results We demonstrate that knockdown of the α-1 isoform of Na/K-ATPase causes a reduction in CTS-induced macrophage infiltration in renal tissue as well reduces the accumulation of immune cells in the peritoneal cavity in vivo. Next, using functional adhesion assay, we demonstrate that CTS-induced increases in the adhesion of macrophages to renal epithelial cells were significantly diminished after reduction of NKA α-1 in either macrophages or renal epithelial cells as well after inhibition of NKA α-1-Src signaling cascade with a specific peptide inhibitor, pNaKtide in vitro. Finally, CTS-induced expression of adhesion markers in both endothelial and immune cells was significantly inhibited in an NKA α-1-Src signaling dependent manner in vitro. Conclusions These findings suggest that CTS potentiates immune cell migration and adhesion to renal epithelium through an NKA α-1-dependent mechanism; our new findings suggest that pharmacological inhibition of this feed-forward loop may be useful in the treatment of renal inflammation associated with renal disease.

Corneal endothelial cell (CEnC) loss is often associated with blinding endothelial corneal dystrophies: dominantly inherited, common (5%) Fuchs endothelial corneal dystrophy (FECD) and recessive, rare congenital hereditary endothelial dystrophy (CHED). Mutations of SLC4A11, an abundant corneal solute transporter, cause CHED and some cases of FECD. The link between defective SLC4A11 solute transport function and CEnC loss is, however, unclear. Cell adhesion assays using SLC4A11-transfected HEK293 cells and primary human CEnC revealed that SLC4A11 promotes adhesion to components of Descemet's membrane (DM), the basement membrane layer to which CEnC bind. An antibody against SLC4A11 extracellular loop 3 (EL3) suppressed cell adhesion, identifying EL3 as the DM-binding site. Earlier studies showed that some SLC4A11 mutations cause FECD and CHED by impairing solute transport activity or cell surface trafficking. Without affecting these functions, FECD-causing mutations in SLC4A11-EL3 compromised cell adhesion capacity. In an energy-minimized SLC4A11-EL3 three-dimensional model, these mutations cluster and are buried within the EL3 structure. A GST fusion protein of SLC4A11-EL3 interacts with principal DM protein, COL8A2, as identified by mass spectrometry. Engineered SLC4A11-EL3-containing protein, STIC (SLC4A11-EL3 Transmembrane-GPA Integrated Chimera), promotes cell adhesion in transfected HEK293 cells and primary human CEnC, confirming the cell adhesion role of EL3. Taken together, the data suggest that SLC4A11 directly binds DM to serve as a cell adhesion molecule (CAM). These data further suggest that cell adhesion defects contribute to FECD and CHED pathology. Observations with STIC point toward a new therapeutic direction in these diseases: replacement of lost cell adhesion capacity., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The cornea, the eye's outermost layer, protects the eye from the environment. The cornea's innermost layer is an endothelium separating the stromal layer from the aqueous humor. A central role of the endothelium is to maintain stromal hydration state. Defects in maintaining this hydration can impair corneal clarity and thus visual acuity. Two endothelial corneal dystrophies, Fuchs Endothelial Corneal Dystrophy (FECD) and Congenital Hereditary Endothelial Dystrophy (CHED), are blinding corneal diseases with varied clinical presentation in patients across different age demographics. Recessive CHED with an early onset (typically age: 0-3 years) and dominantly inherited FECD with a late onset (age: 40-50 years) have similar phenotypes, although caused by defects in several different genes. A range of molecular mechanisms have been proposed to explain FECD and CHED pathology given the involvement of multiple causative genes. This critical review provides insight into the proposed molecular mechanisms underlying FECD and CHED pathology along with common pathways that may explain the link between the defective gene products and provide a new perspective to view these genetic blinding diseases.

Non-alcoholic fatty liver disease (NAFLD) is a growing global health concern. With a propensity to progress towards non-alcoholic steatohepatitis (NASH), cirrhosis, and hepatocellular carcinoma, NAFLD is an important link amongst a multitude of comorbidities including obesity, diabetes, and cardiovascular and kidney disease. As several in vivo models of hyperglycemia and NAFLD are employed to investigate the pathophysiology of this disease process, we aimed to characterize an in vitro model of hyperglycemia that was amenable to address molecular mechanisms and therapeutic targets at the cellular level. Utilizing hyperglycemic cell culturing conditions, we induced steatosis within a human hepatocyte cell line (HepG2 cells), as confirmed by electron microscopy. The deposition and accumulation of lipids within hyperglycemic HepG2 cells is significantly greater than in normoglycemic cells, as visualized and quantified by Nile red staining. Alanine aminotransferase (ALT) and alkaline phosphatase (ALP), diagnostic biomarkers for liver damage and disease, were found to be upregulated in hyperglycemic HepG2 cells as compared with normoglycemic cells. Suppression of CEACAM1, GLUT2, and PON1, and elevation of CD36, PCK1, and G6PK were also found to be characteristic in hyperglycemic HepG2 cells compared with normoglycemic cells, suggesting insulin resistance and NAFLD. These in vitro findings mirror the characteristic genetic and phenotypic profile seen in Leprdb/J mice, a well-established in vivo model of NAFLD. In conclusion, we characterize an in vitro model displaying several key genetic and phenotypic characteristics in common with NAFLD that may assist future studies in addressing the molecular mechanisms and therapeutic targets to combat this disease., Competing Interests: The authors have declared that no competing interests exist.

Rationale: Multiple clinical trials support the effectiveness of cardiac resynchronization therapy (CRT); however, optimal patient selection remains challenging due to substantial treatment heterogeneity among patients who meet the clinical practice guidelines., Objective: To apply machine learning to create an algorithm that predicts CRT outcome using electronic health record (EHR) data avaible before the procedure., Methods and Results: We applied machine learning and natural language processing to the EHR of 990 patients who received CRT at two academic hospitals between 2004-2015. The primary outcome was reduced CRT benefit, defined as <0% improvement in left ventricular ejection fraction (LVEF) 6-18 months post-procedure or death by 18 months. Data regarding demographics, laboratory values, medications, clinical characteristics, and past health services utilization were extracted from the EHR available before the CRT procedure. Bigrams (i.e., two-word sequences) were also extracted from the clinical notes using natural language processing. Patients accrued on average 75 clinical notes (SD, 29) before the procedure including data not captured anywhere else in the EHR. A machine learning model was built using 80% of the patient sample (training and validation dataset), and tested on a held-out 20% patient sample (test dataset). Among 990 patients receiving CRT the mean age was 71.6 (SD, 11.8), 78.1% were male, 87.2% non-Hispanic white, and the mean baseline LVEF was 24.8% (SD, 7.69). Out of 990 patients, 403 (40.7%) were identified as having a reduced benefit from the CRT device (<0% LVEF improvement in 25.2%, death by 18 months in 15.6%). The final model identified 26% of these patients at a positive predictive value of 79% (model performance: Fβ (β = 0.1): 77%; recall 0.26; precision 0.79; accuracy 0.65)., Conclusions: A machine learning model that leveraged readily available EHR data and clinical notes identified a subset of CRT patients who may not benefit from CRT before the procedure., Competing Interests: The authors have declared that no competing interests exists.

Background: The increased mutational burden for rare structural genomic variants in schizophrenia and other neurodevelopmental disorders has so far not yielded therapies targeting the biological effects of specific mutations. We identified two carriers (mother and son) of a triplication of the gene encoding glycine decarboxylase, GLDC, presumably resulting in reduced availability of the N-methyl-D-aspartate receptor coagonists glycine and D-serine and N-methyl-D-aspartate receptor hypofunction. Both carriers had a diagnosis of a psychotic disorder., Methods: We carried out two double-blind, placebo-controlled clinical trials of N-methyl-D-aspartate receptor augmentation of psychotropic drug treatment in these two individuals. Glycine was used in the first clinical trial, and D-cycloserine was used in the second one., Results: Glycine or D-cycloserine augmentation of psychotropic drug treatment each improved psychotic and mood symptoms in placebo-controlled trials., Conclusions: These results provide two independent proof-of-principle demonstrations of symptom relief by targeting a specific genotype and explicitly link an individual mutation to the pathophysiology of psychosis and treatment response., (Copyright © 2019 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.)

Microcystins are potent hepatotoxins that have become a global health concern in recent years. Their actions in at-risk populations with pre-existing liver disease is unknown. We tested the hypothesis that the No Observed Adverse Effect Level (NOAEL) of Microcystin-LR (MC-LR) established in healthy mice would cause exacerbation of hepatic injury in a murine model (Lepr db /J) of Non-alcoholic Fatty Liver Disease (NAFLD). Ten-week-old male Lepr db /J mice were gavaged with 50 μg/kg, 100 μg/kg MC-LR or vehicle every 48 h for 4 weeks ( n = 15-17 mice/group). Early mortality was observed in both the 50 μg/kg (1/17, 6%), and 100 μg/kg (3/17, 18%) MC-LR exposed mice. MC-LR exposure resulted in significant increases in circulating alkaline phosphatase levels, and histopathological markers of hepatic injury as well as significant upregulation of genes associated with hepatotoxicity, necrosis, nongenotoxic hepatocarcinogenicity and oxidative stress response. In addition, we observed exposure dependent changes in protein phosphorylation sites in pathways involved in inflammation, immune function, and response to oxidative stress. These results demonstrate that exposure to MC-LR at levels that are below the NOAEL established in healthy animals results in significant exacerbation of hepatic injury that is accompanied by genetic and phosphoproteomic dysregulation in key signaling pathways in the livers of NAFLD mice.

This work discusses the first set of rheometric measurements carried out on commercially accessible juvenile and aged skin models under large amplitude oscillatory shear deformations. The results were compared with those of native male whole human and dermis-only foreskin specimens, catering to a few ages from 0.5 to 68 years, including specimens from a 23-year-old male abdomen. At large strains, strain thinning was more pronounced for the dermis of the young skins and for their whole skin counterparts. An inverse qualitative tendency was observed for the adult skins and the skin models. This can be explained by the high dermal collagen compactness associated with an incomplete epidermal proliferation. The qualitative Lissajous plots as well as the quantitative dimensionless indices analyzed using the MITlaos software indicated predominant nonlinear intracycle elastic strain stiffening and viscous shear thinning for all the native specimens at the maximum deformation. For the full thickness models, we have evidence of structure collapse and yielding under similar conditions. The whole skin specimen from the 68-year-old male showed smaller age-dependent nonlinear elastic contributions than the dermis, which we relate to the epidermal degeneration taking place during aging. Regardless of the age group, the models manifested more pronounced intercycle and intracycle elastic nonlinearities, and their magnitudes were significantly larger. The nonlinear elastic trends will serve as advanced standards for understanding and delineating the mechanical limits of destructive and non-destructive deformations of such unique biomaterials., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Objectives: To assess baseline patient characteristics and identify factors associated with in-hospital mortality after ventricular assist device (VAD) placement., Methods: Cross-sectional study using the National Inpatient Sample database from January 2010 to December 2014. Analyses were performed with sample weights provided by the National Inpatient Sample, which are reported ± the standard error of the mean., Results: Weighted samples yielded 15,021 ± 1111 patients who received a VAD. The mean age at time of implantation was 56.6 years. Most recipients were white (59.9%) and male (75.0%). Among older patients, in-hospital mortality increased from 17.2% to 48.2% when 1 or more high-risk interventions (cardiac surgery, prolonged mechanical ventilation, hemodialysis, or extracorporeal membrane oxygenation) preceded VAD placement (P < .001). In comparison, in-hospital mortality in younger patients increased from 11.1% to 29.4% when 1 or more of these same procedures preceded VAD placement. The mortality difference associated with these procedures was 19% greater in older patients compared with younger patients (95% confidence interval [CI], 9%-28%). In-hospital mortality among VAD recipients was associated with age older than 65 years (odds ratio [OR], 1.76; 95% CI, 1.29-2.40), female sex (OR, 1.27; 95% CI, 0.97-1.67), and at least 1 high-risk intervention preceding VAD (OR, 5.52; 95% CI, 4.27-7.13)., Conclusions: Older patients who underwent 1 or more intensive treatments before VAD placement had a nearly 50% inpatient mortality and were unlikely to receive a cardiac transplantation. Refining patient selection might help better align VAD with those most likely to benefit., (Copyright © 2018 The American Association for Thoracic Surgery. Published by Elsevier Inc. All rights reserved.)

We have demonstrated that Na/K-ATPase acts as a receptor for reactive oxygen species (ROS), regulating renal Na + handling and blood pressure. TALLYHO/JngJ (TH) mice are believed to mimic the state of obesity in humans with a polygenic background of type 2 diabetes. This present work is to investigate the role of Na/K-ATPase signaling in TH mice, focusing on susceptibility to hypertension due to chronic excess salt ingestion. Age-matched male TH and the control C57BL/6J (B6) mice were fed either normal diet or high salt diet (HS: 2, 4, and 8% NaCl) to construct the renal function curve. Na/K-ATPase signaling including c-Src and ERK1/2 phosphorylation, as well as protein carbonylation (a commonly used marker for enhanced ROS production), were assessed in the kidney cortex tissues by Western blot. Urinary and plasma Na + levels were measured by flame photometry. When compared to B6 mice, TH mice developed salt-sensitive hypertension and responded to a high salt diet with a significant rise in systolic blood pressure indicative of a blunted pressure-natriuresis relationship. These findings were evidenced by a decrease in total and fractional Na + excretion and a right-shifted renal function curve with a reduced slope. This salt-sensitive hypertension correlated with changes in the Na/K-ATPase signaling. Specifically, Na/K-ATPase signaling was not able to be stimulated by HS due to the activated baseline protein carbonylation, phosphorylation of c-Src and ERK1/2. These findings support the emerging view that Na/K-ATPase signaling contributes to metabolic disease and suggest that malfunction of the Na/K-ATPase signaling may promote the development of salt-sensitive hypertension in obesity. The increased basal level of renal Na/K-ATPase-dependent redox signaling may be responsible for the development of salt-sensitive hypertension in polygenic obese TH mice.

The burden of cardiovascular disease and death in chronic kidney disease (CKD) outpaces that of the other diseases and is not adequately described by traditional risk factors alone. Diminished activity of paraoxonase (PON)-1 is associated with increased oxidant stress, a common feature underlying the pathogenesis of CKD. We aimed to assess the prognostic value of circulating PON-1 protein and PON lactonase activity on adverse clinical outcomes across various stages and etiologies of CKD. Circulating PON-1 protein levels and PON lactonase activity were measured simultaneously in patients with CKD as well as a cohort of apparently healthy non-CKD subjects. Both circulating PON-1 protein levels and PON lactonase activity were significantly lower in CKD patients compared to the non-CKD subjects. Similarly, across all stages of CKD, circulating PON-1 protein and PON lactonase activity were significantly lower in patients with CKD compared to the non-CKD controls. Circulating PON lactonase activity, but not protein levels, predicted future adverse clinical outcomes, even after adjustment for traditional risk factors. The combination of lower circulating protein levels and higher activity within the CKD subjects were associated with the best survival outcomes. These findings demonstrate that diminished circulating PON lactonase activity, but not protein levels, predicts higher risk of future adverse clinical outcomes in patients with CKD.

Two blinding corneal dystrophies, pediatric-onset congenital hereditary endothelial dystrophy (CHED) and some cases of late-onset Fuchs endothelial corneal dystrophy (FECD), are caused by SLC4A11 mutations. Three N-terminal SLC4A11 variants: v1, v2 and v3 are expressed in humans. We set out to determine which of these transcripts and what translated products, are present in corneal endothelium as these would be most relevant for CHED and FECD studies. Reverse transcription PCR (RT-PCR) and quantitative RT-PCR revealed only v2 and v3 mRNA in human cornea, but v2 was most abundant. Immunoblots probed with variant-specific antibodies revealed that v2 protein is about four times more abundant than v3 in human corneal endothelium. Bioinformatics and protein analysis using variant-specific antibodies revealed that second methionine in the open reading frame (M36) acts as translation initiation site on SLC4A11 v2 in human cornea. The v2 variants starting at M1 (v2-M1) and M36 (v2-M36) were indistinguishable in their cell surface trafficking and transport function (water flux). Structural homology models of v2-M36 and v3 suggest structural differences but their significance remains unclear. A combination of bioinformatics, RNA quantification and isoform-specific antibodies allows us to conclude that SLC4A11 variant 2 with start site M36 is predominant in corneal endothelium.

Cardiotonic steroids (CTSs) are NKA α-1 (Na + /K + -ATPase α-1) ligands that are increased in volume expanded states and associated with cardiac and renal diseases. Although initiation and resolution of inflammation is an important component of cellular injury and repair in renal disease, it is unknown whether CTS activation of NKA α-1 signaling in this setting regulates this inflammatory response. On this background, we hypothesized that CTS signaling through the NKA α-1-Src kinase complex promotes a proinflammatory response in renal epithelial and immune cells. First, we observed that the CTS telocinobufagin activated multiple proinflammatory cytokines/chemokines in renal epithelial cells, and these effects were attenuated after either NKA α-1 knockdown or with a specific inhibitor of the NKA α-1-Src kinase complex (pNaKtide). Similar findings were observed in immune cells, where we demonstrated that while telocinobufagin induced both oxidative burst and enhanced Nuclear factor kappa-light-chain-enhancer of activated B cells activation in macrophages ( P<0.05), the effects were abolished in NKA α-1 +/- macrophages or by pretreatment with pNaKtide or the Src inhibitor PP2 ( P<0.01). In a series of in vivo studies, we found that 5/6th partial nephrectomy induced significantly less oxidative stress in the remnant kidney of NKA α-1 +/- versus wild-type mice. Similarly, 5/6th partial nephrectomy yielded decreased levels of the urinary oxidative stress marker 8-Oxo-2'-deoxyguanosine in NKA α-1 +/- versus wild-type mice. Finally, we found that in vivo inhibition of the NKA α-1-Src kinase complex with pNaKtide significantly inhibited renal proinflammatory gene expression after 5/6th partial nephrectomy. These findings suggest that the NKA α-1-Src kinase complex plays a central role in regulating the renal inflammatory response induced by elevated CTS both in vitro and in vivo.

Inflammatory Bowel Disease (IBD) represents a collection of gastrointestinal disorders resulting from genetic and environmental factors. Microcystin-leucine arginine (MC-LR) is a toxin produced by cyanobacteria during algal blooms and demonstrates bioaccumulation in the intestinal tract following ingestion. Little is known about the impact of MC-LR ingestion in individuals with IBD. In this study, we sought to investigate MC-LR's effects in a dextran sulfate sodium (DSS)-induced colitis model. Mice were separated into four groups: (a) water only (control), (b) DSS followed by water (DSS), (c) water followed by MC-LR (MC-LR), and (d) DSS followed by MC-LR (DSS + MC-LR). DSS resulted in weight loss, splenomegaly, and severe colitis marked by transmural acute inflammation, ulceration, shortened colon length, and bloody stools. DSS + MC-LR mice experienced prolonged weight loss and bloody stools, increased ulceration of colonic mucosa, and shorter colon length as compared with DSS mice. DSS + MC-LR also resulted in greater increases in pro-inflammatory transcripts within colonic tissue (TNF-α, IL-1β, CD40, MCP-1) and the pro-fibrotic marker, PAI-1, as compared to DSS-only ingestion. These findings demonstrate that MC-LR exposure not only prolongs, but also worsens the severity of pre-existing colitis, strengthening evidence of MC-LR as an under-recognized environmental toxin in vulnerable populations, such as those with IBD.

Objective: Lumbar interbody spacers are widely used in lumbar spinal fusion. The goal of this study is to analyze the biomechanics of a lumbar interbody spacer (Clydesdale Spinal System, Medtronic Sofamor Danek, Memphis, Tennessee, USA) inserted via oblique lumbar interbody fusion (OLIF) or direct lateral interbody fusion (DLIF) approaches, with and without posterior cortical screw and rod (CSR) or pedicle screw and rod (PSR) instrumentation., Methods: Lumbar human cadaveric specimens (L2-L5) underwent nondestructive flexibility testing in intact and instrumented conditions at L3-L4, including OLIF or DLIF, with and without CSR or PSR., Results: OLIF alone significantly reduced range of motion (ROM) in flexion-extension (P = 0.005) but not during lateral bending or axial rotation (P ≥ 0.63). OLIF alone reduced laxity in the lax zone (LZ) during flexion-extension (P < 0.001) but did not affect the LZ during lateral bending or axial rotation (P ≥ 0.14). The stiff zone (SZ) was unaffected in all directions (P ≥ 0.88). OLIF plus posterior instrumentation (cortical, pedicle, or hybrid) reduced the mean ROM in all directions of loading but only significantly so with PSR during lateral bending (P = 0.004), without affecting the compressive stiffness (P > 0.20). The compressive stiffness with the OLIF device without any posterior instrumentation did not differ from that of the intact condition (P = 0.97). In terms of ROM, LZ, or SZ, there were no differences between OLIF and DLIF as standalone devices or OLIF and DLIF with posterior instrumentation (CSR or PSR) (P > 0.5)., Conclusions: OLIF alone significantly reduced mobility during flexion-extension while maintaining axial compressive stiffness compared with the intact condition. Adding posterior instrumentation to the interbody spacer increased the construct stability significantly, regardless of cage insertion trajectory or screw type., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Oligodendrocyte pathology is increasingly implicated in neurodegenerative diseases as oligodendrocytes both myelinate and provide metabolic support to axons. In multiple sclerosis (MS), demyelination in the central nervous system thus leads to neurodegeneration, but the severity of MS between patients is very variable. Disability does not correlate well with the extent of demyelination 1 , which suggests that other factors contribute to this variability. One such factor may be oligodendrocyte heterogeneity. Not all oligodendrocytes are the same-those from the mouse spinal cord inherently produce longer myelin sheaths than those from the cortex 2 , and single-cell analysis of the mouse central nervous system identified further differences 3,4 . However, the extent of human oligodendrocyte heterogeneity and its possible contribution to MS pathology remain unknown. Here we performed single-nucleus RNA sequencing from white matter areas of post-mortem human brain from patients with MS and from unaffected controls. We identified subclusters of oligodendroglia in control human white matter, some with similarities to mouse, and defined new markers for these cell states. Notably, some subclusters were underrepresented in MS tissue, whereas others were more prevalent. These differences in mature oligodendrocyte subclusters may indicate different functional states of oligodendrocytes in MS lesions. We found similar changes in normal-appearing white matter, showing that MS is a more diffuse disease than its focal demyelination suggests. Our findings of an altered oligodendroglial heterogeneity in MS may be important for understanding disease progression and developing therapeutic approaches.

The study reports first ever account of measurements of linear viscoelastic moduli under small amplitude oscillatory shear deformations, for commercially available juvenile and aged in vitro 3D reconstructed skin models. The results were compared with those of native male whole human and dermis-only foreskin samples, catering to a wide age group from 0.5 to 68 years, including samples from a 23-year-old male abdomen. In the strain sweep tests, the dermis of the juvenile/young age group assumed a higher intrinsic elastic modulus than the whole skin. A reverse qualitative trend was noted for the adult/aged age group. Confirmed by the histological examination of the stained cross-sections, this is attributed to the nascent epidermal differentiation and the high fiber density of dermal collagen. The oscillation frequency sweeps exposed a greater dependence of the elasticity on the frequency for the native male dermis foreskin samples as compared to the whole skins, irrespective of age. This is anticipated since the extremely structured epidermis confers higher resistance to the whole skins towards intracycle deformations compared to the dermis, thereby storing smaller elastic energy. The 3D skin models examined in this work exhibited a broader linear viscoelastic region, a larger viscoelasticity, and much higher dynamic moduli, compared to the native skin. The rheological trends are a significant addition to the literature and may be used as a reference for the design of next generation of scaffolds., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Most methods for statistical analysis of RNA-seq data take a matrix of abundance estimates for some type of genomic features as their input, and consequently the quality of any obtained results is directly dependent on the quality of these abundances. Here, we present the junction coverage compatibility score, which provides a way to evaluate the reliability of transcript-level abundance estimates and the accuracy of transcript annotation catalogs. It works by comparing the observed number of reads spanning each annotated splice junction in a genomic region to the predicted number of junction-spanning reads, inferred from the estimated transcript abundances and the genomic coordinates of the corresponding annotated transcripts. We show that although most genes show good agreement between the observed and predicted junction coverages, there is a small set of genes that do not. Genes with poor agreement are found regardless of the method used to estimate transcript abundances, and the corresponding transcript abundances should be treated with care in any downstream analyses., (© 2019 Soneson et al.)

Encapsulation of pharmaceuticals inside nanoporous materials is of increasing interest due to their possible applications as new generation therapeutics, theranostic platforms, or smart devices. Mesoporous silicas are leading materials to be used as nanohosts for pharmaceuticals. Further development of new generation of nanoscale therapeutics requires complete understanding of the complex host-guest interactions of organic molecules confined in nanosized chambers at different length scales. In this context, we present results showing control over formation and phase transition of nanosize crystals of model flexible pharmaceutical molecule tolbutamide confined inside 3.2 nm pores of the MCM-41 host. Using low loading levels (up to 30 wt %), we were able to stabilize the drug in highly dynamic amorphous/disordered state or direct the crystallization of the drug into highly metastable nanocrystalline form V of tolbutamide (at loading levels of 40 and 50 wt %), providing first experimental evidence for crystallization of pharmaceuticals inside the pores as narrow as 3.2 nm.

Fundamental questions regarding collagen biosynthesis, especially with respect to the molecular origins of homotrimeric versus heterotrimeric assembly, remain unanswered. Here, we demonstrate that the presence or absence of a single cysteine in type-I collagen's C-propeptide domain is a key factor governing the ability of a given collagen polypeptide to stably homotrimerize. We also identify a critical role for Ca 2+ in non-covalent collagen C-propeptide trimerization, thereby priming the protein for disulfide-mediated covalent immortalization. The resulting cysteine-based code for stable assembly provides a molecular model that can be used to predict, a priori, the identity of not just collagen homotrimers, but also naturally occurring 2:1 and 1:1:1 heterotrimers. Moreover, the code applies across all of the sequence-diverse fibrillar collagens. These results provide new insight into how evolution leverages disulfide networks to fine-tune protein assembly, and will inform the ongoing development of designer proteins that assemble into specific oligomeric forms.

Mass transfer coefficients of CO 2 are anomalously high in water-lean solvents as compared to aqueous amines. Such phenomena are intrinsic to the molecular and nanoscale structure of concentrated organic CO 2 capture solvents. To decipher the connections, we performed in situ liquid time-of-flight secondary ionization mass spectroscopy on a representative water-lean solvent, 1-((1,3-Dimethylimidazolidin-2-ylidene)amino)propan-2-ol (IPADM-2-BOL). Two-dimensional (2D) and three-dimensional (3D) chemical mapping of the solvent revealed that IPADM-2-BOL exhibited a heterogeneous molecular structure with regions of CO 2 -free solvent coexisting with clusters of zwitterionic carbonate ions. Chemical mapping were consistent with molecular dynamic simulation results, indicating CO 2 diffusing through pockets and channels of unreacted solvent. The observed mesoscopic structure promotes and enhances the diffusion and reactivity of CO 2 , likely prevalent in other water-lean solvents. This finding suggests that if the size, shape and orientation of the domains can be controlled, more efficient CO 2 capture solvents could be developed to enhance mass-transfer and uptake kinetics.

In 1972 Neal Bricker presented the "trade-off" hypothesis in which he detailed the role of physiological adaptation processes in mediating some of the pathophysiology associated with declines in renal function. In the late 1990's Xie and Askari published seminal studies indicating that the Na⁺/K⁺-ATPase (NKA) was not only an ion pump, but also a signal transducer that interacts with several signaling partners. Since this discovery, numerous studies from multiple laboratories have shown that the NKA is a central player in mediating some of these long-term "trade-offs" of the physiological adaptation processes which Bricker originally proposed in the 1970's. In fact, NKA ligands such as cardiotonic steroids (CTS), have been shown to signal through NKA, and consequently been implicated in mediating both adaptive and maladaptive responses to volume overload such as fibrosis and oxidative stress. In this review we will emphasize the role the NKA plays in this "trade-off" with respect to CTS signaling and its implication in inflammation and fibrosis in target organs including the heart, kidney, and vasculature. As inflammation and fibrosis exhibit key roles in the pathogenesis of a number of clinical disorders such as chronic kidney disease, heart failure, atherosclerosis, obesity, preeclampsia, and aging, this review will also highlight the role of newly discovered NKA signaling partners in mediating some of these conditions.

Advances in gene discovery for neurodevelopmental disorders have identified SCN2A dysfunction as a leading cause of infantile seizures, autism spectrum disorder, and intellectual disability. SCN2A encodes the neuronal sodium channel Na V 1.2. Functional assays demonstrate strong correlation between genotype and phenotype. This insight can help guide therapeutic decisions and raises the possibility that ligands that selectively enhance or diminish channel function may improve symptoms. The well-defined function of sodium channels makes SCN2A an important test case for investigating the neurobiology of neurodevelopmental disorders more generally. Here, we discuss the progress made, through the concerted efforts of a diverse group of academic and industry scientists as well as policy advocates, in understanding and treating SCN2A-related disorders., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Osmotic diuresis results from urine loss of large amounts of solutes distributed either in total body water or in the extracellular compartment. Replacement solutions should reflect the volume and monovalent cation (sodium and potassium) content of the fluid lost. Whereas the volume of the solutions used to replace losses that occurred prior to the diagnosis of osmotic diuresis is guided by the clinical picture, the composition of these solutions is predicated on serum sodium concentration and urinary sodium and potassium concentrations at presentation. Water loss is relatively greater than the loss of sodium plus potassium leading to hypernatremia which is seen routinely when the solute responsible for osmotic diuresis (e.g., urea) is distributed in body water. Solutes distributed in the extracellular compartment (e.g., glucose or mannitol) cause, in addition to osmotic diuresis, fluid transfer from the intracellular into the extracellular compartment with concomitant dilution of serum sodium. Serum sodium concentration corrected to euglycemia should be substituted for actual serum sodium concentration when calculating the composition of the replacement solutions in hyperglycemic patients. While the patient is monitored during treatment, the calculation of the volume and composition of the replacement solutions for losses of water, sodium and potassium from ongoing osmotic diuresis should be based directly on measurements of urine volume and urine sodium and potassium concentrations and not by means of any predictive formulas. Monitoring of clinical status, serum sodium, potassium, glucose, other relevant laboratory values, urine volume, and urine sodium and potassium concentrations during treatment of severe osmotic diuresis is of critical importance.

Background: Recent studies have highlighted a critical role for CD40 in the pathogenesis of renal injury and fibrosis. However, little is currently understood about the regulation of CD40 in this setting., Methods: We use novel Na/K-ATPase cell lines and inhibitors in order to demonstrate the regulatory function of Na/K-ATPase with regards to CD40 expression and function. We utilize 5/6 partial nephrectomy as well as direct infusion of a Na/K-ATPase ligand to demonstrate this mechanism exists in vivo., Results: We demonstrate that knockdown of the α1 isoform of Na/K-ATPase causes a reduction in CD40 while rescue of the α1 but not the α2 isoform restores CD40 expression in renal epithelial cells. Second, because the major functional difference between α1 and α2 is the ability of α1 to form a functional signaling complex with Src, we examined whether the Na/K-ATPase/Src complex is important for CD40 expression. We show that a gain-of-Src binding α2 mutant restores CD40 expression while loss-of-Src binding α1 reduces CD40 expression. Furthermore, loss of a functional Na/K-ATPase/Src complex also disrupts CD40 signaling. Importantly, we show that use of a specific Na/K-ATPase/Src complex antagonist, pNaKtide, can attenuate cardiotonic steroid (CTS)-induced induction of CD40 expression in vitro., Conclusions: Because the Na/K-ATPase/Src complex is also a key player in the pathogenesis of renal injury and fibrosis, our new findings suggest that Na/K-ATPase and CD40 may comprise a pro-fibrotic feed-forward loop in the kidney and that pharmacological inhibition of this loop may be useful in the treatment of renal fibrosis.

Small supernumerary marker chromosomes (sSMC) are chromosomal fragments difficult to characterize genomically. Here, we detail a proband with schizoaffective disorder and a mother with bipolar disorder with psychotic features who present with a marker chromosome that segregates with disease. We explored the architecture of this marker and investigated its temporal origin. Array comparative genomic hybridization (aCGH) analysis revealed three duplications and three triplications that spanned the short arm of chromosome 9, suggestive of a chromoanasynthesis-like event. Segregation of marker genotypes, phased using sSMC mosaicism in the mother, provided evidence that it was generated during a germline-level event in the proband's maternal grandmother. Whole-genome sequencing (WGS) was performed to resolve the structure and junctions of the chromosomal fragments, revealing further complexities. While structural variations have been previously associated with neuropsychiatric disorders and marker chromosomes, here we detail the precise architecture, human life-cycle genesis, and propose a DNA replicative/repair mechanism underlying formation., (© 2018 Wiley Periodicals, Inc.)

Introduction: Vaccination against influenza on an annual basis is widely recommended, yet recent studies suggest consecutive vaccination may reduce vaccine effectiveness (VE)., Purpose: To assess whether when examining the entirety of existing data consecutive influenza vaccination reduces VE compared to current season influenza vaccination., Data Sources: MEDLINE, EMBASE and the Cochrane Central Register of Controlled Trials (CENTRAL) from inception to April 26, 2017; citations of included studies., Study Selection: Randomized, controlled trials (RCTs) and observational studies of children, adults and/or the elderly that reported laboratory-confirmed influenza infection over 2 or more consecutive influenza seasons were eligible., Data Extraction: Data related to study characteristics, participant demographics, cases of influenza infection by vaccination group and risk of bias assessment was extracted in duplicate., Data Synthesis: Five RCTs involving 11,987 participants did not show a significant reduction in VE when participants vaccinated in two consecutive seasons (VE 71%, 95% CI 62-78%) were compared to those vaccinated in the current season (VE 58%, 95% CI 48-66%) (odds ratio [OR] 0.88, 95% CI 0.62-1.26, p = 0.49, I 2  = 39%). Twenty-eight observational studies involving 28,627 participants also did not show a reduction (VE for two consecutive seasons 41%, 95% CI 30-51% compared to VE for current season 47%, 95% CI 39-54%; OR 1.14, 95% CI 0.98-1.32, p = 0.09, I 2  = 63%). Results from subgroup analyses by influenza type/subtype, vaccine type, age, vaccine match and co-morbidity support these findings; however, dose-response results were inconsistent. Certainty in the evidence was assessed to be very low due to unexplained heterogeneity and imprecision., Limitations: The inclusion of studies with relatively small sample sizes and low event rates contributed to the imprecision of summary VE and OR estimates, which were based on unadjusted data., Conclusion: Available evidence does not support a reduction in VE with consecutive influenza vaccination, but the possibility of reduced effectiveness cannot be ruled out due to very low certainty in this evidence., Funding Source: CIHR Foundation Grant (PROSPERO: CRD42017059893)., (Copyright © 2018. Published by Elsevier Ltd.)

Chemokines are secreted proteins that regulate a range of processes in eukaryotic organisms. Interestingly, different chemokine receptors control distinct biological processes, and the same receptor can direct different cellular responses, but the basis for this phenomenon is not known. To understand this property of chemokine signaling, we examined the function of the chemokine receptors Cxcr4a, Cxcr4b, Ccr7, Ccr9 in the context of diverse processes in embryonic development in zebrafish. Our results reveal that the specific response to chemokine signaling is dictated by cell-type-specific chemokine receptor signal interpretation modules (CRIM) rather than by chemokine-receptor-specific signals. Thus, a generic signal provided by different receptors leads to discrete responses that depend on the specific identity of the cell that receives the signal. We present the implications of employing generic signals in different contexts such as gastrulation, axis specification and single-cell migration., Competing Interests: DM, JS, ER No competing interests declared, LS Reviewing editor, eLife, (© 2018, Malhotra et al.)