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Abstract Measurement of cellular resting membrane potential (RMP) is important in understanding ion channels and their role in regulation of cell function across a wide range of cell types. However, methods available for the measurement of RMP (including patch clamp, microelectrodes, and potential-sensitive fluorophores) are expensive, slow, open to operator bias, and often result in cell destruction. We present non-contact, label-free membrane potential estimation which uses dielectrophoresis to determine the cytoplasm conductivity slope as a function of medium conductivity. By comparing this to patch clamp data available in the literature, we have demonstratet the accuracy of this approach using seven different cell types, including primary suspension cells (red blood cells, platelets), cultured suspension cells (THP-1), primary adherent cells (chondrocytes, human umbilical mesenchymal stem cells), and adherent (HeLa) and suspension (Jurkat) cancer cell lines. Analysis of the effect of ion channel inhibitors suggests the effects of pharmaceutical agents (TEA on HeLa; DMSO and neuraminidase on red blood cells) can also be measured. Comparison with published values of membrane potential suggest that the differences between our estimates and values recorded by patch clamp are accurate to within published margins of error. The method is low-cost, non-destructive, operator-independent and label-free, and has previously been shown to allow cells to be recovered after measurement.

Abstract Background This study characterized vessel wall imaging (VWI) features of Moyamoya disease (MMD) in a predominantly adult population at a North American center. Methods Consecutive patients with VWI were included. Twelve arterial segments were analyzed for wall thickening, degree and pattern of contrast enhancement, and remodeling. Results Overall, 286 segments were evaluated in 24 patients (mean age = 36.0 years [range = 1–58]). Of 172 affected segments, 163 (95%) demonstrated negative remodeling. Complete vessel wall obliteration was most frequent in the proximal M1 (17/48, 35%). Affected segments enhanced in 72/172 (42%) (n = 15 for grade II; n = 54 for concentric and n = 18 for eccentric); 20 of 24 (83%) patients had at least one enhancing segment. Both enhancing and non-enhancing segments were present in 19/20 (95%) patients. Vessel wall enhancement was most common in the proximal segments and correlated to the degree of stenosis (p

Objective: To retrospectively evaluate the safety and efficacy of percutaneous image-guided mediastinal mass core-needle biopsy. Patients and Methods: Retrospective review of an institutionally maintained biopsy registry identified 337 computed tomography– or ultrasound-guided percutaneous mediastinal mass core needle biopsies between October 2002 and August 2017 in a single quaternary referral center. Mean patient age was 51 (range, 18 to 93) years. Procedural techniques, anticoagulation/antiplatelet therapy, and tumor anatomical characteristics were reviewed. Classification and gradation of complications was based on the Clavien-Dindo system. Diagnostic yield was defined as the ratio of diagnostic biopsy to all biopsies performed. Results: Mean tumor size was 59.2 (range, 10 to 180) mm with 89.9% (n=303) of lesions located in the prevascular (anterior) mediastinum. There was a single major complication (0.3%) of a symptomatic pneumothorax requiring intervention. There were seven (2.1%) minor complications, including three bleeding complications. A transpleural approach was the only variable associated with an increased complication rate (P

Abstract Compared to controlled laboratory conditions, plant growth in the field is rarely optimal since it is frequently challenged by large fluctuations in light and temperature which lower the efficiency of photosynthesis and lead to photo‐oxidative stress. Plants grown under natural conditions therefore place an increased onus on the regulatory mechanisms that protect and repair the delicate photosynthetic machinery. Yet, the exact changes in thylakoid proteome composition which allow plants to acclimate to the natural environment remain largely unexplored. Here, we use quantitative label‐free proteomics to demonstrate that field‐grown Arabidopsis plants incorporate aspects of both the low and high light acclimation strategies previously observed in laboratory‐grown plants. Field plants showed increases in the relative abundance of ATP synthase, cytochrome b6f, ferredoxin‐NADP+ reductases (FNR1 and FNR2) and their membrane tethers TIC62 and TROL, thylakoid architecture proteins CURT1A, CURT1B, RIQ1, and RIQ2, the minor monomeric antenna complex CP29.3, rapidly‐relaxing non‐photochemical quenching (qE)‐related proteins PSBS and VDE, the photosystem II (PSII) repair machinery and the cyclic electron transfer complexes NDH, PGRL1B, and PGR5, in addition to decreases in the amounts of LHCII trimers composed of LHCB1.1, LHCB1.2, LHCB1.4, and LHCB2 proteins and CP29.2, all features typical of a laboratory high light acclimation response. Conversely, field plants also showed increases in the abundance of light harvesting proteins LHCB1.3 and CP29.1, zeaxanthin epoxidase (ZEP) and the slowly‐relaxing non‐photochemical quenching (qI)‐related protein LCNP, changes previously associated with a laboratory low light acclimation response. Field plants also showed distinct changes to the proteome including the appearance of stress‐related proteins ELIP1 and ELIP2 and changes to proteins that are largely invariant under laboratory conditions such as state transition related proteins STN7 and TAP38. We discuss the significance of these alterations in the thylakoid proteome considering the unique set of challenges faced by plants growing under natural conditions.

Carotenoids are crucial photosynthetic pigments utilized for light harvesting, energy transfer, and photoprotection. Although most of the enzymes involved in carotenoid biosynthesis in chlorophototrophs are known, some are yet to be identified or fully characterized in certain organisms. A recently characterized enzyme in oxygenic phototrophs is 15-cis-zeta(ζ)-carotene isomerase (Z-ISO), which catalyzes the cis-to-trans isomerization of the central 15–15′ cis double bond in 9,15,9′-tri-cis-ζ-carotene to produce 9,9′-di-cis-ζ-carotene during the four-step conversion of phytoene to lycopene. Z-ISO is a heme B-containing enzyme best studied in angiosperms. Homologs of Z-ISO are present in organisms that use the multi-enzyme poly-cis phytoene desaturation pathway, including algae and cyanobacteria, but appear to be absent in green bacteria. Here we confirm the identity of Z-ISO in the model unicellular cyanobacterium Synechocystis sp. PCC 6803 by showing that the protein encoded by the slr1599 open reading frame has ζ-carotene isomerase activity when produced in Escherichia coli. A Synechocystis Δslr1599 mutant synthesizes a normal quota of carotenoids when grown under illumination, where the photolabile 15–15′ cis double bond of 9,15,9′-tri-cis-ζ-carotene is isomerized by light, but accumulates this intermediate and fails to produce ‘mature’ carotenoid species during light-activated heterotrophic growth, demonstrating the requirement of Z-ISO for carotenoid biosynthesis during periods of darkness. In the absence of a structure of Z-ISO, we analyze AlphaFold models of the Synechocystis, Zea mays (maize), and Arabidopsis thaliana enzymes, identifying putative protein ligands for the heme B cofactor and the substrate-binding site.

Bacterial C-type haem-copper oxidases in the cbb3 family are widespread in microaerophiles, which exploit their high oxygen-binding affinity for growth in microoxic niches. In microaerophilic pathogens, C-type oxidases can be essential for infection, yet little is known about their biogenesis compared to model bacteria. Here, we have identified genes involved in cbb3-oxidase (Cco) assembly and activity in the Gram-negative pathogen Campylobacter jejuni, the commonest cause of human food-borne bacterial gastroenteritis. Several genes of unknown function downstream of the oxidase structural genes ccoNOQP were shown to be essential (cj1483c and cj1486c) or important (cj1484c and cj1485c) for Cco activity; Cj1483 is a CcoH homologue, but Cj1484 (designated CcoZ) has structural similarity to MSMEG_4692, involved in Qcr-oxidase supercomplex formation in Mycobacterium smegmatis. Blue-native polyacrylamide gel electrophoresis of detergent solubilised membranes revealed three major bands, one of which contained CcoZ along with Qcr and oxidase subunits. Deletion of putative copper trafficking genes ccoI (cj1155c) and ccoS (cj1154c) abolished Cco activity, which was partially restored by addition of copper during growth, while inactivation of cj0369c encoding a CcoG homologue led to a partial reduction in Cco activity. Deletion of an operon encoding PCuAC (Cj0909) and Sco (Cj0911) periplasmic copper chaperone homologues reduced Cco activity, which was partially restored in the cj0911 mutant by exogenous copper. Phenotypic analyses of gene deletions in the cj1161c–1166c cluster, encoding several genes involved in intracellular metal homeostasis, showed that inactivation of copA (cj1161c), or copZ (cj1162c) led to both elevated intracellular Cu and reduced Cco activity, effects exacerbated at high external Cu. Our work has therefore identified (i) additional Cco subunits, (ii) a previously uncharacterized set of genes linking copper trafficking and Cco activity, and (iii) connections with Cu homeostasis in this important pathogen.

Background Mental health after spontaneous coronary artery dissection (SCAD), a cause of myocardial infarction in young women, remains largely unexplored. We assessed the prevalence and severity of psychiatric symptoms after SCAD. Methods and Results Individuals with confirmed SCAD who consented to the Mayo Clinic “Virtual” Multicenter SCAD Registry were sent the Posttraumatic Stress Disorder Diagnostic Scale for Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition, Patient Health Questionnaire‐9, Generalized Anxiety Disorder‐7, Anxiety Sensitivity Index, Connor‐Davidson Resilience Scale, 36‐Item Short‐Form Health Survey, and an SCAD‐specific questionnaire. Among 782 patients contacted, 512 surveys were returned. Most respondents were women (97.5%), with median age at time of SCAD and survey completion of 47 and 52 years, respectively. Eighty‐two percent had at least one trauma, with mild or more posttraumatic stress disorder symptoms in 28%. Symptoms of anxiety and depression were observed in 41% and 32%, respectively. On multivariable analysis, those of younger age at first SCAD and low resiliency scored higher on measures of trauma, anxiety, and depression. Those with higher anxiety sensitivity had more severe anxiety and posttraumatic stress disorder symptoms. Emotional and social quality of life was higher in those with high resiliency scores. Time from SCAD event to survey completion was associated with lower Generalized Anxiety Disorder‐7 score severity. Conclusions Survivors of SCAD have significant rates of posttraumatic stress disorder, depression, and anxiety, which are associated with lower quality of life specifically among those with lower resiliency. Given the prevalence and potential impact, screening and treatment for the psychological distress is advised. Behavioral interventions targeted toward resiliency training may be beneficial for this patient population.

Abstract Background Microalgae accumulate lipids when exposed to stressful conditions such as nutrient limitation that can be used to generate biofuels. Nitrogen limitation or deprivation is a strategy widely employed to elicit this response. However, this strategy is associated with a reduction in the microalgal growth, leading to overall poor lipid productivities. Here, we investigated the combined effect of a reduced source of nitrogen (ammonium) and super-saturating light intensities on the growth and induction of lipid accumulation in two model but diverse microalgal species, Phaeodactylum tricornutum and Nannochloropsis oceanica. We hypothesized that the lower energy cost of assimilating ammonium would allow the organisms to use more reductant power for lipid biosynthesis without compromising growth and that this would be further stimulated by the effect of high light (1000 µmol m−2 s−1) stress. We studied the changes in growth and physiology of both species when grown in culture media that either contained nitrate or ammonium as the nitrogen source, and an additional medium that contained ammonium with tungsten in place of molybdenum and compared this with growth in media without nitrogen. We focused our investigation on the early stages of exposure to the treatments to correspond to events relevant to induction of lipid accumulation in these two species. Results At super-saturating light intensities, lipid productivity in P. tricornutum increased twofold when grown in ammonium compared to nitrogen free medium that increased further when tungsten was present in the medium in place of molybdenum. Conversely, N. oceanica growth and physiology was not compromised by the high light intensities used, and the use of ammonium had a negative effect on the lipid productivity, which was even more marked when tungsten was present. Conclusions Whilst the use of ammonium and super-saturating light intensities in P. tricornutum was revealed to be a good strategy for increasing lipid biosynthesis, no changes in the lipid productivity of N. oceanica were observed, under these conditions. Both results provide relevant direction for the better design of processes to produce biofuels in microalgae by manipulating growth conditions without the need to subject them to genetic engineering manipulation.

Culturally responsive assessments have been proposed as potential tools to ensure equity and fairness for examinees from all backgrounds including those from traditionally underserved or minoritized groups. However, these assessments are relatively new and, with few exceptions, are yet to be implemented in large scale. Consequently, there is a lack of guidance on how one can compute comparable scores on various versions of these assessments. In this paper, the multigroup multidimensional Rasch model is repurposed for modeling data originating from various versions of a culturally responsive assessment and for analyzing such data to compute comparable scores. Two simulation studies are performed to evaluate the performance of the model for data simulated from hypothetical culturally responsive assessments and to find the conditions under which the computed scores are accurate. Recommendations are made for measurement practitioners interested in culturally responsive assessments.

We investigated the effects of a major revision of the differential and integral calculus curriculum, the primary goal of which was to improve STEM retention. The revamped curriculum has greater emphasis on the power of computing to help visualize patterns and gain insights to better prepare students for STEM majors, and less emphasis on traditional and theoretical calculus content, such as the limit definition of the derivative. We investigated a comparison between 338 students who had taken the traditional sequence and 328 who had taken the revamped sequence during the Fall 2020 and Spring 2021 semesters. STEM retention, i.e., following through with the initial intent to major in a STEM field, was enhanced by 7% in the revamped group. STEM majors in the traditional group were found to over-perform in their other STEM classes by a 0.18 GPA margin, while non-STEM majors in the revamped group over-performed in their other STEM classes by a 0.17 GPA margin. Both differences are statistically significant. Focus groups were also conducted to gather and synthesize student perceptions. Results may encourage similar innovations to core math curricula in other universities to foster enhanced STEM retention.

Measurements of fasting glucose (FG) or glycated hemoglobin A1c (HbA1c) are two clinically approved approaches commonly used to determine glycemia, both of which are influenced by genetic factors. Obtaining accurate measurements of FG or HbA1c is not without its challenges, though. Measuring glycated serum protein (GSP) offers an alternative approach for assessing glycemia. The aim of this study was to estimate the heritability of GSP and GSP expressed as a percentage of total serum albumin (%GA) using a variance component approach and localize genomic regions (QTLs) that harbor genes likely to influence GSP and %GA trait variation in a large extended multigenerational pedigree from Jiri, Nepal (n=1,800). We also performed quantitative bivariate analyses to assess the relationship between GSP or %GA and several cardiometabolic traits. Additive genetic effects significantly influence variation in GSP and %GA levels (p values: 1.15×10−5 and 3.39×10−5, respectively). We localized a significant (LOD score=3.18) and novel GSP QTL on chromosome 11q, which has been previously linked to type 2 diabetes. Two common (MAF>0.4) SNPs within the chromosome 11 QTL were associated with GSP (adjusted pvalue

Obesity is one of the most prevalent metabolic diseases in the Western world and correlates directly with insulin resistance, which may ultimately culminate in type 2 diabetes (T2D). We sought to ascertain whether the human metalloproteinase A Disintegrin and Metalloproteinase 19 (ADAM19) correlates with parameters of the metabolic syndrome in humans and mice. To determine the potential novel role of ADAM19 in the metabolic syndrome, we first conducted microarray studies on peripheral blood mononuclear cells from a well-characterised human cohort. Secondly, we examined the expression of ADAM19 in liver and gonadal white adipose tissue using an in vivo diet induced obesity mouse model. Finally, we investigated the effect of neutralising ADAM19 on diet induced weight gain, insulin resistance in vivo, and liver TNF-α levels. Significantly, we show that, in humans, ADAM19 strongly correlates with parameters of the metabolic syndrome, particularly BMI, relative fat, HOMA-IR, and triglycerides. Furthermore, we identified that ADAM19 expression was markedly increased in the liver and gonadal white adipose tissue of obese and T2D mice. Excitingly, we demonstrate in our diet induced obesity mouse model that neutralising ADAM19 therapy results in weight loss, improves insulin sensitivity, and reduces liver TNF-α levels. Our novel data suggest that ADAM19 is pro-obesogenic and enhances insulin resistance. Therefore, neutralisation of ADAM19 may be a potential therapeutic approach to treat obesity and T2D.

The high-density-lipoprotein-(HDL-) associated esterase paraoxonase 1 (PON1) is a likely contributor to the antioxidant and antiatherosclerotic capabilities of HDL. Two nonsynonymous mutations in the structural gene, PON1, have been associated with variation in activity levels, but substantial interindividual differences remain unexplained and are greatest for substrates other than the eponymous paraoxon. PON1 activity levels were measured for three substrates—organophosphate paraoxon, arylester phenyl acetate, and lactone dihydrocoumarin—in 767 Mexican American individuals from San Antonio, Texas. Genetic influences on activity levels for each substrate were evaluated by association with approximately one million single nucleotide polymorphism (SNPs) while conditioning on PON1 genotypes. Significant associations were detected at five loci including regions on chromosomes 4 and 17 known to be associated with atherosclerosis and lipoprotein regulation and loci on chromosome 3 that regulate ubiquitous transcription factors. These loci explain 7.8% of variation in PON1 activity with lactone as a substrate, 5.6% with the arylester, and 3.0% with paraoxon. In light of the potential importance of PON1 in preventing cardiovascular disease/events, these novel loci merit further investigation.