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The objective of this study was to optimize the methodology for spectrophotometric determination of sperm concentration in Atlantic croaker Micropogonias undulatus L. milt and to estimate its potential for short-term cold-storage. The spectrophotometric determination of sperm concentration was evaluated using milt samples from six males serially diluted in Hank's balanced salt solution at 200 mOsm kg−1 (HBSS). The predictive power of regression models between sperm concentration and absorbance was determined from 200 to 500 nm and found to be highest within the visible spectrum despite a peak of milt absorbance at 288 nm. Absorbance reading at 400 nm was selected for further analysis to maximize the absorbance of the sample hence the sensitivity of the method while minimizing the impact of potential sample contamination with blood. The standard-curve of correlation between sperm absorbance at 400 nm and concentration was validated and held an accuracy ranging between −7.40% and +4.56% across males. Total sperm motility duration and the proportion of motile spermatozoa were significantly higher in milt samples diluted 1:3 in HBSS than in the undiluted control during up to 30 h of cold-storage.The methodologies investigated in this study can be applied to optimize sperm usage and achieve predictable artificial fertilization protocols in Atlantic croaker. [ABSTRACT FROM AUTHOR]

KCNH2 encodes Kv11.1 and underlies the rapidly activating delayed rectifier K+ current (IKr) in the heart. Loss-of-function KCNH2 mutations cause the type 2 long QT syndrome (LQT2), and most LQT2-linked missense mutations inhibit the trafficking of Kv11.1 channels. Drugs that bind to Kv11.1 and block IKr (e.g., E-4031) can act as pharmacological chaperones to increase the trafficking and functional expression for most LQT2 channels (pharmacological correction). We previously showed that LQT2 channels are selectively stored in a microtubuledependent compartment within the endoplasmic reticulum (ER). We tested the hypothesis that pharmacological correction promotes the trafficking of LQT2 channels stored in this compartment. Confocal analyses of cells expressing the trafficking-deficient LQT2 channel G601S showed that the microtubule-dependent ER compartment is the transitional ER. Experiments with E-4031 and the protein synthesis inhibitor cycloheximide suggested that pharmacological correction promotes the trafficking of G601S stored in this compartment. Treating cells in E-4031 or ranolazine (a drug that blocks IKr and has a short half-life) for 30 min was sufficient to cause pharmacological correction. Moreover, the increased functional expression of G601S persisted 4-5 h after drug washout. Coexpression studies with a dominant- negative form of Rab11B, a small GTPase that regulates Kv11.1 trafficking, prevented the pharmacological correction of G601S trafficking from the transitional ER. These data suggest that pharmacological correction quickly increases the trafficking of LQT2 channels stored in the transitional ER via a Rab11B-dependent pathway, and we conclude that the pharmacological chaperone activity of drugs like ranolazine might have therapeutic potential. [ABSTRACT FROM AUTHOR]

Reconstituted cell-free (CF) protein expression systems hold the promise of overcoming the traditional barriers associated with in vivo systems. This is particularly true for membrane proteins, which are often cytotoxic and due to the nature of the membrane, difficult to work with. To evaluate the potential of cell-free expression, we cloned 120 membrane proteins from E. coli and compared their expression profiles in both an E. coli in vivo system and an E. coli-derived cell-free system. Our results indicate CF is a more robust system and we were able to express 63% of the targets in CF, compared to 44% in vivo. To benchmark the quality of CF produced protein, five target membrane proteins were purified and their homogeneity assayed by gel filtration chromatography. Finally, to demonstrate the ease of amino acid labeling with CF, a novel membrane protein was substituted with selenomethionine, purified, and shown to have 100% incorporation of the unnatural amino acid. We conclude that CF is a novel, robust expression system capable of expressing more proteins than an in vivo system and suitable for production of membrane proteins at the milligram level. [ABSTRACT FROM AUTHOR]

KCNH2 (hERG1) encodes the α-subunit proteins for the rapidly activating delayed rectifier K+ current (IKr), a major K+ current for cardiac myocyte repolarization. In isolated myocytes IKr frequently is small in amplitude or absent, yet KCNH2 channels and IKr are targets for drug block or mutations to cause long QT syndrome. We hypothesized that KCNH2 channels and IKr are uniquely sensitive to enzymatic damage. To test this hypothesis, we studied heterologously expressed K+, Na+, and L-type Ca2+ channels, and in ventricular myoctyes IKr, slowly activating delayed rectifier K+ current (IKs), and inward rectifier K+ current (IKl), by using electrophysiological and biochemical methods. 1) Specific exogenous serine proteases (protease XIV, XXIV, or proteinase K) selectively degraded KCNH2 current (IKCNH2) and its mature channel protein without damaging cell integrity and with minimal effects on the other channel currents; 2) immature KCNH2 channel protein remained intact; 3) smaller molecular mass KCNH2 degradation products appeared; 4) protease XXIV selectively abolished IKr; and 5) reculturing HEK-293 cells after protease exposure resulted in the gradual recovery of IKCN2 and its mature channel protein over several hours. Thus the channel protein for IKCNH2 and IKr is uniquely sensitive to proteolysis. Analysis of the degradation products suggests selective proteolysis within the S5-pore extracellular linker, which is structurally unique among Kv channels. These data provide 1) a new mechanism to account for low IKr density in some isolated myocytes, 2) evidence that most complexly glycosylated KCNH2 channel protein is in the plasma membrane, and 3) new insight into the rate of biogenesis of KCNH2 channel protein within cells. [ABSTRACT FROM AUTHOR]

KCNH2 (hERG1) encodes the α-subunit proteins for the rapidly activating delayed rectifier K+ current (IKr), a major K+ current for cardiac myocyte repolarization. In isolated myocytes IKr frequently is small in amplitude or absent, yet KCNH2 channels and IKr are targets for drug block or mutations to cause long QT syndrome. We hypothesized that KCNH2 channels and IKr are uniquely sensitive to enzymatic damage. To test this hypothesis, we studied heterologously expressed K+, Na+, and L-type Ca2+ channels, and in ventricular myoctyes IKr, slowly activating delayed rectifier K+ current (IKs), and inward rectifier K+ current (IKl), by using electrophysiological and biochemical methods. 1) Specific exogenous serine proteases (protease XIV, XXIV, or proteinase K) selectively degraded KCNH2 current (IKCNH2) and its mature channel protein without damaging cell integrity and with minimal effects on the other channel currents; 2) immature KCNH2 channel protein remained intact; 3) smaller molecular mass KCNH2 degradation products appeared; 4) protease XXIV selectively abolished IKr; and 5) reculturing HEK-293 cells after protease exposure resulted in the gradual recovery of IKCN2 and its mature channel protein over several hours. Thus the channel protein for IKCNH2 and IKr is uniquely sensitive to proteolysis. Analysis of the degradation products suggests selective proteolysis within the S5-pore extracellular linker, which is structurally unique among Kv channels. These data provide 1) a new mechanism to account for low IKr density in some isolated myocytes, 2) evidence that most complexly glycosylated KCNH2 channel protein is in the plasma membrane, and 3) new insight into the rate of biogenesis of KCNH2 channel protein within cells. [ABSTRACT FROM AUTHOR]

Mutations in the human ether-a-go-go-related gene (HERG) cause long QT syndrome. We previously showed that the HERG N470D mutation expressed as homotetrameric channels causes a protein trafficking defect, and this can be corrected by the HERG channel blocking drug E-4031. The N470D mutant also has been reported to cause dominant negative suppression of HERG current when coexpressed with wild-type channel subunits. The aims of this study were 1) to investigate the molecular mechanism responsible for the dominant negative effect of the N470D mutant co-expressed with wild-type subunits and 2) to test whether the trafficking defective heteromeric channels could be pharmacologically rescued by E-4031. Using a combination of immunoprecipitation and Western blot methods, we showed that N470D mutant and wild-type HERG subunits were physically associated in the endoplasmic reticulum as heteromeric channels. The coassembly resulted in the retention of both wild-type and N470D subunits in the endoplasmic reticulum. Culturing cells in E-4031 increased the cell surface expression of these channels, although with an altered electrophysiological phenotype. These results suggest that the dominant negative effect of the N470D wild-type coassembled channels is caused by retention of heteromeric channels in the endoplasmic reticulum and that the trafficking defect of these channels can be corrected by specific pharmacological strategies. [ABSTRACT FROM AUTHOR]

Presents a study that used the approaches of site-directed mutagenesis and biochemical modification to inhibit asparagine (N)-linked glycosylation and examined the role of glycosylation in the cell surface expression and turnover of human ether- à -go-go-related gene (HERG). Information on HERG; Details of the inhibition of N-linked glycosylation by tunicamycin; Cell surface expression of wild-type HERG and N-linked glycosylation.

[Figure: see text]. [ABSTRACT FROM AUTHOR]

A two-step reaction sequence yielding 2-aminooxazolines under mild conditions was developed. Both electron-rich and electron-deficient styrenes as well as functionalized primary and secondary amides could be used in the reaction to afford 2-aminooxazolines in reasonable yields. [ABSTRACT FROM AUTHOR]

Gunnison's prairie dogs, Cynomys gunnisoni , frequently utter a repetitive series (a 'bout') of alarm calls (called 'barks') in response to a variety of terrestrial predators. Previous work has suggested that the structure of barks is finely tuned in ways that provide highly specific information about the identity of the predator. We re-evaluated these findings by presenting individually marked adult females with taxidermy mounts of three terrestrial predators of prairie dogs: a coyote, Canis latrans , a bobcat, Lynx rufus , and an American badger, Taxidea taxus , as well as one control stimulus (a cardboard box). Significant variation among individuals was evident from analyses of 10 measures associated with the general pattern of barking (e.g. latency to begin barking, bout duration, average number of barks/bout, overall bark rate, interbout interval) and also from eight structural features of individual barks (e.g. frequency and duration measures of the fundamental frequency band). When controlling for the identity of alarm callers, we found little evidence for differences in these same variables in response to different stimuli. We conclude that, like several other species of ground-dwelling squirrels, Gunnison's prairie dogs have an individually distinctive alarm call that does not differ for different predators. Our results conflict with earlier claims of extreme specificity in the alarm calls of Gunnison's prairie dogs. Highlights • We tested whether Gunnison's prairie dog alarm calls show referential specificity. • Controlling for caller ID, we found little evidence of call variation to four stimuli. • However, individuals' alarm calls were distinct from one other. • Previous studies not considering individual variation need to be re-evaluated. [ABSTRACT FROM AUTHOR]

The article discusses research on the putative stem cells found in the regenerated tissues through markers related to mesenchymal stem cells. It references the study "Putative Stem Cells in Regenerating Human Periodontium," by N. H. et al, published in the July 4, 2008 issue of "Journal of Periodontal Research." The researchers explained that the presence of cells with periodontal ligament (PDL) stem cells qualities in the tissue taken from the so-called guided tissue regeneration (GTR)-treated defects indicates the role of stem cells in healing.

The electrocardiogram (ECG) empowered clinician scientists to measure the electrical activity of the heart noninvasively to identify arrhythmias and heart disease. Shortly after the standardization of the 12-lead ECG for the diagnosis of heart disease, several families with autosomal recessive (Jervell and Lange-Nielsen Syndrome) and dominant (Romano–Ward Syndrome) forms of long QT syndrome (LQTS) were identified. An abnormally long heart rate-corrected QT-interval was established as a biomarker for the risk of sudden cardiac death. Since then, the International LQTS Registry was established; a phenotypic scoring system to identify LQTS patients was developed; the major genes that associate with typical forms of LQTS were identified; and guidelines for the successful management of patients advanced. In this review, we discuss the molecular and cellular mechanisms for LQTS associated with missense variants in KCNQ1 (LQT1) and KCNH2 (LQT2). We move beyond the "benign" to a "pathogenic" binary classification scheme for different KCNQ1 and KCNH2 missense variants and discuss gene- and mutation-specific differences in K+ channel dysfunction, which can predispose people to distinct clinical phenotypes (e.g., concealed, pleiotropic, severe, etc.). We conclude by discussing the emerging computational structural modeling strategies that will distinguish between dysfunctional subtypes of KCNQ1 and KCNH2 variants, with the goal of realizing a layered precision medicine approach focused on individuals. [ABSTRACT FROM AUTHOR]

Idiopathic ventricular fibrillation (IVF) is an unrefined diagnosis representing a heterogeneous patient group without a structural or genetic definition. IVF treatment is not mechanistic-based due to the lack of experimental patient-models. We sought to create a methodology to assess cellular arrhythmia mechanisms for IVF as a proof-of-concept study. Using IVF patient-specific induced pluripotent stem cell-derived cardiomyocytes, we integrate electrophysiological optical mapping with computational modeling to characterize the cellular phenotype. This approach flips the traditional paradigm using a biophysically detailed computational model to solve the problem inversely. Insight into the cellular mechanisms of this patient's IVF phenotype could also serve as a therapeutic testbed., Competing Interests: Funding Support and Author Disclosures This work was supported by NIH R01HL170521 (LLE, EG), NIH R01HL163987 (Dr Eckhardt), NIH R01HL139738 (Dr Eckhardt), 2R01HL141214 (Dr Glukhov), AHA846898, and AHA 24SCEFIA1255230 (Dr Lang), 24CDA1258695 (Dr Ni). This work was also funded in part by the Gary and Marie Weiner Professor in Cardiovascular Medicine Research (Dr Eckhardt). All authors have reported that they have no relationships relevant to the contents of this paper to disclose., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Lamin A/C (LMNA) is an important component of nuclear lamina. Mutations cause arrhythmia, heart failure, and sudden cardiac death. While LMNA-associated cardiomyopathy typically has an aggressive course that responds poorly to conventional heart failure therapies, there is variability in severity and age of penetrance between and even within specific mutations, which is poorly understood at the cellular level. Further, this heterogeneity has not previously been captured to mimic the heterozygous state, nor have the hundreds of clinical LMNA mutations been represented. Herein, we have overexpressed cardiopathic LMNA variants in HEK cells and utilized state-of-the-art quantitative proteomics to compare the global proteomic profiles of (1) aggregating Q353 K alone, (2) Q353 K coexpressed with WT, (3) aggregating N195 K coexpressed with WT, and (4) nonaggregating E317 K coexpressed with WT to help capture some of the heterogeneity between mutations. We analyzed each data set to obtain the differentially expressed proteins (DEPs) and applied gene ontology (GO) and KEGG pathway analyses. We found a range of 162 to 324 DEPs from over 6000 total protein IDs with differences in GO terms, KEGG pathways, and DEPs important in cardiac function, further highlighting the complexity of cardiac laminopathies. Pathways disrupted by LMNA mutations were validated with redox, autophagy, and apoptosis functional assays in both HEK 293 cells and in induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) for LMNA N195 K. These proteomic profiles expand our repertoire for mutation-specific downstream cellular effects that may become useful as druggable targets for personalized medicine approach for cardiac laminopathies.

This study described spatiotemporal changes in health insurance coverage, healthcare access, and reasons for non-insurance among racial/ethnic minority populations in the United States during the COVID-19 pandemic using four national survey datasets. Getis-Ord Gi* statistic and scan statistics were used to analyze geospatial clusters of health insurance coverage by race/ethnicity. Logistic regression was used to estimate odds of reporting inability to access healthcare across two pandemic time periods by race/ethnicity. Racial/ethnic differences in insurance were observed from 2010 through 2019, with the lowest rates being among Hispanic/Latino, African American, American Indian/Alaska Native, and Native Hawaiian/Pacific Islander populations. Pre-pandemic insurance coverage rates were geographically clustered. The percentage of adults citing change in employment status as the reason for non-insurance increased by about 7% after the start of the pandemic, with a small decrease observed among African American adults. Almost half of adults reported reduced healthcare access in June 2020, with 38.7% attributing reduced access to the pandemic; however, by May 2021, the percent of respondents reporting reduced access for any reason and due to the pandemic fell to 26.9% and 12.7%, respectively. In general, racial/ethnic disparities in health insurance coverage and healthcare access worsened during the pandemic. Although coverage and access improved over time, pre-COVID disparities persisted with African American and Hispanic/Latino populations being the most affected by insurance loss and reduced healthcare access. Cost, unemployment, and eligibility drove non-insurance before and during the pandemic.

Hypoxic-ischemic injury has been linked with increased risk for developing Alzheimer's disease (AD). The underlying mechanism of this association is poorly understood. Here, we report distinct roles for hypoxia-inducible factor-1α (Hif-1α) in the regulation of BACE1 and γ-secretase activity, two proteases involved in the production of amyloid-beta (Aβ). We have demonstrated that Hif-1α upregulates both BACE1 and γ-secretase activity for Aβ production in brain hypoxia-induced either by cerebral hypoperfusion or breathing 10% O 2 . Hif-1α binds to γ-secretase, which elevates the amount of active γ-secretase complex without affecting the level of individual subunits in hypoxic-ischemic mouse brains. Additionally, the expression of full length Hif-1α increases BACE1 and γ-secretase activity in primary neuronal culture, whereas a transcriptionally incompetent Hif-1α variant only activates γ-secretase. These findings indicate that Hif-1α transcriptionally upregulates BACE1 and nontranscriptionally activates γ-secretase for Aβ production in hypoxic-ischemic conditions. Consequently, Hif-1α-mediated Aβ production may be an adaptive response to hypoxic-ischemic injury, subsequently leading to increased risk for AD. Preventing the interaction of Hif-1α with γ-secretase may therefore be a promising therapeutic strategy for AD treatment., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Over the last two decades, an exponentially expanding number of genetic variants have been identified associated with inherited cardiac conditions. These tremendous gains also present challenges in deciphering the clinical relevance of unclassified variants or variants of uncertain significance (VUS). This review provides an overview of the advancements (and challenges) in functional and computational approaches to characterize variants and help keep pace with VUS identification related to inherited heart diseases., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Anderson, Munawar, Reilly, Kamp, January, Delisle and Eckhardt.)

Background: This article evaluates the predictive accuracy of the CareMOSAIC Risk Assessment for discharge disposition in Medicare patients undergoing total joint arthroplasty., Methods: Retrospectively collected data from a single institution on 499 consecutive Medicare patients who underwent primary total hip arthroplasty or total knee arthroplasty were reviewed. The CareMOSAIC Risk Assessment was completed by each patient during the preoperative period. The CareMOSAIC Risk Assessment scores were calculated via the CareMOSAIC software, and the scores indicate a risk category for each patient as it relates to post-acute care discharge needs., Results: The CareMOSAIC Risk Assessment with a binary logistic regression area under the receiver operating characteristic curve of 0.798 appears to be a reliable tool for predicting discharge disposition. The assessment had a positive predictive value of 90.0% and negative predictive value of 76.3% for discharge disposition., Conclusions: The CareMOSAIC Risk Assessment effectively predicts the discharge disposition for Medicare patients undergoing total hip or total knee arthroplasty., (© 2021 The Authors.)

Hundreds of LMNA variants have been associated with several distinct disease phenotypes. However, genotype-phenotype relationships remain largely undefined and the impact for most variants remains unknown. We performed a functional analysis for 178 variants across five structural domains using two different overexpression models. We found that lamin A aggregation is a major determinant for skeletal and cardiac laminopathies. An in vitro solubility assay shows that aggregation-prone variants in the immunoglobulin-like domain correlate with domain destabilization. Finally, we demonstrate that myopathic-associated LMNA variants show aggregation patterns in induced pluripotent stem cell derived-cardiomyocytes (iPSC-CMs) in contrast to non-myopathic LMNA variants. Our data-driven approach (1) reveals that striated muscle laminopathies are predominantly protein misfolding diseases, (2) demonstrates an iPSC-CM experimental platform for characterizing laminopathic variants in human cardiomyocytes, and (3) supports a functional assay to aid in assessing pathogenicity for myopathic variants of uncertain significance., (© 2021. The Author(s).)

Purpose: DNA sequencing technology has unmasked a vast number of uncharacterized single-nucleotide variants in disease-associated genes, and efficient methods are needed to determine pathogenicity and enable clinical care., Methods: We report an E. coli-based solubility assay for assessing the effects of variants on protein domain stability for three disease-associated proteins., Results: First, we examined variants in the Kv11.1 channel PAS domain (PASD) associated with inherited long QT syndrome type 2 and found that protein solubility correlated well with reported in vitro protein stabilities. A comprehensive solubility analysis of 56 Kv11.1 PASD variants revealed that disruption of membrane trafficking, the dominant loss-of-function disease mechanism, is largely determined by domain stability. We further validated this assay by using it to identify second-site suppressor PASD variants that improve domain stability and Kv11.1 protein trafficking. Finally, we applied this assay to several cancer-linked P53 tumor suppressor DNA-binding domain and myopathy-linked Lamin A/C Ig-like domain variants, which also correlated well with reported protein stabilities and functional analyses., Conclusion: This simple solubility assay can aid in determining the likelihood of pathogenicity for sequence variants due to protein misfolding in structured domains of disease-associated genes as well as provide insights into the structural basis of disease.

The GTPase OPA1 and the AAA-protease OMA1 serve well-established roles in mitochondrial stress responses and mitochondria-initiated cell death. In addition to its role in mitochondrial membrane fusion, cristae structure, and bioenergetic function, OPA1 controls apoptosis by sequestering cytochrome c (cyt c) in mitochondrial cristae. Cleavage of functional long OPA1 (L-OPA1) isoforms by OMA1 inactivates mitochondrial fusion and primes apoptosis. OPA1 cleavage is regulated by the prohibitin (PHB) complex, a heteromeric, ring-shaped mitochondrial inner membrane scaffolding complex composed of PHB1 and PHB2. In neurons, PHB plays a protective role against various stresses, and PHB deletion destabilizes OPA1 causing neurodegeneration. While deletion of OMA1 prevents OPA1 destabilization and attenuates neurodegeneration in PHB2 KO mice, how PHB levels regulate OMA1 is still unknown. Here, we investigate the effects of modulating neuronal PHB levels on OMA1 stability and OPA1 cleavage. We demonstrate that PHB promotes OMA1 turnover, effectively decreasing the pool of OMA1. Further, we show that OMA1 binds to cardiolipin (CL), a major mitochondrial phospholipid. CL binding promotes OMA1 turnover, as we show that deleting the CL-binding domain of OMA1 decreases its turnover rate. Since PHB is known to stabilize CL, these data suggest that PHB modulates OMA1 through CL. Furthermore, we show that PHB decreases cyt c release induced by tBID and attenuates caspase 9 activation in response to hypoxic stress in neurons. Taken together, our results suggest that PHB-mediated CL stabilization regulates stress responses and cell death through OMA1 turnover and cyt c release.

Mutations in coiled-coil-helix-coiled-coil-helix domain containing 10 (CHCHD10), a mitochondrial protein of unknown function, cause a disease spectrum with clinical features of motor neuron disease, dementia, myopathy and cardiomyopathy. To investigate the pathogenic mechanisms of CHCHD10, we generated mutant knock-in mice harboring the mouse-equivalent of a disease-associated human S59L mutation, S55L in the endogenous mouse gene. CHCHD10 S55L mice develop progressive motor deficits, myopathy, cardiomyopathy and accelerated mortality. Critically, CHCHD10 accumulates in aggregates with its paralog CHCHD2 specifically in affected tissues of CHCHD10 S55L mice, leading to aberrant organelle morphology and function. Aggregates induce a potent mitochondrial integrated stress response (mtISR) through mTORC1 activation, with elevation of stress-induced transcription factors, secretion of myokines, upregulated serine and one-carbon metabolism, and downregulation of respiratory chain enzymes. Conversely, CHCHD10 ablation does not induce disease pathology or activate the mtISR, indicating that CHCHD10 S55L -dependent disease pathology is not caused by loss-of-function. Overall, CHCHD10 S55L mice recapitulate crucial aspects of human disease and reveal a novel toxic gain-of-function mechanism through maladaptive mtISR and metabolic dysregulation.

The mitochondrial protein prohibitin (PHB) has emerged as an important modulator of neuronal survival in different injury modalities . We previously showed that viral gene transfer of PHB protects CA1 neurons from delayed neurodegeneration following transient forebrain ischemia through mitochondrial mechanisms. However, since PHB is present in all cell types, it is not known if its selective expression in neurons is protective, and if the protection occurs also in acute focal ischemic brain injury, the most common stroke type in humans. Therefore, we generated transgenic mice overexpressing human PHB1 specifically in neurons (PHB1 Tg). PHB1 Tg mice and littermate controls were subjected to transient middle cerebral artery occlusion (MCAo). Infarct volume and sensory-motor impairment were assessed three days later. Under the control of a neuronal promoter (CaMKIIα), PHB1 expression was increased by 50% in the forebrain and hippocampus in PHB1 Tg mice. The brain injury produced by MCAo was reduced by 63 ± 11% in PHB1 Tg mice compared to littermate controls. This reduction was associated with improved sensory-motor performance, suggesting that the salvaged brain remains functional. Approaches to enhance PHB expression may be useful to ameliorate the devastating impact of cerebral ischemia on the brain.

Background: Heterologous functional validation studies of putative long-QT syndrome subtype 2-associated variants clarify their pathological potential and identify disease mechanism(s) for most variants studied. The purpose of this study is to clarify the pathological potential for rare nonsynonymous KCNH2 variants seemingly associated with sudden infant death syndrome., Methods: Genetic testing of 292 sudden infant death syndrome cases identified 9 KCNH2 variants: E90K, R181Q, A190T, G294V, R791W, P967L, R1005W, R1047L, and Q1068R. Previous studies show R181Q-, P967L-, and R1047L-Kv11.1 channels function similar to wild-type Kv11.1 channels, whereas Q1068R-Kv11.1 channels accelerate inactivation gating. We studied the biochemical and biophysical properties for E90K-, G294V-, R791W-, and R1005W-Kv11.1 channels expressed in human embryonic kidney 293 cells; examined the electronic health records of patients who were genotype positive for the sudden infant death syndrome-linked KCNH2 variants; and simulated their functional impact using computational models of the human ventricular action potential., Results: Western blot and voltage-clamping analyses of cells expressing E90K-, G294V-, R791W-, and R1005W-Kv11.1 channels demonstrated these variants express and generate peak Kv11.1 current levels similar to cells expressing wild-type-Kv11.1 channels, but R791W- and R1005W-Kv11.1 channels accelerated deactivation and activation gating, respectively. Electronic health records of patients with the sudden infant death syndrome-linked KCNH2 variants showed that the patients had median heart rate-corrected QT intervals <480 ms and none had been diagnosed with long-QT syndrome or experienced cardiac arrest. Simulating the impact of dysfunctional gating variants predicted that they have little impact on ventricular action potential duration., Conclusions: We conclude that these rare Kv11.1 missense variants are not long-QT syndrome subtype 2-causative variants and therefore do not represent the pathogenic substrate for sudden infant death syndrome in the variant-positive infants., (© 2018 American Heart Association, Inc.)

Background and Purpose: Ischemic brain injury is characterized by 2 temporally distinct but interrelated phases: ischemia (primary energy failure) and reperfusion (secondary energy failure). Loss of cerebral blood flow leads to decreased oxygen levels and energy crisis in the ischemic area, initiating a sequence of pathophysiological events that after reoxygenation lead to ischemia/reperfusion (I/R) brain damage. Mitochondrial impairment and oxidative stress are known to be early events in I/R injury. However, the biochemical mechanisms of mitochondria damage in I/R are not completely understood., Methods: We used a mouse model of transient focal cerebral ischemia to investigate acute I/R-induced changes of mitochondrial function, focusing on mechanisms of primary and secondary energy failure., Results: Ischemia induced a reversible loss of flavin mononucleotide from mitochondrial complex I leading to a transient decrease in its enzymatic activity, which is rapidly reversed on reoxygenation. Reestablishing blood flow led to a reversible oxidative modification of mitochondrial complex I thiol residues and inhibition of the enzyme. Administration of glutathione-ethyl ester at the onset of reperfusion prevented the decline of complex I activity and was associated with smaller infarct size and improved neurological outcome, suggesting that decreased oxidation of complex I thiols during I/R-induced oxidative stress may contribute to the neuroprotective effect of glutathione ester., Conclusions: Our results unveil a key role of mitochondrial complex I in the development of I/R brain injury and provide the mechanistic basis for the well-established mitochondrial dysfunction caused by I/R. Targeting the functional integrity of complex I in the early phase of reperfusion may provide a novel therapeutic strategy to prevent tissue injury after stroke., (© 2018 The Authors.)

Reconstituted cell-free (CF) protein expression systems hold the promise of overcoming the traditional barriers associated with in vivo systems. This is particularly true for membrane proteins, which are often cytotoxic and due to the nature of the membrane, difficult to work with. To evaluate the potential of cell-free expression, we cloned 120 membrane proteins from E. coli and compared their expression profiles in both an E. coli in vivo system and an E. coli-derived cell-free system. Our results indicate CF is a more robust system and we were able to express 63% of the targets in CF, compared to 44% in vivo. To benchmark the quality of CF produced protein, five target membrane proteins were purified and their homogeneity assayed by gel filtration chromatography. Finally, to demonstrate the ease of amino acid labeling with CF, a novel membrane protein was substituted with selenomethionine, purified, and shown to have 100% incorporation of the unnatural amino acid. We conclude that CF is a novel, robust expression system capable of expressing more proteins than an in vivo system and suitable for production of membrane proteins at the milligram level.