Hypoxia extends lifespan and neurological function in a mouse model of aging.

There is widespread interest in identifying interventions that extend healthy lifespan. Chronic continuous hypoxia delays the onset of replicative senescence in cultured cells and extends lifespan in yeast, nematodes, and fruit flies. Here, we asked whether chronic continuous hypoxia is beneficial in mammalian aging. We utilized the Ercc1 Δ/- mouse model of accelerated aging given that these mice are born developmentally normal but exhibit anatomic, physiological, and biochemical features of aging across multiple organs. Importantly, they exhibit a shortened lifespan that is extended by dietary restriction, the most potent aging intervention across many organisms. We report that chronic continuous 11% oxygen commenced at 4 weeks of age extends lifespan by 50% and delays the onset of neurological debility in Ercc1 Δ/- mice. Chronic continuous hypoxia did not impact food intake and did not significantly affect markers of DNA damage or senescence, suggesting that hypoxia did not simply alleviate the proximal effects of the Ercc1 mutation, but rather acted downstream via unknown mechanisms. To the best of our knowledge, this is the first study to demonstrate that "oxygen restriction" can extend lifespan in a mammalian model of aging.

Circulating N-lactoyl-amino acids and N-formyl-methionine reflect mitochondrial dysfunction and predict mortality in septic shock.

INTRODUCTION: Sepsis is a highly morbid condition characterized by multi-organ dysfunction resulting from dysregulated inflammation in response to acute infection. Mitochondrial dysfunction may contribute to sepsis pathogenesis, but quantifying mitochondrial dysfunction remains challenging. OBJECTIVE: To assess the extent to which circulating markers of mitochondrial dysfunction are increased in septic shock, and their relationship to severity and mortality. METHODS: We performed both full-scan and targeted (known markers of genetic mitochondrial disease) metabolomics on plasma to determine markers of mitochondrial dysfunction which distinguish subjects with septic shock (n = 42) from cardiogenic shock without infection (n = 19), bacteremia without sepsis (n = 18), and ambulatory controls (n = 19) - the latter three being conditions in which mitochondrial function, proxied by peripheral oxygen consumption, is presumed intact. RESULTS: Nine metabolites were significantly increased in septic shock compared to all three comparator groups. This list includes N-formyl-L-methionine (f-Met), a marker of dysregulated mitochondrial protein translation, and N-lactoyl-phenylalanine (lac-Phe), representative of the N-lactoyl-amino acids (lac-AAs), which are elevated in plasma of patients with monogenic mitochondrial disease. Compared to lactate, the clinical biomarker used to define septic shock, there was greater separation between survivors and non-survivors of septic shock for both f-Met and the lac-AAs measured within 24 h of ICU admission. Additionally, tryptophan was the one metabolite significantly decreased in septic shock compared to all other groups, while its breakdown product kynurenate was one of the 9 significantly increased. CONCLUSION: Future studies which validate the measurement of lac-AAs and f-Met in conjunction with lactate could define a sepsis subtype characterized by mitochondrial dysfunction.

Scalable manufacture of therapeutic mesenchymal stromal cell products on customizable microcarriers in vertical wheel bioreactors that improve direct visualization, product harvest, and cost.

BACKGROUND AIMS: Human mesenchymal stromal cells (hMSCs) and their secreted products show great promise for treatment of musculoskeletal injury and inflammatory or immune diseases. However, the path to clinical utilization is hampered by donor-tissue variation and the inability to manufacture clinically relevant yields of cells or their products in a cost-effective manner. Previously we described a method to produce chemically and mechanically customizable gelatin methacryloyl (GelMA) microcarriers for culture of hMSCs. Herein, we demonstrate scalable GelMA microcarrier-mediated expansion of induced pluripotent stem cell (iPSC)-derived hMSCs (ihMSCs) in 500 mL and 3L vertical wheel bioreactors, offering several advantages over conventional microcarrier and monolayer-based expansion strategies. METHODS: Human mesenchymal stromal cells derived from induced pluripotent cells were cultured on custom-made spherical gelatin methacryloyl microcarriers in single-use vertical wheel bioreactors (PBS Biotech). Cell-laden microcarriers were visualized using confocal microscopy and elastic light scattering methodologies. Cells were assayed for viability and differentiation potential in vitro by standard methods. Osteogenic cell matrix derived from cells was tested in vitro for osteogenic healing using a rodent calvarial defect assay. Immune modulation was assayed with an in vivo peritonitis model using Zymozan A. RESULTS: The optical properties of GelMA microcarriers permit noninvasive visualization of cells with elastic light scattering modalities, and harvest of product is streamlined by microcarrier digestion. At volumes above 500 mL, the process is significantly more cost-effective than monolayer culture. Osteogenic cell matrix derived from ihMSCs expanded on GelMA microcarriers exhibited enhanced in vivo bone regenerative capacity when compared to bone morphogenic protein 2, and the ihMSCs exhibited superior immunosuppressive properties in vivo when compared to monolayer-generated ihMSCs. CONCLUSIONS: These results indicate that the cell expansion strategy described here represents a superior approach for efficient generation, monitoring and harvest of therapeutic MSCs and their products.

Skeletal muscle mitochondrial correlates of critical power and W' in healthy active individuals.

The asymptote (critical power; CP) and curvature constant (W') of the hyperbolic power-duration relationship can predict performance within the severe-intensity exercise domain. However, the extent to which these parameters relate to skeletal muscle mitochondrial content and respiratory function is not known. Fifteen males (peak O2 uptake, 52.2 ± 8.7 mL kg-1 min-1; peak work rate, 366 ± 40 W; and gas exchange threshold, 162 ± 41 W) performed three to five constant-load tests to task failure for the determination of CP (246 ± 44 W) and W' (18.6 ± 4.1 kJ). Skeletal muscle biopsies were obtained from the vastus lateralis to determine citrate synthase (CS) activity, as a marker of mitochondrial content, and the ADP-stimulated respiration (P) and maximal electron transfer (E) through mitochondrial complexes (C) I-IV. The CP was positively correlated with CS activity (absolute CP, r = 0.881, P < 0.001; relative CP, r = 0.751, P = 0.001). The W' was not correlated with CS activity (P > 0.05). Relative CP was positively correlated with mass-corrected CI + IIE (r = 0.659, P = 0.038), with absolute CP being inversely correlated with CS activity-corrected CIVE (r = -0.701, P = 0.024). Relative W' was positively correlated with CS activity-corrected CI + IIP (r = 0.713, P = 0.021) and the phosphorylation control ratio (r = 0.661, P = 0.038). There were no further correlations between CP or W' and mitochondrial respiratory variables. These findings support the assertion that skeletal muscle mitochondrial oxidative capacity is positively associated with CP and that this relationship is strongly determined by mitochondrial content.

Black Pastors' Experiences of Occupational and Life Stress During COVID-19 in the USA.

This study sought to identify the occupational stressors Black pastors experience, who serve in Black Church denominations and Black nondenominational churches. A total of 218 pastors completed the survey out of 2786 for a response rate of 10.1%. Black pastors identified their most challenging stressors as member dynamics, financial stress, leading a church to fulfill its mission, and pastor's workload. Black women pastors faced the additional stressor of having their pastoral leadership challenged by male congregants. Black pastors faced more stressors during the COVID-19 pandemic including church closures, transitioning to virtual services, unexpected deaths, and an increased workload with 72.5% of pastors reporting moderate to extreme stress levels. Approximately 77% of pastors acknowledged experiencing from moderate to extreme stress levels during social protests for the deaths of Black people by law enforcement. Black pastors further acknowledged experiencing an additional three to six life stressors outside of their pastoral roles.

MitoCarta3.0: an updated mitochondrial proteome now with sub-organelle localization and pathway annotations.

The mammalian mitochondrial proteome is under dual genomic control, with 99% of proteins encoded by the nuclear genome and 13 originating from the mitochondrial DNA (mtDNA). We previously developed MitoCarta, a catalogue of over 1000 genes encoding the mammalian mitochondrial proteome. This catalogue was compiled using a Bayesian integration of multiple sequence features and experimental datasets, notably protein mass spectrometry of mitochondria isolated from fourteen murine tissues. Here, we introduce MitoCarta3.0. Beginning with the MitoCarta2.0 inventory, we performed manual review to remove 100 genes and introduce 78 additional genes, arriving at an updated inventory of 1136 human genes. We now include manually curated annotations of sub-mitochondrial localization (matrix, inner membrane, intermembrane space, outer membrane) as well as assignment to 149 hierarchical 'MitoPathways' spanning seven broad functional categories relevant to mitochondria. MitoCarta3.0, including sub-mitochondrial localization and MitoPathway annotations, is freely available at http://www.broadinstitute.org/mitocarta and should serve as a continued community resource for mitochondrial biology and medicine.

Examining the Relationship of Clergy Distress, Spiritual Well-Being, Stress Management and Irritation to Life Satisfaction among Black Pastors in the USA.

This study sought to determine the level of clergy distress and other psychological characteristics of Black pastors and their relationship to life satisfaction through a convenience sample of 2786 Black pastors in historically Black Protestant denominations and nondenominational Black churches. The response rate equaled 10.1% (283/2786) while the survey completion rate equaled 77% (218/283). These 218 Black pastors were serving as either senior pastors (86.3%) or co-pastors (13.7%). This study found clergy distress in Black pastors did not differ based on gender or age but differed by church size and denomination. Clergy distress (r = - .187, p = .023) and irritation (r = - .293, p = .003) possessed significant relationships with satisfaction with life as expected, but stress management (r = .039, p = .641), spiritual well-being in daily life (r = .140, p = .140), and spiritual well-being in ministry (r = - .064, p = .475) did not, which was surprising. Notably strong relationships existed between stress management and spiritual well-being in daily life (r = .469, p = .003) and stress management and irritation (r = - .359, p = .003). These two important relationships may offer some guideposts for Black pastors in developing strategies to combat the impact of both clergy distress and irritation. The study concludes with implications for Black pastors and suggestions for future research.

Development and Validation of a Deep Learning Strategy for Automated View Classification of Pediatric Focused Assessment With Sonography for Trauma.

OBJECTIVE: Pediatric focused assessment with sonography for trauma (FAST) is a sequence of ultrasound views rapidly performed by clinicians to diagnose hemorrhage. A technical limitation of FAST is the lack of expertise to consistently acquire all required views. We sought to develop an accurate deep learning view classifier using a large heterogeneous dataset of clinician-performed pediatric FAST. METHODS: We developed and conducted a retrospective cohort analysis of a deep learning view classifier on real-world FAST studies performed on injured children less than 18 years old in two pediatric emergency departments by 30 different clinicians. FAST was randomly distributed to training, validation, and test datasets, 70:20:10; each child was represented in only one dataset. The primary outcome was view classifier accuracy for video clips and still frames. RESULTS: There were 699 FAST studies, representing 4925 video clips and 1,062,612 still frames, performed by 30 different clinicians. The overall classification accuracy was 97.8% (95% confidence interval [CI]: 96.0-99.0) for video clips and 93.4% (95% CI: 93.3-93.6) for still frames. Per view still frames were classified with an accuracy: 96.0% (95% CI: 95.9-96.1) cardiac, 99.8% (95% CI: 99.8-99.8) pleural, 95.2% (95% CI: 95.0-95.3) abdominal upper quadrants, and 95.9% (95% CI: 95.8-96.0) suprapubic. CONCLUSION: A deep learning classifier can accurately predict pediatric FAST views. Accurate view classification is important for quality assurance and feasibility of a multi-stage deep learning FAST model to enhance the evaluation of injured children.

Stria terminalis microstructure in humans predicts variability in orienting towards threat.

Current concepts of the extended amygdala posit that basolateral to central amygdala projections mediate fear-conditioned autonomic alerting, whereas projections to the bed nucleus of the stria terminalis mediate sustained anxiety. Using diffusion tensor imaging tractography in humans, we show that microstructure of the stria terminalis correlates with an orienting bias towards threat in a saccade decision task, providing the first evidence that this circuit supports decisions guiding evaluation of threatening stimuli.

Heat shock protein 72 regulates hepatic lipid accumulation.

Induction of the chaperone heat shock protein 72 (HSP72) through heat treatment (HT), exercise, or overexpression improves glucose tolerance and mitochondrial function in skeletal muscle. Less is known about HSP72 function in the liver where lipid accumulation can result in insulin resistance and nonalcoholic fatty liver disease (NAFLD). The purpose of this study was 1) to determine whether weekly in vivo HT induces hepatic HSP72 and improves glucose tolerance in rats fed a high-fat diet (HFD) and 2) to determine the ability of HSP72 to protect against lipid accumulation and mitochondrial dysfunction in primary hepatocytes. Male Wistar rats were fed an HFD for 15 wk and were given weekly HT (41°C, 20 min) or sham treatments (37°C, 20 min) for the final 7 wk. Glucose tolerance and insulin sensitivity were assessed, along with HSP72 induction and triglyceride storage, in the skeletal muscle and liver. The effect of an acute loss of HSP72 in primary hepatocytes was examined via siRNA. Weekly in vivo HT improved glucose tolerance, elevated muscle and hepatic HSP72 protein content, and reduced muscle triglyceride storage. In primary hepatocytes, mitochondrial morphology was changed, and fatty acid oxidation was reduced in small interfering HSP72 (siHSP72)-treated hepatocytes. Lipid accumulation following palmitate treatment was increased in siHSP72-treated hepatocytes. These data suggest that HT may improve systemic metabolism via induction of hepatic HSP72. Additionally, acute loss of HSP72 in primary hepatocytes impacts mitochondrial health as well as fat oxidation and storage. These findings suggest therapies targeting HSP72 in the liver may prevent NAFLD.

Association between Ki-67 expression and clinical outcomes among patients with clinically node-negative, thick primary melanoma who underwent nodal staging.

BACKGROUND: Patients with thick primary melanomas (≥4 mm) have highly variable survival outcomes. Cell proliferation marker Ki-67 has been identified as promising biomarker in thick melanoma but has not been evaluated since the wide spread adoption of sentinel lymph node biopsy. We revisit its prognostic relevance in the sentinel node era. METHODS: We studied patients with thick (≥4 mm) primary melanoma prospectively enrolled in a clinicopathological biospecimen database from 2002 to 2015, and evaluated the prognostic value of Ki-67 expression while controlling for features included in the existing staging criteria. RESULTS: We analyzed 68 patients who underwent lymph node sampling and who had an available tumor for Ki-67 immunohistochemical (IHC) staining. The median tumor thickness was 6.0 mm; the median follow-up was 2.6 years. In multivariable analysis including nodal status and primary tumor ulceration, Ki-67 expression was an independent predictor of worse recurrence-free survival (HR 2.19, P = 0.024) and overall survival (HR 2.49, P = 0.028). Natural log-transformed tumor thickness (ln [thickness]) was also significantly associated with worse OS (HR 2.39, P = 0.010). CONCLUSION: We identify Ki-67 and ln (thickness) as potential biomarkers for patients with thick melanoma who have undergone nodal staging. If validated in additional studies, these biomarkers could be integrated into the staging criteria to improve risk-stratification.

The effect of hunger and satiety in the judgment of ethical violations.

Human history is studded with instances where instinctive motivations take precedence over ethical choices. Nevertheless, the evidence of any linking between motivational states and morality has never been systematically explored. Here we addressed this topic by testing a possible linking between appetite and moral judgment. We compared moral disapproval ratings (MDR) for stories of ethical violations in participants under fasting and after having eaten a snack. Our results show that subjective hunger, measured via self-reported rating, reduces MDR for ethical violations. Moreover, the higher the disgust sensitivity the higher the MDR for ethical violations. This study adds new insights to research on physiological processes influencing morality by showing that appetite affects moral disapproval of ethical violations.

An Initial Investigation of Individual Rate-of-Play Preferences and Associations with EGM Gambling Behavior.

Electronic gambling machines (EGMs) show a strong association with gambling problems. The high speed of gaming offered by modern EGMs allows playing numerous games in a short span of time, which is thought to contribute to attentional distraction, increased spending and prolonged play. However, the relationship between EGM speeds and potentially risk-related gambling behavior remains unclear. We introduce a novel approach to investigating the role of gaming speed in EGM gambling behavior by examining 'individual rate-of-play' (I-ROP) during simulated EGM gambling. A community sample of male regular gamblers (N = 72) played virtual slot machines in pairs offering sequentially adjusted game speeds towards the estimation of a behaviorally expressed preference speed, or I-ROP. This initial experiment aimed to explore the variability of I-ROPs during simulated EGM gambling, and examine behavior while playing EGMs at speeds relative to their I-ROP. Estimated I-ROPs ranged from less than one half second to over seven seconds and were negatively associated with cognitive ability, but not related to problem gambling severity, impulsiveness, or gambling-related cognitions. Subsequent gambling sessions on EGMs offering individually calibrated faster and slower gaming speeds were associated with greater and reduced risk-related gambling behaviors respectively. I-ROPs represent a potentially informative construct for exploring influences of gaming speed on gambling behavior, and may lend insight into potential risk-related behavior an individual vulnerability with respect to commercially available EGMs that warrants additional research.

Pelvic Retroperitoneal Dissection: A Hands-on Primer.

This article is a personal perspective of female surgical pelvic anatomy and recommended surgical dissection techniques.

Vulnerability to depression is associated with a failure to acquire implicit social appraisals.

Major depressive disorder (MDD) is associated with disrupted relationships with partners, family, and peers. These problems can precipitate the onset of clinical illness, influence severity and the prospects for recovery. Here, we investigated whether individuals who have recovered from depression use interpersonal signals to form favourable appraisals of others as social partners. Twenty recovered-depressed adults (with >1 adult episode of MDD but euthymic and medication-free for six months) and 23 healthy, never-depressed adults completed a task in which the gaze direction of some faces reliably cued the location a target (valid faces), whereas other faces cued the opposite location (invalid faces). No participants reported awareness of this contingency, and both groups were significantly faster to categorise targets following valid compared with invalid gaze cueing faces. Following this task, participants judged the trustworthiness of the faces. Whereas the healthy never-depressed participants judged the valid faces to be significantly more trustworthy than the invalid faces; this implicit social appraisal was absent in the recovered-depressed participants. Individuals who have recovered from MDD are able to respond appropriately to joint attention with other people but appear to not use joint attention to form implicit trust appraisals of others as potential social partners.

Chinese Beliefs in Luck are Linked to Gambling Problems via Strengthened Cognitive Biases: A Mediation Test.

Problematic patterns of gambling and their harms are known to have culturally specific expressions. For ethnic Chinese people, patterns of superstitious belief in this community appear to be linked to the elevated rates of gambling-related harms; however, little is known about the mediating psychological mechanisms. To address this issue, we surveyed 333 Chinese gamblers residing internationally and used a mediation analysis to explore how gambling-related cognitive biases, gambling frequency and variety of gambling forms ('scope') mediate the association between beliefs in luck and gambling problems. We found that cognitive biases and scope were significant mediators of this link but that the former is a stronger mediator than the latter. The mediating erroneous beliefs were not specific to any particular type of cognitive bias. These results suggest that Chinese beliefs in luck are expressed as gambling cognitive biases that increase the likelihood of gambling problems, and that biases that promote gambling (and its harms) are best understood within their socio-cultural context.

Second language feedback abolishes the "hot hand" effect during even-probability gambling.

Research into language-emotion interactions has revealed intriguing cognitive inhibition effects by emotionally negative words in bilinguals. Here, we turn to the domain of human risk taking and show that the experience of positive recency in games of chance-the "hot hand" effect-is diminished when game outcomes are provided in a second language rather than the native language. We engaged late Chinese-English bilinguals with "play" or "leave" decisions upon presentation of equal-odds bets while manipulating language of feedback and outcome value. When positive game outcomes were presented in their second language, English, participants subsequently took significantly fewer gambles and responded slower compared with the trials in which equivalent feedback was provided in Chinese, their native language. Positive feedback was identified as driving the cross-language difference in preference for risk over certainty: feedback for previous winning outcomes presented in Chinese increased subsequent risk taking, whereas in the English context no such effect was observed. Complementing this behavioral effect, event-related brain potentials elicited by feedback words showed an amplified response to Chinese relative to English in the feedback-related negativity window, indicating a stronger impact in the native than in the second language. We also observed a main effect of language on P300 amplitude and found it correlated with the cross-language difference in risk selections, suggesting that the greater the difference in attention between languages, the greater the difference in risk-taking behavior. These results provide evidence that the hot hand effect is at least attenuated when an individual operates in a non-native language.

Separating full-length protein from aggregating proteolytic products using filter flow-through purification.

Separation of full-length protein from proteolytic products is challenging, since the properties used to isolate the protein can also be present in proteolytic products. Many separation techniques risk non-specific protein adhesion and/or require a lot of time, enabling continued proteolysis and aggregation after lysis. We demonstrate that proteolytic products aggregate for two different proteins. As a result, full-length protein can be rapidly separated from these fragments by filter flow-through purification, resulting in a substantial protein purity enhancement. This rapid approach is likely to be useful for intrinsically disordered proteins, whose repetitive sequence composition and flexible nature can facilitate aggregation.

Through-space (19)F-(19)F spin-spin coupling in ortho-fluoro Z-azobenzene.

We report through-space (TS) (19)F-(19)F coupling for ortho-fluoro-substituted Z-azobenzenes. The magnitude of the TS-coupling constant ((TS) JFF ) ranged from 2.2-5.9 Hz. Using empirical formulas reported in the literature, these coupling constants correspond to non-bonded F-F distances (dFF) of 3.0-3.5 Å. These non-bonded distances are significantly smaller than those determined by X-ray crystallography or density functional theory, which argues that simple models of (19)F-(19)F TS spin-spin coupling solely based dFF are not applicable. (1)H, (13)C and (19)F data are reported for both the E and Z isomers of ten fluorinated azobenzenes. Density functional theory [B3YLP/6-311++G(d,p)] was used to calculate (19) F chemical shifts, and the calculated values deviated 0.3-10.0 ppm compared with experimental values.

The role of spinal GABAergic circuits in the control of phrenic nerve motor output.

While supraspinal mechanisms underlying respiratory pattern formation are well characterized, the contribution of spinal circuitry to the same remains poorly understood. In this study, we tested the hypothesis that intraspinal GABAergic circuits are involved in shaping phrenic motor output. To this end, we performed bilateral phrenic nerve recordings in anesthetized adult rats and observed neurogram changes in response to knocking down expression of both isoforms (65 and 67 kDa) of glutamate decarboxylase (GAD65/67) using microinjections of anti-GAD65/67 short-interference RNA (siRNA) in the phrenic nucleus. The number of GAD65/67-positive cells was drastically reduced on the side of siRNA microinjections, especially in the lateral aspects of Rexed's laminae VII and IX in the ventral horn of cervical segment C4, but not contralateral to microinjections. We hypothesize that intraspinal GABAergic control of phrenic output is primarily phasic, but also plays an important role in tonic regulation of phrenic discharge. Also, we identified respiration-modulated GABAergic interneurons (both inspiratory and expiratory) located slightly dorsal to the phrenic nucleus. Our data provide the first direct evidence for the existence of intraspinal GABAergic circuits contributing to the formation of phrenic output. The physiological role of local intraspinal inhibition, independent of descending direct bulbospinal control, is discussed.