GTP-dependent membrane fusion.

Shape changes and topological remodeling of membranes are essential for the identity of organelles and membrane trafficking. Although all cellular membranes have common features, membranes of different organelles create unique environments that support specialized biological functions. The endoplasmic reticulum (ER) is a prime example of this specialization, as its lipid bilayer forms an interconnected system of cisternae, vesicles, and tubules, providing a highly compartmentalized structure for a multitude of biochemical processes. A variety of peripheral and integral membrane proteins that facilitate membrane curvature generation, fission, and/or fusion have been identified over the past two decades. Among these, the dynamin-related proteins (DRPs) have emerged as key players. Here, we review recent advances in our functional and molecular understanding of fusion DRPs, exemplified by atlastin, an ER-resident DRP that controls ER structure, function, and signaling.

CyVerse: Cyberinfrastructure for open science.

CyVerse, the largest publicly-funded open-source research cyberinfrastructure for life sciences, has played a crucial role in advancing data-driven research since the 2010s. As the technology landscape evolved with the emergence of cloud computing platforms, machine learning and artificial intelligence (AI) applications, CyVerse has enabled access by providing interfaces, Software as a Service (SaaS), and cloud-native Infrastructure as Code (IaC) to leverage new technologies. CyVerse services enable researchers to integrate institutional and private computational resources, custom software, perform analyses, and publish data in accordance with open science principles. Over the past 13 years, CyVerse has registered more than 124,000 verified accounts from 160 countries and was used for over 1,600 peer-reviewed publications. Since 2011, 45,000 students and researchers have been trained to use CyVerse. The platform has been replicated and deployed in three countries outside the US, with additional private deployments on commercial clouds for US government agencies and multinational corporations. In this manuscript, we present a strategic blueprint for creating and managing SaaS cyberinfrastructure and IaC as free and open-source software.

Awareness, social cognition, and commitment: Developing a social justice orientation in psychology training programs.

This study investigates how the awareness of social inequities and racism may serve as a foundation for psychology trainees' social justice self-efficacy beliefs, outcome expectations, interests, and commitment. Using the social-cognitive justice developmental framework proposed by Miller et al. (2009), a total of 222 participants were recruited from accredited applied psychology programs across the United States. Participants completed measures assessing their levels of two dimensions of critical consciousness: Egalitarianism and awareness of inequality (Diemer et al., 2017), their colorblind racial attitudes (Neville et al., 2000), and their social justice self-efficacy, outcome expectations, interests, and commitment (Miller et al., 2009). A hypothesized path model was fit to the data. Alternative models were also considered. Results indicated that participants who endorsed egalitarianism and were more aware of social inequities showed greater awareness of racism and, in turn, were more likely to endorse a higher orientation and commitment to social justice. Limitations and implications for future research and training are discussed. (PsycInfo Database Record (c) 2022 APA, all rights reserved).

Pediatric Lawnmower Injuries.

OBJECTIVE: The aim of this study was to evaluate demographics, characteristics, and mechanisms of injuries caused by lawnmowers in children. METHODS: Chart review from 1990 to 2010 at a level I pediatric trauma center identified patients younger than 18 years with lawnmower injuries. Demographics and characteristics of the injuries were analyzed by descriptive statistical analysis. RESULTS: The study identified 88 subjects, with 80% males and 42% of the subjects younger than 5 years. When the lawnmower type was specified, riding lawnmowers caused the majority of injuries (72%). The most common mechanism of injury was related to slipping under lawnmower/being run over (51%). The most common injuries were lacerations (36%), fractures (27%), and amputations (22%); lower extremities were injured more frequently than other body areas (62%). The majority of patients (76%) required hospitalization with a mean length of stay (LOS) of 9.7 days and a mean number of procedures of 4. Complications included 6 infections, 1 tissue necrosis, and 1 death from hemorrhagic shock. Riding-lawnmower injuries were associated with younger children (P < 0.0001). Riding lawnmowers and younger age were associated with longer hospital LOS (P = 0.01, 0.006) and increased number of procedures (P = 0.03, 0.003, respectively). CONCLUSIONS: Lawnmower injuries are still prevalent in children despite national safety recommendations. Injuries seen with riding lawnmowers were associated with younger age, higher number of procedures, longer LOS, and more severe injuries.

The Atlastin C-terminal tail is an amphipathic helix that perturbs the bilayer structure during endoplasmic reticulum homotypic fusion.

Fusion of tubular membranes is required to form three-way junctions found in reticular subdomains of the endoplasmic reticulum. The large GTPase Atlastin has recently been shown to drive endoplasmic reticulum membrane fusion and three-way junction formation. The mechanism of Atlastin-mediated membrane fusion is distinct from SNARE-mediated membrane fusion, and many details remain unclear. In particular, the role of the amphipathic C-terminal tail of Atlastin is still unknown. We found that a peptide corresponding to the Atlastin C-terminal tail binds to membranes as a parallel α helix, induces bilayer thinning, and increases acyl chain disorder. The function of the C-terminal tail is conserved in human Atlastin. Mutations in the C-terminal tail decrease fusion activity in vitro, but not GTPase activity, and impair Atlastin function in vivo. In the context of unstable lipid bilayers, the requirement for the C-terminal tail is abrogated. These data suggest that the C-terminal tail of Atlastin locally destabilizes bilayers to facilitate membrane fusion.

Mental illness and intensification of diabetes medications: an observational cohort study.

BACKGROUND: Mental health condition (MHC) comorbidity is associated with lower intensity care in multiple clinical scenarios. However, little is known about the effect of MHC upon clinicians' decisions about intensifying antiglycemic medications in diabetic patients with poor glycemic control. We examined whether delay in intensification of antiglycemic medications in response to an elevated Hemoglobin A1c (HbA1c) value is longer for patients with MHC than for those without MHC, and whether any such effect varies by specific MHC type. METHODS: In this observational study of diabetic Veterans Health Administration (VA) patients on oral antiglycemics with poor glycemic control (HbA1c ≥8) (N =52,526) identified from national VA databases, we applied Cox regression analysis to examine time to intensification of antiglycemics after an elevated HbA1c value in 2003-2004, by MHC status. RESULTS: Those with MHC were no less likely to receive intensification: adjusted Hazard Ratio [95% CI] 0.99 [0.96-1.03], 1.13 [1.04-1.23], and 1.12 [1.07-1.18] at 0-14, 15-30 and 31-180 days, respectively. However, patients with substance use disorders were less likely than those without substance use disorders to receive intensification in the first two weeks following a high HbA1c, adjusted Hazard Ratio 0.89 [0.81-0.97], controlling for sex, age, medical comorbidity, other specific MHCs, and index HbA1c value. CONCLUSIONS: For most MHCs, diabetic patients with MHC in the VA health care system do not appear to receive less aggressive antiglycemic management. However, the subgroup with substance use disorders does appear to have excess likelihood of non-intensification; interventions targeting this high risk subgroup merit attention.

Ectopic Expression of Neurod1 Is Sufficient for Functional Recovery following a Sensory-Motor Cortical Stroke.

Stroke is the leading cause of adult disability worldwide. The majority of stroke survivors are left with devastating functional impairments for which few treatment options exist. Recently, a number of studies have used ectopic expression of transcription factors that direct neuronal cell fate with the intention of converting astrocytes to neurons in various models of brain injury and disease. While there have been reports that question whether astrocyte-to-neuron conversion occurs in vivo, here, we have asked if ectopic expression of the transcription factor Neurod1 is sufficient to promote improved functional outcomes when delivered in the subacute phase following endothelin-1-induced sensory-motor cortex stroke. We used an adeno-associated virus to deliver Neurod1 from the short GFAP promoter and demonstrated improved functional outcomes as early as 28 days post-stroke and persisting to at least 63 days post-stroke. Using Cre-based cell fate tracking, we showed that functional recovery correlated with the expression of neuronal markers in transduced cells by 28 days post-stroke. By 63 days post-stroke, the reporter-expressing cells comprised ~20% of all the neurons in the perilesional cortex and expressed markers of cortical neuron subtypes. Overall, our findings indicate that ectopic expression of Neurod1 in the stroke-injured brain is sufficient to enhance neural repair.

Human atlastin-3 is a constitutive ER membrane fusion catalyst.

Homotypic membrane fusion catalyzed by the atlastin (ATL) GTPase sustains the branched endoplasmic reticulum (ER) network in metazoans. Our recent discovery that two of the three human ATL paralogs (ATL1/2) are C-terminally autoinhibited implied that relief of autoinhibition would be integral to the ATL fusion mechanism. An alternative hypothesis is that the third paralog ATL3 promotes constitutive ER fusion with relief of ATL1/2 autoinhibition used conditionally. However, published studies suggest ATL3 is a weak fusogen at best. Contrary to expectations, we demonstrate here that purified human ATL3 catalyzes efficient membrane fusion in vitro and is sufficient to sustain the ER network in triple knockout cells. Strikingly, ATL3 lacks any detectable C-terminal autoinhibition, like the invertebrate Drosophila ATL ortholog. Phylogenetic analysis of ATL C-termini indicates that C-terminal autoinhibition is a recent evolutionary innovation. We suggest that ATL3 is a constitutive ER fusion catalyst and that ATL1/2 autoinhibition likely evolved in vertebrates as a means of upregulating ER fusion activity on demand.

Sleep-disordered breathing in Vietnam veterans with posttraumatic stress disorder.

OBJECTIVE: : To study the prevalence of sleep-disordered breathing (SDB) in Vietnam- era veterans. METHODS: : This was an observational study of Vietnam-era veterans using unattended, overnight polysomnography, cognitive testing, and genetic measures. RESULTS: : A sample of 105 Vietnam-era veterans with posttraumatic stress disorder: 69% had an Apnea Hypopnea Index >10. Their mean body mass index was 31, "obese" by Centers for Disease Control and Prevention criteria, and body mass index was significantly associated with Apnea Hypopnea Index (Spearman r = 0.41, N = 97, p < 0.0001). No significant effects of sleep-disordered breathing or apolipoprotein status were found on an extensive battery of cognitive tests. CONCLUSION: : There is a relatively high prevalence of SDB in these patients which raises the question of to what degree excess cognitive loss in older PTSD patients may be due to a high prevalence of SDB.

Regioselective reduction of 3-substituted N-acylpyrazinium salts toward the synthesis of 1,2-dihydropyrazines.

The regioselective reduction of 3-substituted N-acylpyrazinium salts with n-Bu(3)SnH has been developed for the synthesis of 3-substituted 1,2-dihydropyrazines in yields of 56-94%. Substitution of the pyrazinium salts with electron-donating groups favors the formation of the 1,2-isomers as a result of their better stability over the 1,6-isomers. Under mild acidic conditions, 3-methoxy substituted 1,2-dihydropyrazine was easily hydrolyzed in excellent yield to Δ(5)-2-oxopiperazine.

In vitro metabolism of piperaquine is primarily mediated by CYP3A4.

Piperaquine (PQ) is part of a first-line treatment regimen for Plasmodium falciparum malaria recommended by the World Health Organization (WHO). We aimed to determine the major metabolic pathway(s) of PQ in vitro. A reliable, validated tandem mass spectrometry method was developed. Concentrations of PQ were measured after incubation with both human liver microsomes (HLMs) and expressed cytochrome P450 enzymes (P450s). In pooled HLMs, incubations with an initial PQ concentration of 0.3 µM resulted in a 34.8 ± 4.9% loss of substrate over 60 min, corresponding to a turnover rate of 0.009 min(-1) (r(2) = 0.9223). Miconazole, at nonspecific P450 inhibitory concentrations, resulted in almost complete inhibition of PQ metabolism. The greatest inhibition was demonstrated with selective CYP3A4 (100%) and CYP2C8 (66%) inhibitors. Using a mixture of recombinant P450 enzymes, turnover for PQ metabolism was estimated as 0.0099 min(-1); recombinant CYP3A4 had a higher metabolic rate (0.017 min(-1)) than recombinant CYP2C8 (p < .0001). Inhibition of CYP3A4-mediated PQ loss was greatest using the selective inhibitor ketoconazole (9.1 ± 3.5% loss with ketoconazole vs 60.7 ± 5.9% with no inhibitor, p < .0001). In summary, the extent of inhibition of in vitro metabolism with ketoconazole (83%) denotes that PQ appears to be primarily catalyzed by CYP3A4. Further studies to support these findings through the identification and characterization of PQ metabolites are planned.

Membrane fusion by Drosophila atlastin does not require GTP hydrolysis.

Atlastin (ATL) GTPases undergo trans dimerization and a power strokelike crossover conformational rearrangement to drive endoplasmic reticulum membrane fusion. Fusion depends on GTP, but the role of nucleotide hydrolysis has remained controversial. For instance, nonhydrolyzable GTP analogs block fusion altogether, suggesting a requirement for GTP hydrolysis in ATL dimerization and crossover, but this leaves unanswered the question of how the ATL dimer is disassembled after fusion. We recently used the truncated cytoplasmic domain of wild-type Drosophila ATL (DATL) and a novel hydrolysis-deficient D127N variant in single turnover assays to reveal that dimerization and crossover consistently precede GTP hydrolysis, with hydrolysis coinciding more closely with dimer disassembly. Moreover, while nonhydrolyzable analogs can bind the DATL G domain, they fail to fully recapitulate the GTP-bound state. This predicted that nucleotide hydrolysis would be dispensable for fusion. Here we report that the D127N variant of full-length DATL drives both outer and inner leaflet membrane fusion with little to no detectable hydrolysis of GTP. However, the trans dimer fails to disassemble and subsequent rounds of fusion fail to occur. Our findings confirm that ATL mediated fusion is driven in the GTP-bound state, with nucleotide hydrolysis serving to reset the fusion machinery for recycling.

Reconstitution of human atlastin fusion activity reveals autoinhibition by the C terminus.

ER network formation depends on membrane fusion by the atlastin (ATL) GTPase. In humans, three paralogs are differentially expressed with divergent N- and C-terminal extensions, but their respective roles remain unknown. This is partly because, unlike Drosophila ATL, the fusion activity of human ATLs has not been reconstituted. Here, we report successful reconstitution of fusion activity by the human ATLs. Unexpectedly, the major splice isoforms of ATL1 and ATL2 are each autoinhibited, albeit to differing degrees. For the more strongly inhibited ATL2, autoinhibition mapped to a C-terminal α-helix is predicted to be continuous with an amphipathic helix required for fusion. Charge reversal of residues in the inhibitory domain strongly activated its fusion activity, and overexpression of this disinhibited version caused ER collapse. Neurons express an ATL2 splice isoform whose sequence differs in the inhibitory domain, and this form showed full fusion activity. These findings reveal autoinhibition and alternate splicing as regulators of atlastin-mediated ER fusion.

Informatics response to address the COVID-19 pandemic in a safety net healthcare system.

In service of particularly vulnerable populations, safety net healthcare systems must nimbly leverage health information technology (IT), including electronic health records (EHRs), to coordinate the medical and public health response to the novel coronavirus (COVID-19). Six months after the San Francisco Department of Public Health implemented a new EHR across its hospitals and citywide clinics, California declared a state of emergency in response to COVID-19. This paper describes how the IT and informatics teams supported San Francisco Department of Public Health's goals of expanding the safety net healthcare system capacity, meeting the needs of specific vulnerable populations, increasing equity in COVID-19 testing access, and expanding public health analytics and research capacity. Key enabling factors included critical partnerships with operational leaders, early identification of priorities, a clear governance structure, agility in the face of rapidly changing circumstances, and a commitment to vulnerable populations.

Nucleotide-dependent self-assembly of Nucleoside Diphosphate Kinase (NDPK) in vitro.

In addition to their role in nucleotide homeostasis, members of the Nucleoside Diphosphate Kinase (NDPK) family have been implicated in tumor metastasis, cell migration and vesicle trafficking. Although its role in most cases depends on nucleotide catalysis, a precise understanding of how the catalytic activity of NDPK supports its function in diverse processes is lacking. Here we report that wild type, but not catalytically inactive (H118C) NDPKB, undergoes dynamic self-assembly into ordered 20-25 nm diameter filaments in vitro. Self-assembly is nucleoside triphosphate dependent, GTP being most effective at promoting polymer formation. In addition, polymerization appears to depend on formation of the phosphoryl-Histidine intermediate of the enzyme, suggesting a previously unappreciated conformational change in NDPK during its catalytic cycle. We hypothesize that the observed nucleotide-dependent self-assembly property of NDPKB may reflect a key feature of NDPK enzymes that enables their function in diverse processes.

Nucleoside diphosphate kinase B (NDKB) scaffolds endoplasmic reticulum membranes in vitro.

The mechanisms that structure the mammalian endoplasmic reticulum (ER) network are not fully understood. Here we show that salt extraction of semi-intact normal rat kidney (NRK) fibroblasts and subsequent incubation of the extracted cells with ATP resulted in dramatic ER network retraction. Under these conditions, addition of a single protein, Nucleoside Diphosphate Kinase B (NDKB), was sufficient to reverse the retraction and to promote ER network extension. The underlying mechanism of membrane extension involved direct lipid binding, as NDKB bound phosphatidylinositol (PtdIns)(4)P, PtdIns(4,5)P(2) and phosphatidic acid (PA); binding to these anionic lipids required clusters of basic residues on the surface of the NDKB hexamer; and amino acid changes in NDKB that blocked lipid binding also blocked ER network extension. Remarkably, purified NDKB transformed a uniform population of synthetic lipid vesicles into extensive membrane networks, and this also required its phospholipid-binding activity. Altogether these results identify a protein sufficient to scaffold extended membrane networks, and suggest a possible role for NDKB-like proteins, as well as phosphoinositides and/or acidic phospholipids, in modulating ER network morphogenesis.

Systematic review on outcome of free functioning muscle transfers for elbow flexion in brachial plexus injuries.

Elbow flexion is widely regarded as the most important function to restore in brachial plexus injuries. Free functioning muscle transfer surgery is indicated in patients with delayed presentation or failure of other primary procedures. Results of the transfer surgeries have been reported in the form of case series, but no further studies are available. This systematic review aims to provide a deeper understanding of this complex surgery and consists of 19 articles that include 364 patients. Data on injury characteristics, surgical techniques, complications as well as outcome measures were analysed. Our results show that functional muscle transfer for elbow flexion enables 87% and 65% of patients to achieve a useful power grade of ≥ 3 and ≥ 4, respectively, although other important outcome factors should be considered.

Distress and pain during pelvic examinations: effect of sexual violence.

OBJECTIVE: To estimate the range and severity of distress and pain during pelvic examinations among female veterans with and without histories of sexual violence, and to examine whether posttraumatic stress disorder explains additional variance in examination-related distress and pain above that accounted for by exposure to sexual violence. METHODS: We employed a cross-sectional cohort design in which 67 selected female veterans completed self-administered questionnaires to assess history of sexual violence and experiences of distress and pain associated with the pelvic examination. A subsample of 49 completed an assessment for posttraumatic stress disorder approximately 2 weeks later. RESULTS: Distress associated with the pelvic examination was highest for women with prior sexual violence and posttraumatic stress disorder (median 5.49), next highest for women with sexual violence only (median 2.44), and lowest for women with neither (median 0), P=.015. Higher ratings of pain were also found among women with sexual violence (median 2.5) compared with those without (median 0), P=.04. However, posttraumatic stress disorder was not linked with increased pain from speculum insertion beyond that accounted for by sexual violence; limited power may have precluded detection of this effect. CONCLUSION: Distress and pain during pelvic examinations may indicate a history of previous sexual violence, particularly in those with posttraumatic stress disorder. Extra sensitivity to the special needs of this population is warranted and may contribute positively to the quality of patients' experiences. LEVEL OF EVIDENCE: II.

Subclinical anxiety symptoms, sleep, and daytime dysfunction in older adults with primary insomnia.

"Both Insomnia complaints and anxiety-related distress are common in older adults, and are associated with poor daytime functioning. We investigated whether subclinical levels of anxiety were associated with sleep disturbance and daytime functioning in older adults who met diagnostic criteria for primary insomnia, and therefore but did not meet criteria for depression or an anxiety disorder. After adjustment for depressive symptoms, elevated state anxiety was associated with higher levels of wake after sleep onset (measured by both actigraphy and sleep log) and shorter sleep sleep onset latency (measured by sleep log). Higher levels of trait anxiety were associated with greater wake after sleep onset (measured by sleep log). Elevated state and trait anxiety were associated with worse and social functioning, and higher levels of trait anxiety were associated with worse role functioning. Thus, subclinical anxiety symptoms may be an important target for clinical intervention to improve sleep and functioning in older adults with primary insomnia."