bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanically Induced Nuclear Shuttling of β-Catenin Requires Co-transfer of Actin
Authors: Sen Buer , Xie Zhihui , Howard Sean , Styner Maya , van Wijnen Andre J , Uzer Gunes , Rubin Janet
Published: February 2022

Mesenchymal stem cells (MSCs) respond to environmental forces with both cytoskeletal re-structuring and activation of protein chaperones of mechanical information, β-catenin, and yes-associated protein 1 (YAP1). To function, MSCs must differentiate between dynamic forces such as cyclic strains of extracellular matrix due to physical activity and static strains due to ECM stiffening. To delineate how MSCs recognize and respond differently to both force types, we compared effects of dynamic (200 cycles × 2%) and static (1 × 2% hold) strain on nuclear translocation of β-catenin and YAP1 at 3 hours after force application. Dynamic strain induced nuclear accumulation of β-catenin, and increased cytoskeletal actin structure and cell stiffness, but had no effect on nuclear YAP1 levels. Critically, both nuclear actin and nuclear stiffness increased along with dynamic strain-induced β-catenin transport. Augmentation of cytoskeletal structure using either static strain or lysophosphatidic acid did not increase nuclear content of β-catenin or actin, but induced robust nuclear increase in YAP1. As actin binds β-catenin, we considered whether β-catenin, which lacks a nuclear localization signal, was dependent on actin to gain entry to the nucleus. Knockdown of cofilin-1 (Cfl1) or importin-9 (Ipo9), which co-mediate nuclear transfer of G-actin, prevented dynamic strain-mediated nuclear transfer of both β-catenin and actin. In sum, dynamic strain induction of actin re-structuring promotes nuclear transport of G-actin, concurrently supporting nuclear access of β-catenin via mechanisms used for actin transport. Thus, dynamic and static strain activate alternative mechanoresponses reflected by differences in the cellular distributions of actin, β-catenin, and YAP1.
Brown Rot Disease in Stored Nectarines: Modeling the Combined Effects of Preharvest and Storage Conditions
Authors: Casagrande Enrico , Génard Michel , Lurol Sébastien , Charles Florence , Bevacqua Daniele , Martinetti Davide , Lescourret Françoise
Published: February 2022

Brown rot in stored stone fruits, caused by Monilinia spp., may be due to preharvest and storage factors, but the combined effect of these factors has yet to be investigated. We set up two experiments to monitor the progression of brown rot during the storage of nectarines subjected to various preharvest and storage conditions. We assessed the effects of different agricultural practices (irrigation regimen × fruit load) and harvest dates on brown rot progress during storage in 2018 and the effect of different storage temperatures in 2019. We found that the cumulative incidence of brown rot during storage increased with individual fruit mass, which was influenced by agricultural practices, and for later harvest dates. It also increased with storage temperature. We observed that during storage no secondary infections developed in nectarines not in direct contact with fruits infected with Monilinia laxa. These findings led to the identification of candidate variables describing the brown rot risk on nectarines during storage, such as individual fruit mass, meteorological conditions before fruit harvest, prevalence of brown rot at harvest, and storage temperature. We used these variables to build a mathematical model for estimating the time-to-appearance of brown rot symptoms in stored nectarines. This model fitted the experimental data well, highlighting the need to pay greater attention to the interaction between preharvest and storage conditions. This model could be used to evaluate management strategies for reducing the impact of brown rot in nectarines during storage.
Being at EASE: Student Perceptions of Interprofessional Practice Following an Experiential Interprofessional Application for Sensitive Patient Encounters
Authors: Osborne Victoria A. , Rosenbloom Sylvie , Glenn Constance H.
Published: February 2022

Learning to interact and collaborate with other professions is key to optimal patient care and is best achieved when started during one’s education. This study implemented an interprofessional education simulation with family nurse practitioner and social work students utilizing standardized patients who presented with sensitive issues. The Student Perceptions of Interprofessional Clinical Education-Revised and Readiness for Interprofessional Learning Scale questionnaires were given before and after the activity. Significant changes were noted in both student groups regarding attitudes and perceptions of interprofessional collaboration. Interprofessional collaboration is crucial. Introducing interprofessional education in the education curriculum of health professionals can help future providers achieve essential competencies.
Small intestinal bacterial overgrowth
Authors: Perkins Amanda
Published: February 2022

Prevent detrimental patient outcomes by learning how to spot this often underdiagnosed condition.
Evaluating a patient safety learning laboratory to create an interdisciplinary ecosystem for health care innovation
Authors: Atkinson Mariam Krikorian , Benneyan James C. , Bambury Elizabeth A. , Schiff Gordon D. , Phillips Russell S. , Hunt Lindsay S. , Belleny Deanna , Singer Sara J.
Published: February 2022

Background

In response to the complexity, challenges, and slow pace of innovation, health care organizations are adopting interdisciplinary team approaches. Systems engineering, which is oriented to creating new, scalable processes that perform with higher reliability and lower costs, holds promise for driving innovation in the face of challenges to team performance. A patient safety learning laboratory (lab) can be an essential aspect of fostering interdisciplinary team innovation across multiple projects and organizations by creating an ecosystem focused on deploying systems engineering methods to accomplish process redesign.

Purpose

We sought to identify the role and activities of a learning ecosystem that support interdisciplinary team innovation through evaluation of a patient safety learning lab.

Methods

Our study included three participating learning lab project teams. We applied a mixed-methods approach using a convergent design that combined data from qualitative interviews of team members conducted as teams neared the completion of their redesign projects, as well as evaluation questionnaires administered throughout the 4-year learning lab.

Results

Our results build on learning theories by showing that successful learning ecosystems continually create alignment between interdisciplinary teams’ activities, organizational context, and innovation project objectives. The study identified four types of alignment, interpersonal/interprofessional, informational, structural, and processual, and supporting activities for alignment to occur.

Conclusion

Interdisciplinary learning ecosystems have the potential to foster health care improvement and innovation through alignment of team activities, project goals, and organizational contexts.

Practice Implications

This study applies to interdisciplinary teams tackling multilevel system challenges in their health care organization and suggests that the work of such teams benefits from the four types of alignment. Alignment on all four dimensions may yield best results.
Protean Clinical Features and Diagnostic Dilemmas of Pancreatic Tuberculosis
Authors: Shera Irfan Ali , Khan Mosin Saleem , Gupta Rubal
Published: February 2022

Tuberculosis (TB) is a systemic disease with protean manifestations and an important health problem. Approximately 15% of cases involve extrapulmonary sites with pancreatic TB (PTB) being rare. Clinically and radiologically, it may mimic a pancreatic malignancy, thus posing a clinical dilemma. The vague symptomatology and lack of clinical findings make it trickier to diagnose. We present a case series of patients with PTB and their tricky diagnosis and management. Endoscopic ultrasound–guided fine needle aspiration cytology along with various ancillary tests were used for the correct diagnosis. Endoscopic ultrasound–guided fine needle aspiration cytology provided the diagnosis, especially with the help of an expert cytologist experienced in the diagnosis of TB, thereby avoiding unnecessary laparotomy. Response to antituberculous therapy for 6 to 12 months is predictable and complete in these patients. Endoscopic ultrasound–guided fine needle aspiration cytology is the modality of choice for diagnosing PTB. However, patients still need to be followed up carefully for subjective and objective response to therapy to rule out the rare possibility of TB coexisting with malignancy, especially in endemic areas.
Faculty, Preceptor, and Students' Perceptions of the Need for Trauma-Informed Education
Authors: Kuzma Elizabeth K. , Cannon Lindsay M. , Coolidge Elizabeth M. , Harris Megan , Buckley Courtney , Chapin Emily , Coley Katrina , Arbogast-Wilson Harbor Mariel
Published: February 2022

Background:

The widespread physical, mental, and emotional health impacts of trauma are well established. Trauma-informed care (TIC) is an approach that uses knowledge about trauma and its effects to create safe care environments.

Purpose:

Using a concurrent mixed-methods design, this study assessed faculty, preceptor, and students' perceptions about the need for TIC content in nursing education.

Methods:

Semistructured interviews were conducted with 15 faculty, and cross-sectional survey data were collected from a nonprobability sample of 99 nursing students at a large Midwestern university to evaluate the need for education on TIC.

Results:

Faculty and preceptors stressed the importance of education on TIC and discussed barriers and facilitators to implementation. Nursing students reported that it is important to learn about TIC, yet do not feel prepared to provide TIC.

Conclusions:

The results illustrate the need for nursing content on TIC and provide recommendations for trauma-informed educational practices.
Hypogonadism: a neglected comorbidity in young and middle-aged HIV-positive men on effective combination antiretroviral therapy
Authors: Lachâtre Marie , Pasquet Armelle , Ajana Faïza , Soudan Benoit , Quertainmont Yann , Lion Georges , Durand Emmanuel , Bocket Laurence , Mole Martine , Cornavin Pauline , Catalan Pilartxo , Senneville Éric , Goujard Cécile , Boufassa Faroudy , Cheret Antoine
Published: February 2022

Objective:

Male hypogonadism is poorly characterized in young-to-middle-aged people with HIV (PWH). We used a reliable free testosterone assay to assess the prevalence and predictive factors for male hypogonadism in PWH on effective combined antiretroviral therapy (cART).

Design:

A French cross-sectional study from January 2013 to June 2016.

Methods:

We included HIV-1-infected men aged between 18 and 50years with HIV loads of 50 RNA copies/ml or less, on effective cART for at least 6 months. Hypogonadism was defined, according to guidelines, as a mean calculated serum free testosterone concentration less than 70pg/ml (Vermeulen equation). Sociodemographic, anthropo-metric, bone-densitometry, hormonal, immunovirological, metabolic, and therapeutic parameters were collected. The IIEF-5, HAM-D, and AMS scales, respectively, assessed erectile function, depression, and quality of life.

Results:

Overall, 240 patients were enrolled, 231 were analyzed. Low free testosterone concentrations (<70pg/ml) were recorded in 20 patients (8.7%), and were exclusively of secondary origin. In multivariable analysis, the risk factors predictive of male hypogonadism were age more than 43 years [adjusted odds ratio (aOR) 3.17, 95% confidence interval (95% CI) 1.02–9.86;P = 0.04], total fat percentage more than 19% (aOR3.5, 95% CI 1.18–10.37; P = 0.02), and treatment including efavirenz (aOR3.77, 95% CI 1.29–10.98;P = 0.02). A nadir CD4+ T-cell count more than 200 cells/μl (aOR 0.22, 95% CI 0.07–0.65;P < 0.01) were protective.

Conclusion:

Male hypogonadism remains common in young-to-middle-aged PWH with stably suppressed viral replication. Treatment including efavirenz, being over 43 years old, and having a total body fat percentage greater than 19% could be used as criteria for identifying PWH at risk. Early screening for male hypogonadism might improve care by identifying patients requiring testosterone replacement.
Decreased hepatitis B virus vaccine response among HIV-positive infants compared with HIV-negative infants in Botswana
Authors: Shaver Zachary M. , Anderson Motswedi , Bhebhe Lynnette , Baruti Kabo , Choga Wonderful T. , Ngidi Julia , Mbangiwa Tshepiso , Tau Modiri , Setlhare Ditiro R. , Melamu Pinkie , Phinius Bonolo B. , Musonda Rosemary , Mine Madisa , Moyo Sikhulile , Gaseitsiwe Simani
Published: February 2022

Objectives:

We sought to determine vaccine antibody titres and the prevalence of hepatitis B surface antigen (HBsAg) in both HIV-positive and HIV-negative infants born to HIV-positive mothers in Botswana.

Design:

This was a retrospective cross-sectional study using 449 archived dried blood spot samples from both HIV-positive and HIV-negative infants collected between 2016 and 2018.

Methods:

We screened dried blood spot samples for HBsAg and determined hepatitis B surface antibody titres. We determined hepatitis B virus (HBV) genotypes by amplifying 415 base-pairs of the surface region.

Results:

HIV-positive infants mounted a significantly lower immune response to the HBV vaccine (P 
< 0.001). Furthermore, a lower proportion of HIV-positive infants had protective hepatitis B surface antibody titres (74.5%) than HIV-negative infants (89.2%) (P < 0.001). HIV-positive infants were older and 50.9% of them had completed vaccination (P = 0.018). Of the 449 infant samples tested, three (0.67%) were positive for HBsAg. Of the three HBsAg-positive infants, two had protective titres (>10 mIU/ml). Two of the three HBV-positive infants were infected with genotype D3 and had no drug-resistance or escape mutations.

Conclusion:

Vaccine response was lower among HIV-positive infants compared with HIV-negative infants. HBV infections were observed in both HIV-positive and HIV-negative infants in Botswana. Studies to investigate additional preventive strategies to reduce HBV mother-to-child transmission are recommended.
Changes in Performance of Apical Suspension at the Time of Surgery for Prolapse: Assessment of the Influence of the American Urogynecologic Society and American College of Obstetricians and Gynecologists Practice Bulletin
Authors: Luchristt Douglas , Zemtsov Gregory , Jelovsek J. Eric
Published: February 2022

Objectives

To assess the association of publication of the American Urogynecologic Society (AUGS)/American College of Obstetricians and Gynecologists (ACOG) Practice Bulletin on pelvic organ prolapse and performance of an apical suspension at the time of surgery for pelvic organ prolapse.

Methods

Surgical procedures performed with a primary diagnosis of uterovaginal or female genital prolapse, cystocele, or enterocele were isolated from the 2011 to 2019 American College of Surgeons National Surgical Quality Improvement Program Database. An autoregressive interrupted time series regression estimated the overall temporal trend in performance of an apical suspension and assessed for a change in trend associated with publication of the AUGS/ACOG Practice Bulletin in April 2017. A stratified analysis was also performed depending on performance of a concomitant hysterectomy, and sensitivity analysis was performed using only diagnoses of uterovaginal or vaginal vault prolapse.

Results

There were 72,194 individuals identified; 83.4% had a diagnosis of uterovaginal or female genital prolapse, 15.2% cystocele and 1.4% enterocele. Only 36.6% of cases had an apical suspension. Prior to the practice bulletin publication, performance of an apical suspension grew at 0.19% per quarter (95% confidence interval [CI], 0.07–0.31), with a trend toward increased utilization (+0.12%; 95% CI, −0.06 to 0.30) after publication. The increase was greater among cases with a concomitant hysterectomy (+0.35%; 95% CI, 0.08–0.62). Sensitivity analyses found similar changes in trend.

Conclusions

Performance of apical suspensions during surgery for prolapse remains low and is increasing at less than 1% per year. The AUGS/ACOG practice guidelines were associated with minimal changes in this pattern. Incentives or other strategies may be needed to further encourage standard of care management of prolapse.
Incidence of Midurethral Sling Revision or Removal by Its Timing With Prolapse Surgery
Authors: Boyd Sarah Samuel , Long Jaime B. , Agbese Edeanya , Leslie Douglas
Published: February 2022

Objective

The aim of the study was to evaluate incidence of midurethral sling removal/revision based on timing with surgery for pelvic organ prolapse.

Methods

This was a retrospective cohort study of women who underwent midurethral sling placement in a claims-based database of women 65 years or older. Three groups were identified using the Current Procedural Terminology codes: (1) isolated sling, (2) concomitant sling, and (3) prolapse surgery and staged sling after prolapse surgery. In the staged group, placement of sling was identified within 18 months after index prolapse surgery. Fascial grafts were excluded. Sling removal/revision was identified across 3 years after sling surgery using Current Procedural Terminology code 57287. Rates of sling removal/revision were calculated by group. Comparisons were made using the χ2 test and analysis of variance. Cumulative incidence of removal/revision was evaluated using the Kaplan-Meier curves. Cox proportional hazards was performed to evaluate factors influencing removal/revision.

Results

We identified 39,381 isolated MUSs, 25,389 concomitant, and 886 staged. The rate of sling removal/revision was 3.52%. Rates of removal/revision differed between groups (7% staged vs 3.94% concomitant vs 3.17% isolated sling, P < 0.001). Compared with the staged group, the rate of removal/revision was lower in the isolated sling group (relative risk, 0.4550; 95% confidence interval [CI], 0.358–0.568) and the concomitant group (relative risk, 0.5666; 95% CI, 0.4450–0.7287). After adjusting for patient characteristics, sling revision or removal remained significantly less in the isolated MUS (hazard ratio, 0.50; 95% CI, 0.39–0.65) and concomitant (odds ratio, 0.55; 95% CI, 0.43–0.71) groups.

Conclusions

Sling removal/revision is higher when it is staged after prolapse surgery compared with isolated and concomitant placement. Future studies are needed to confirm these findings in a controlled population.
Hospital sink traps as a potential source of the emerging multidrug-resistant pathogen Cupriavidus pauculus: characterization and draft genome sequence of strain MF1
Authors: Butler James , Kelly Sean D. , Muddiman Katie J. , Besinis Alexandros , Upton Mathew
Published: February 2022

Introduction.

Cupriavidus pauculus

is historically found in soil and water but has more recently been reported to cause human infection and death. Hospital sink traps can serve as a niche for bacterial persistence and a platform for horizontal gene transfer, with evidence of dissemination of pathogens in hospital plumbing systems driving nosocomial infection.

Gap Statement. This paper presents the first

C. pauculus

strain isolated from a hospital sink trap. There are only six genome assemblies available on NCBI for

C. pauculus

; two of these are PacBio/Illumina hybrids. This paper presents the first ONT/Illumina hybrid assembly, with five contigs. The other assemblies available consist of 37, 38, 111 and 227 contigs. This paper also presents data on biofilm formation and lethal dose in Galleria mellonella; there is little published information describing these aspects of virulence.

Aim. The aims were to identify the isolate found in a hospital sink trap, characterize its genome, and assess whether it could pose a risk to human health.

Methodology. The genome was sequenced, and a hybrid assembly of short and long reads produced. Antimicrobial susceptibility was determined by the broth microdilution method. Virulence was assessed by measuring in vitro biofilm formation compared to

Pseudomonas aeruginosa

and in vivo lethality in Galleria mellonella larvae.

Results. The isolate was confirmed to be a strain of

C. pauculus

, with a 6.8 Mb genome consisting of 6468 coding sequences and an overall G+C content of 63.9 mol%. The genome was found to contain 12 antibiotic resistance genes, 8 virulence factor genes and 33 metal resistance genes. The isolate can be categorized as resistant to meropenem, amoxicillin, amikacin, gentamicin and colistin, but susceptible to cefotaxime, cefepime, imipenem and ciprofloxacin. Clear biofilm formation was seen in all conditions over 72 h and exceeded that of

P. aeruginosa

when measured at 37 °C in R2A broth. Lethality in G. mellonella larvae over 48 h was relatively low.

Conclusion. The appearance of a multidrug-resistant strain of

C. pauculus

in a known pathogen reservoir within a clinical setting should be considered concerning. Further work should be completed to compare biofilm formation and in vivo virulence between clinical and environmental strains, to determine how easily environmental strains may establish human infection. Infection control teams and clinicians should be aware of the emerging nature of this pathogen and further work is needed to minimize the impact of contaminated hospital plumbing systems on patient outcomes.
Anterior Segment Parameters on Optical Coherence Tomography in Healthy Children
Authors: Nadeem Sana
Published: February 2022

Purpose

To assess the anterior segment parameters in healthy children, using high resolution spectral domain optical coherence tomography (SD-OCT).

Design

Prospective, observational study

Methods

108 eyes of 54 healthy children ≤16 years were included after a thorough eye examination. Significant refractive error (>±5 DS), history of previous glaucoma, ocular pathology, intraocular surgery, trauma or systemic ailments were excluded. The anterior segment analysis was done by Optopol Revo 80® high resolution SD-OCT. Central corneal thickness (CCT), Angle opening distance (AOD), Anterior chamber (AC) angle, Trabecular iris space area (TISA), Trabecular iris angle (TIA), iris thickness at 2 mm, internal AC diameter and lens vault were evaluated.

Results

Mean age was 11.38±3.18 years with a female predominance; 29 (53.7%). Mean IOP was 13.63±2.90 mmHg, mean axial length was 22.82±0.89 mm, mean spherical equivalent was -0.14±1.28 D, mean CCT was 532.6±46.09mm and mean CDR was 0.31±0.19. The mean internal AC diameter was 11609.15±2354 µm. The nasal AC angle was 53.54±11.82° and significantly wider than the temporal AC angle; 50.37±10.26° [p=0.008]. The nasal AOD500 was 0.9±0.33 mm and not significantly greater than the temporal AOD500, 0.85±0.28 mm [p=0.09]. The nasal AOD750 was 1.21±0.45 mm, significantly greater than the temporal AOD750, 1.06±0.32 mm [p=0.001]. The nasal TIA was 41.75°; not significantly greater than the temporal TIA, 40.24° [p=0.246]. The nasal TISA500 was 0.33±0.15 mm2; not significantly wider than the temporal TISA500, 0.31±0.10 mm2 [p=0.056]. The nasal TISA750 was 0. 59±0.22 mm2; not significantly wider than the temporal TISA750, 0.56±0.21 mm2 [p=0.14]. The nasal iris thickness at 2 mm from pupil was 483.54±129.56 µm; not significantly different from the temporal iris thickness, 505.8±138.85 µm [p=0.08]. The mean lens vault was - 519.58±359µm.

Conclusions

Our study data depicts the normal anterior segment parameters in healthy children. This will be useful in diagnosing and managing ocular pediatric pathology.

Key Messages

Anterior segment OCT is still an emerging technique especially in pediatric eye assessment

Anterior segment evaluation via OCT has not been reported in our pediatric population

Our study describes a detailed assessment of anterior segment in pediatric eyes

Reporting results of normative data helps in diagnosing ocular pathology
Wall teichoic acids facilitate the release of toxins from the surface of Staphylococcus aureus
Authors: Brignoli Tarcisio , Douglas Edward , Duggan Seána , Fagunloye Olayemi Grace , Adhikari Rajan , Aman M. Javad , Massey Ruth C.
Published: February 2022

A major feature of the pathogenicity of Staphylococcus aureus is its ability to secrete cytolytic toxins. This process involves the translocation of the toxins from the cytoplasm, through the bacterial membrane and the cell wall to the external environment. The process of their movement through the membrane is relatively well defined, involving both general and toxin-specific secretory systems. Movement of the toxins through the cell wall was considered to involve the passive diffusion of the proteins through the porous cell wall structures, however, recent work suggests that this is more complex, and here we demonstrate a role for the wall teichoic acids (WTA) in this process. Utilising a genome-wide association approach we identified a polymorphism in the locus encoding the WTA biosynthetic machinery as associated with the cytolytic activity of the bacteria. We verified this association using an isogenic mutant set and found that WTA is required for the release of several cytolytic toxins from the bacterial cells. We show this effect is mediated by a change in the electrostatic charge across the cell envelope that results from the loss of WTA. As a major target for the development of novel therapeutics, it is important that we fully understand the entire process of cytolytic toxin production and release. These findings open up a new aspect to this process that requires in-depth investigation, while also demonstrating that clinical isolates can utilise WTA production to vary their cytotoxicity, thereby altering their pathogenic capabilities.

Importance

The production and release of cytolytic toxins is a critical aspect to the pathogenicity of many bacterial pathogens. In this study we demonstrate a role for wall teichoic acids, molecules that are anchored to the peptidoglycan of the bacterial cell wall, in the release of toxins from S. aueus cells into the extracellular environment. Our findings suggest this effect is mediated by a gradient of electrostatic charge the presence of the negatively charged WTA molecules create across the cell envelope. This work brings an entirely new aspect to our understanding of the cytotoxicity of S. aureus and demonstrates a further means by which this major human pathogen can adapt its pathogenic capabilities.
Personalized structural biology reveals the molecular mechanisms underlying heterogeneous epileptic phenotypes caused by de novo KCNC2 variants
Authors: Mukherjee Souhrid , Cassini Thomas A. , Hu Ningning , Yang Tao , Li Bian , Shen Wangzhen , Moth Christopher W. , Rinker David C. , Sheehan Jonathan H. , Cogan Joy D. ,   Undiagnosed Diseases Network, Newman John H. , Hamid Rizwan , Macdonald Robert L. , Roden Dan M. , Meiler Jens , Kuenze Georg , Phillips John A. , Capra John A.
Published: February 2022

Background

Next-generation whole exome sequencing (WES) is ubiquitous as an early step in the diagnosis of rare diseases and the interpretation of variants of unknown significance (VUS). Developmental and epileptic encephalopathies (DEE) are a group of rare devastating epilepsies, many of which have unknown causes. Increasing WES in the clinic has identified several rare monogenic DEEs caused by ion channel variants. However, WES often fails to provide actionable insight, due to the challenges of proposing functional hypotheses for candidate variants. Here, we describe a “personalized structural biology” (PSB) approach that addresses this challenge by leveraging recent innovations in the determination and analysis of protein 3D structures.

Results

We illustrate the power of the PSB approach in an individual from the Undiagnosed Diseases Network (UDN) with DEE symptoms who has a novel de novo VUS in KCNC2 (p.V469L), the gene that encodes the Kv3.2 voltage-gated potassium channel. A nearby KCNC2 variant (p.V471L) was recently suggested to cause DEE-like phenotypes. We find that both variants are located in the conserved hinge region of the S6 helix and likely to affect protein function. However, despite their proximity, computational structural modeling suggests that the V469L variant is likely to sterically block the channel pore, while the V471L variant is likely to stabilize the open state. Biochemical and electrophysiological analyses demonstrate heterogeneous loss-of-function and gain-of-function effects, respectively, as well as differential inhibition in response to 4-aminopyridine (4-AP) treatment. Using computational structural modeling and molecular dynamics simulations, we illustrate that the pore of the V469L variant is more constricted increasing the energetic barrier for K+ permeation, whereas the V471L variant stabilizes the open conformation

Conclusions

Our results implicate KCNC2 as a causative gene for DEE and guided the interpretation of a UDN case. They further delineate the molecular basis for the heterogeneous clinical phenotypes resulting from two proximal pathogenic variants. This demonstrates how the PSB approach can provide an analytical framework for individualized hypothesis-driven interpretation of protein-coding VUS suspected to contribute to disease.
Hospitalisation for COVID-19 predicts long lasting cerebrovascular impairment: A prospective observational cohort study
Authors: Tsvetanov Kamen A , Spindler Lennart R B , Stamatakis Emmanuel A , Newcombe Virginia FJ , Lupson Victoria C , Chatfield Doris A , Manktelow Anne E , Outtrim Joanne G , Elmer Anne , Kingston Nathalie , Bradley John R , Bullmore Edward T , Rowe James B , Menon David K ,   The Cambridge NeuroCOVID Group,   The NIHR COVID-19 BioResource,   The Cambridge NIHR Clinical Research Facility,   The CITIID-NIHR BioResource COVID-19 Collaboration
Published: February 2022

Human coronavirus disease 2019 (COVID-19) due to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has multiple neurological consequences, but its long-term effect on brain health is still uncertain. The cerebrovascular consequences of COVID-19 may also affect brain health. Here we assess cerebrovascular health in 45 hospitalised patients using the resting state fluctuation amplitudes (RSFA) from functional magnetic resonance imaging, in relation to disease severity and in contrast with 42 controls. Widespread changes in frontoparietal RSFA were related to the severity of the acute COVID-19 episode, as indexed by COVID-19 WHO Progression Scale, inflammatory and coagulatory biomarkers. This relationship was not explained by chronic cardiorespiratory dysfunction, age, or sex. Exploratory analysis suggests that the level of cerebrovascular dysfunction is associated with cognitive, mental, and physical health at follow-up. The principal findings were consistent across univariate and multivariate approaches. The results indicate chronic cerebrovascular impairment following severe acute COVID-19, with the potential for long-term consequences on cognitive function and mental wellbeing.
Core and Flanking bHLH-PAS:DNA interactions mediate specificity and drive obesity
Authors: Bersten DC , Sullivan AE , McDougal D , Breen J , Fitzsimmons RL , Muscat GEO , Pederson S , Bruning JB , Fan CM , Thomas PQ , Russell DL , Peet DJ , Whitelaw ML
Published: February 2022

The basic-Helix-Loop-Helix Per-Arnt-Sim (PAS) homology domain (bHLH-PAS) transcription factor (TF) family comprises critical biological sensors of physiological (hypoxia, tryptophan metabolites, neuronal activity, and appetite) and environmental (diet derived metabolites and environmental pollutants) stimuli to regulate genes involved in signal adaptation and homeostasis1. bHLH TFs bind DNA as homo or heterodimers via E-box (CANNTG) response elements, however the DNA binding specificity of the PAS domain-containing bHLH subfamily remains unresolved1. We systematically analysed cognate DNA binding hierarchies of prototypical bHLH-PAS family members (ARNT, ARNT2, HIF1α, HIF2α, AhR, NPAS4, SIM1) and demonstrate distinct core (NNCGTG) specificities for different heterodimer classes. The results also show that bHLH-PAS TFs bind over a large footprint 12-15bp and recognise preferential DNA sequences flanking the core. For example, specificity beyond otherwise identical core binding by SIM1 and the HIFs is mediated through N-terminal HIFα-DNA interactions. We also reveal an intimate relationship between DNA shape and both core and flanking TF binding allowing motif sequence flexibility and underpinning TF binding specificity. Furthermore, DNA-shape affinity relationships revealed that novel downstream PAS-A-loop DNA interactions are associated with AT-rich sequences that lead to high-affinity binding, and that loss of this function underpins a monogenic cause of human hyperphagic obesity in a recapitulated SIM1.R171H knock-in mouse model.

Importantly, models of protein-DNA binding accurately predict in vivo occupancy, while response element methylation blocks DNA binding and predicts cell type specific chromatin occupancy. These data provide a definitive and accurate map of bHLH-PAS TF specificity and target selectivity through novel flanking protein-DNA interactions that are crucial for in vivo biological function.
Augmented glucose dependency of autoreactive B cells provides a treatment target for lupus
Authors: Wilson John J. , Wei Jian , Daamen Andrea R. , Sears John D. , Bechtel Elaine , Mayberry Colleen L. , Stafford Grace A. , Bechtold Lesley , Grammer Amrie C. , Lipsky Peter E. , Roopenian Derry C. , Chang Chih-Hao
Published: February 2022

Heightened glycolysis is inherent to immune/inflammatory disorders, but little is known of its role in the pathogenesis of systemic lupus erythematosus (lupus). Here, we profile key autoimmune populations in acute and chronic lupus-prone models and their response to glycolytic inhibition. We demonstrate that glycolysis is specifically required for autoreactive germinal center B cells (GCB), but not for T follicular helper cells (Tfh) to survive. This augmented reliance on glucose oxidation to maintain ATP production in pathogenic GCB renders them highly susceptible to oxidative stress-induced apoptosis triggered by glycolysis blockade via 2-deoxyglucose (2DG). We show that 2DG can preferentially reduce GCB in lupus-prone mice, while sparing other autoreactive populations, including Tfh, but still significantly improving lifespan and kidney function. Furthermore, the subset of GCB expressing B-cell maturation antigen (BCMA) exhibits an exaggerated dependence on glycolysis to sustain their growth. Depletion of these cells with a proliferation-inducing ligand-based CAR T-cells leads to greatly prolonged lifespan of mice with severe autoimmune activation. These results reveal that glycolysis dependent GCB, especially those expressing BCMA, are key lupus mediators and highlight that they can be selectively targeted to improve disease outcomes for lupus patients.
Investigating RNA-RNA interactions through computational and biophysical analysis
Authors: Mrozowich Tyler , Park Sean M. , Waldl Maria , Henrickson Amy , Tersteeg Scott , Nelson Corey R. , Deklerk Anneke , Demeler Borries , Hofacker Ivo L. , Wolfinger Michael T. , Patel Trushar R.
Published: February 2022

Numerous viruses utilize essential long-range RNA-RNA genome interactions, specifically flaviviruses. Using Japanese encephalitis virus (JEV) as a model system, we computationally predicted and then biophysically validated and characterized its long-range RNA-RNA genomic interaction. Using multiple RNA computation assessment programs, we determine the primary RNA-RNA interacting site among JEV isolates and numerous related viruses. Followingin vitrotranscription of RNA, we provide, for the first time, characterization of an RNA-RNA interaction using multi-angle light scattering (SEC-MALS) and analytical ultra-centrifugation (AUC). Next, we report the first RNA-RNA interaction study quantified by microscale thermophoresis (MST), demonstrating that the 5’ and 3’ TR of JEV interact with nM affinity, which is significantly reduced when the conserved cyclization sequence is not present. Furthermore, we perform computational kinetic analyses validating the cyclization sequence as the primary driver of this RNA-RNA interaction. Finally, we examined the 3-dimensional structure of the interaction using small-angle X-ray scattering, revealing a flexible yet stable interaction. This pathway can be adapted and utilized to study various viral and human long-non-coding RNA-RNA interactions, and determine their binding affinities, a critical pharmacological property of designing potential therapeutics.

Graphical Abstract
USP22 controls type III interferon signaling and SARS-CoV-2 infection through activation of STING
Authors: Karlowitz Rebekka , Stanifer Megan L. , Roedig Jens , Andrieux Geoffroy , Bojkova Denisa , Smith Sonja , Kowald Lisa , Schubert Ralf , Boerries Melanie , Cinatl Jindrich , Boulant Steeve , van Wijk Sjoerd J. L.
Published: February 2022

Pattern recognition receptors (PRRs) and interferons (IFNs) serve as essential antiviral defense against SARS-CoV-2, the causative agent of the COVID-19 pandemic. Type III IFN (IFN-λ) exhibit cell-type specific and long-lasting functions in autoinflammation, tumorigenesis and antiviral defense. Here, we identify the deubiquitinating enzyme USP22 as central regulator of basal IFN-λ secretion and SARS-CoV-2 infections in native human intestinal epithelial cells (hIECs). USP22-deficient hIECs strongly upregulate genes involved in IFN signaling and viral defense, including numerous IFN-stimulated genes (ISGs), with increased secretion of IFN-λ and enhanced STAT1 signaling, even in the absence of exogenous IFNs or viral infection. Interestingly, USP22 controls basal and 2’3’-cGAMP-induced STING activation and loss of STING reversed STAT activation and ISG and IFN-λ expression. Intriguingly, USP22-deficient hIECs are protected against SARS-CoV-2 infection, viral replication and the formation of de novo infectious particles, in a STING-dependent manner. These findings reveal USP22 as central host regulator of STING and type III IFN signaling, with important implications for SARS-CoV-2 infection and antiviral defense.
Pyruvate supports RET-dependent mitochondrial ROS production necessary to control Mycobacterium avium infection in human primary macrophages
Authors: Røst Lisa Marie , Louet Claire , Bruheim Per , Flo Trude Helen , Gidon Alexandre
Published: February 2022

Macrophages deploy a variety of antimicrobial programs to contain mycobacterial infection. Upon activation, they undergo extensive metabolic reprogramming to meet an increase in energy demand, but also to support immune effector functions such as secretion of cytokines and antimicrobial activities. Here, we report that mitochondrial import of pyruvate is linked to production of mitochondrial ROS and control of Mycobacterium avium infection in human primary macrophages. Using chemical inhibition, targeted mass spectrometry and single cell image analysis, we show that macrophages infected with M. avium switched to aerobic glycolysis without any major imbalances in the tricarboxylic acid cycle or changes in the energy charge. Instead, we found that pyruvate import contributed to hyperpolarization of mitochondria in infected cells and increased production of mitochondrial reactive oxygen species by the complex I via reverse electron transport, which reduced the macrophage burden of M. avium. While mycobacterial infections are extremely difficult to treat and notoriously resistant to antibiotics, this work stresses out that compounds specifically inducing mitochondrial reactive oxygen species could present themself as valuable adjunct treatments.
Interphase chromosomes of the Aedes aegypti mosquito are liquid crystalline and can sense mechanical cues
Authors: Contessoto Vinícius G. , Dudchenko Olga , Aiden Erez Lieberman , Wolynes Peter G. , Onuchic José N. , Di Pierro Michele
Published: February 2022

We use data-driven physical simulations to study the three-dimensional architecture of the Aedes aegypti genome. Hi-C maps exhibit both a broad diagonal and compart-mentalization with telomeres and centromeres clustering together. Physical modeling reveals that these observations correspond to an ensemble of 3D chromosomal structures that are folded over and partially condensed. Clustering of the centromeres and telomeres near the nuclear lamina appears to be a necessary condition for the formation of the observed structures. Further analysis of the mechanical properties of the genome reveals that the chromosomes of Aedes aegypti, by virtue of their atypical structural organization, are highly sensitive to the deformation of the nuclei. This last finding provides a possible physical mechanism linking mechanical cues to gene regulation.
Modeling of Alpha-1 Antitrypsin Deficiency with Syngeneic Human iPSC-Hepatocytes Reveals Metabolic Dysregulation and Cellular Heterogeneity in PiMZ and PiZZ Hepatocytes
Authors: Kaserman Joseph E , Werder Rhiannon B. , Wang Feiya , Matte Taylor , Higgins Michelle I. , Dodge Mark , Lindstrom-Vautrin Jonathan , Hinds Anne , Bullitt Esther , Caballero Ignacio S. , Shi Xu , Gerszten Robert E. , Brunetti-Pierri Nicola , Liesa Marc , Villacorta-Martin Carlos , Hollenberg Anthony N. , Kotton Darrell N. , Wilson Andrew A.
Published: February 2022

Individuals homozygous for the pathogenic “Z” mutation in alpha-1 antitrypsin deficiency (AATD) are known to be at increased risk for chronic liver disease. That some degree of risk is similarly conferred by the heterozygous state, estimated to affect 2% of the US population, has also become clear. A lack of model systems that recapitulate heterozygosity in human hepatocytes has limited the ability to study the impact of expressing a single ZAAT allele on hepatocyte biology. Here, through the application of CRISPR-Cas9 editing, we describe the derivation of syngeneic induced pluripotent stem cells (iPSCs) engineered to determine the effects of ZAAT heterozygosity in iPSC-derived hepatocytes (iHeps) relative to homozygous mutant (ZZ) or corrected (MM) cells. We find that heterozygous MZ iHeps exhibit an intermediate disease phenotype and share with ZZ iHeps alterations in AAT protein processing and downstream perturbations in hepatic metabolic function including ER and mitochondrial morphology, reduced mitochondrial respiration, and branch-specific activation of the unfolded protein response in subpopulations of cells. Our cellular model of MZ heterozygosity thus provides evidence that expression of a single Z allele is sufficient to disrupt hepatocyte homeostatic function and suggest a mechanism underlying the increased risk of liver disease observed among MZ individuals.
IZUMO1 is a sperm fusogen
Authors: Brukman Nicolas G. , Nakajima Kohdai P. , Valansi Clari , Flyak Kateryna , Li Xiaohui , Higashiyama Tetsuya , Podbilewicz Benjamin
Published: February 2022

Mammalian sperm-egg adhesion depends on the trans-interaction between the sperm-specific type I glycoprotein IZUMO1 and its oocyte-specific GPI-anchored receptor JUNO. However, the mechanisms and proteins (fusogens) which mediate the following step of gamete fusion remain unknown. Using live imaging and content mixing assays in a heterologous system and structure-guided mutagenesis, we unveil an unexpected function for IZUMO1 in cell-to-cell fusion. We show that IZUMO1 alone is sufficient to induce fusion, and that this ability is retained in a mutant unable to bind JUNO. On the other hand, a triple mutation in exposed aromatic residues prevents this fusogenic activity without impairing JUNO interaction. Our findings suggest a second, crucial function for IZUMO1 as a unilateral mouse gamete fusogen.

Highlights

IZUMO1 expression in somatic cells in culture induces cell-to-cell fusion

The fusogenic activity of IZUMO1 is unilateral

Cell fusion is independent of the binding of IZUMO1 to JUNO

IZUMO1-mediated cell merger depends on its transmembrane domain, and three solvent-exposed aromatic residues

Graphical abstract
Trpc6 gain-of-function disease mutation enhances phosphatidylserine exposure in murine platelets
Authors: Boekell Kimber L. , Brown Brittney J. , Talbot Brianna E. , Schlondorff Johannes S.
Published: February 2022

Platelets enhance coagulation by exposing phosphatidylserine (PS) on their cell surface in response to strong agonist activation. Transient receptor potential channels, including TRPC6, have been implicated in the calcium influx central to this process. Here, we characterize the effect of a Trpc6 gain-of-function (GOF) disease-associated, and a dominant negative (DN), mutation on murine platelet activation. Platelets from mice harboring Trpc6E896K/E896K (GOF) and Trpc6DN/DN mutations were subject to in vitro analysis. Trpc6E896K/E896K and Trpc6DN/DN mutant platelets show enhanced and absent calcium influx, respectively, upon addition of the TRPC3/6 agonist GSK1702934A (GSK). GSK was sufficient to induce integrin αIIbβ3 activation, P-selection and PS exposure, talin cleavage, and MLC2 phosphorylation in Trpc6E896K/E896K, but not in wild-type, platelets. Thrombin-induced calcium influx and PS exposure were enhanced, and clot retraction delayed, by GOF TRPC6, while no differences were noted between wild-type and Trpc6DN/DN platelets. In contrast, Erk activation upon GSK treatment was absent in Trpc6DN/DN, and enhanced in Trpc6E896K/E896K, platelets, compared to wild-type. The positive allosteric modulator, TRPC6-PAM-C20, and fluoxetine maintained their ability to enhance and inhibit, respectively, GSK-mediated calcium influx in Trpc6E896K/E896K platelets. The data demonstrate that gain-of-function mutant TRPC6 channel can enhance platelet activation, including PS exposure, while confirming that TRPC6 is not necessary for this process. Furthermore, they suggest that Trpc6 GOF disease mutants do not simply increase wild-type TRPC6 responses, but can affect pathways not usually modulated by TRPC6 channel activity, displaying a true gain-of-function phenotype.
Scale of analysis drives the observed ratio of spatial to non-spatial variance in microbial water quality: insights from two decades of citizen science data
Authors: Weller Daniel L. , Weller Donald E. , Strawn Laura K. , Love Tanzy M. T.
Published: February 2022

While fecal indicator bacteria (FIB) testing is used to monitor surface waters for potential health hazards, recent studies report substantial variation in FIB levels and that observed variation appeared dependent on scale of analysis (SOA). Citizen science data and random effects models were used to quantify variance in FIB levels attributable to spatial versus temporal factors. Separately, Bayesian models were used to quantify the ratio of spatial to nonspatial variance in FIB levels, and identify associations between environmental factors and FIB levels. Separate analyses were performed for each stream, lake, and watershed as well as at the state-level (three SOAs). As SOA increased (from waterway to watershed to statewide models), variance attributable to spatial sources generally increased and variance attributable to temporal sources generally decreased. While relationships between FIB levels and environmental factors, such as flow conditions, were constant across SOA, the effect of land cover was both highly dependent on SOA and consistently smaller than the effect of outfalls. This study demonstrates the importance of scale when designing monitoring programs or using FIB monitoring to inform management. Moreover, these data represent a comprehensive survey of water quality in Upstate NY, and the study highlights a valuable publicly available database.