Modeling membranes in situ.

Molecular dynamics simulations of cellular membranes have come a long way-from simple model lipid bilayers to multicomponent systems capturing the crowded and complex nature of real cell membranes. In this opinionated minireview, we discuss the current challenge to simulate the dynamics of membranes in their native environment, in situ, with the prospect of reaching the level of whole cells and cell organelles using an integrative modeling framework.

Psychosocial Distress Screening Among Interprofessional Palliative Care Teams: A Narrative Review.

With increased need for palliative care and limited staffing resources, non-social workers are increasingly responsible for screening for urgent psychosocial distress. The National Consensus Project guidelines call for all palliative care team members to be competent in screening across domains. Yet, in contrast to an abundance of evidence-informed tools for palliative social work assessments, standardization for interprofessional psychosocial screening is lacking. This lack of standardized practice may lead to harmful disparities in care delivery. The purpose of this narrative review is to examine current literature on evidence-informed practices for psychosocial screening within palliative care. Google Scholar, a university Summon library search engine, and prominent palliative care journals were searched using the same phrases to locate articles for inclusion. Each article was reviewed and synthesized across common themes. Although an abundance of validated screening tools exists for outpatient oncology-specific settings, there is minimal guidance on psychosocial screening tools intended for specialty palliative care. The most oft-cited tools have been met with concern for validity across diverse palliative care populations and settings. Additional research is needed to operationalize and measure brief psychosocial screening tools that can be validated for use by interprofessional palliative care teams, a stepping-stone for increased equity in palliative care practice.

Diabetic Ocular Surface Has Defects in Oxygen Uptake Revealed by Optic Fiber Microsensor.

Purpose: Diabetes mellitus causes diabetic keratopathy (DK). This and other ocular surface disorders are underdiagnosed and problematic for affected patients as well as recipients of diabetic donor corneas. Thus, it is important to find noninvasive means to facilitate determination of the potentially vision-threatening DK. It has been reported that diabetic corneas uptake significantly less oxygen (O2) than healthy controls. However, an integral assessment of the ocular surface is missing. Methods: Using an optic-fiber O2 micro-sensor (optrode) we demonstrated recently that the healthy ocular surface displays a unique spatiotemporal map of O2 consumption. We hypothesize that diabetes impairs the spatiotemporal profile of O2 uptake at the ocular surface. Results: Using streptozotocin (STZ)-induced diabetic mice, we found diminished O2 uptake and loss of the unique pattern across the ocular surface. A diabetic cornea consumes significantly less O2 at the bulbar conjunctiva and limbus, but not the central and peripheral cornea, compared to controls. Further, we show that, contrary to the healthy cornea, the diabetic cornea does not increase the O2 consumption at the limbus in the evening as the normal control. Conclusions: Altogether, our measurements reveal a previously unknown impairment in O2 uptake at the diabetic cornea, making it a potential tool to diagnose ocular surface abnormalities and suggesting a new etiology mechanism.

Amyloidosis and Considerations for the Hand Surgeon.

Amyloidosis can lead to cardiac, renal, and other multiorgan failure. New treatments have become available that can prolong survival but rely on early diagnosis. Manifestations of amyloidosis in hand surgery include carpal tunnel syndrome, trigger finger, peripheral neuropathy, and spontaneous distal biceps rupture. Often, these can predate systemic amyloidosis, offering hand surgeons an opportunity to diagnose patients with amyloidosis before systemic disease, refer them for treatment, and potentially alter disease course and prolong survival. In this review, we describe the pathophysiology and two most common subtypes of amyloidosis seen by hand surgeons. We provide guidance on biopsy practices and referral for patients with amyloidosis. Lastly, we provide a brief overview of the treatments for amyloidosis and their effect on disease course.

Trauma-Informed Care for Inpatient Palliative Care Social Work: Applying Existing Models at the Bedside.

Coexisting serious illness and posttraumatic stress place hospitalized individuals at risk for complex pain, anxiety, and retraumatization. Hospital palliative care social workers increasingly recognize the value of trauma-informed care (TIC) for reducing harm in the inpatient setting. Despite this recognition, there is limited operationalization of TIC principles for inpatient interventions. This paper integrates each TIC principle with inpatient psychosocial interventions to advance trauma-informed competencies among inpatient palliative care social workers and to provide a foundation for future TIC implementation research.

Reducing Sialylation Enhances Electrotaxis of Corneal Epithelial Cells.

Corneal wound healing is a complex biological process that integrates a host of different signals to coordinate cell behavior. Upon wounding, there is the generation of an endogenous wound electric field that serves as a powerful cue to guide cell migration. Concurrently, the corneal epithelium reduces sialylated glycoforms, suggesting that sialylation plays an important role during electrotaxis. Here, we show that pretreating human telomerase-immortalized corneal epithelial (hTCEpi) cells with a sialyltransferase inhibitor, P-3FAX-Neu5Ac (3F-Neu5Ac), improves electrotaxis by enhancing directionality, but not speed. This was recapitulated using Kifunensine, which inhibits cleavage of mannoses and therefore precludes sialylation on N-glycans. We also identified that 3F-Neu5Ac enhanced the responsiveness of the hTCEpi cell population to the electric field and that pretreated hTCEpi cells showed increased directionality even at low voltages. Furthermore, when we increased sialylation using N-azidoacetylmannosamine-tetraacylated (Ac4ManNAz), hTCEpi cells showed a decrease in both speed and directionality. Importantly, pretreating enucleated eyes with 3F-Neu5Ac significantly improved re-epithelialization in an ex vivo model of a corneal injury. Finally, we show that in hTCEpi cells, sialylation is increased by growth factor deprivation and reduced by PDGF-BB. Taken together, our results suggest that during corneal wound healing, reduced sialylated glycoforms enhance electrotaxis and re-epithelialization, potentially opening new avenues to promote corneal wound healing.

Structural basis of peptidoglycan synthesis by E. coli RodA-PBP2 complex.

Peptidoglycan (PG) is an essential structural component of the bacterial cell wall that is synthetized during cell division and elongation. PG forms an extracellular polymer crucial for cellular viability, the synthesis of which is the target of many antibiotics. PG assembly requires a glycosyltransferase (GT) to generate a glycan polymer using a Lipid II substrate, which is then crosslinked to the existing PG via a transpeptidase (TP) reaction. A Shape, Elongation, Division and Sporulation (SEDS) GT enzyme and a Class B Penicillin Binding Protein (PBP) form the core of the multi-protein complex required for PG assembly. Here we used single particle cryo-electron microscopy to determine the structure of a cell elongation-specific E. coli RodA-PBP2 complex. We combine this information with biochemical, genetic, spectroscopic, and computational analyses to identify the Lipid II binding sites and propose a mechanism for Lipid II polymerization. Our data suggest a hypothesis for the movement of the glycan strand from the Lipid II polymerization site of RodA towards the TP site of PBP2, functionally linking these two central enzymatic activities required for cell wall peptidoglycan biosynthesis.

PLSCR1 is a cell-autonomous defence factor against SARS-CoV-2 infection.

Understanding protective immunity to COVID-19 facilitates preparedness for future pandemics and combats new SARS-CoV-2 variants emerging in the human population. Neutralizing antibodies have been widely studied; however, on the basis of large-scale exome sequencing of protected versus severely ill patients with COVID-19, local cell-autonomous defence is also crucial1-4. Here we identify phospholipid scramblase 1 (PLSCR1) as a potent cell-autonomous restriction factor against live SARS-CoV-2 infection in parallel genome-wide CRISPR-Cas9 screens of human lung epithelia and hepatocytes before and after stimulation with interferon-γ (IFNγ). IFNγ-induced PLSCR1 not only restricted SARS-CoV-2 USA-WA1/2020, but was also effective against the Delta B.1.617.2 and Omicron BA.1 lineages. Its robust activity extended to other highly pathogenic coronaviruses, was functionally conserved in bats and mice, and interfered with the uptake of SARS-CoV-2 in both the endocytic and the TMPRSS2-dependent fusion routes. Whole-cell 4Pi single-molecule switching nanoscopy together with bipartite nano-reporter assays found that PLSCR1 directly targeted SARS-CoV-2-containing vesicles to prevent spike-mediated fusion and viral escape. A PLSCR1 C-terminal ß-barrel domain-but not lipid scramblase activity-was essential for this fusogenic blockade. Our mechanistic studies, together with reports that COVID-associated PLSCR1 mutations are found in some susceptible people3,4, identify an anti-coronavirus protein that interferes at a late entry step before viral RNA is released into the host-cell cytosol.

Missed opportunities for diagnosis of hepatitis B and C in individuals diagnosed with decompensated cirrhosis or hepatocellular carcinoma.

BACKGROUND AND AIM: This study aimed to assess utilization of health-care services in people with decompensated cirrhosis (DC) or hepatocellular carcinoma (HCC) and a "late diagnosis" of hepatitis B or hepatitis C. METHODS: Hepatitis B and C cases during 1997-2016 in Victoria, Australia, were linked with hospitalizations, deaths, liver cancer diagnoses, and medical services. A late diagnosis was defined as hepatitis B or hepatitis C notification occurring after, at the same time, or within 2 years preceding an HCC/DC diagnosis. Services provided during the 10-year period before HCC/DC diagnosis were assessed, including general practitioner (GP) or specialist visits, emergency department presentations, hospital admissions, and blood tests. RESULTS: Of the 25 766 notified cases of hepatitis B, 751 (2.9%) were diagnosed with HCC/DC, and hepatitis B was diagnosed late in 385 (51.3%). Of 44 317 cases of hepatitis C, 2576 (5.8%) were diagnosed with HCC/DC, and hepatitis C was diagnosed late in 857 (33.3%). Although late diagnosis dropped over time, missed opportunities for timely diagnosis were observed. Most people diagnosed late had visited a GP (97.4% for hepatitis B, 98.9% for hepatitis C) or had a blood test (90.9% for hepatitis B, 88.6% for hepatitis C) during the 10 years before HCC/DC diagnosis. The median number of GP visits was 24 and 32, and blood tests 7 and 8, for hepatitis B and C, respectively. CONCLUSIONS: Late diagnosis of viral hepatitis remains a concern, with the majority having frequent health-care service provision in the preceding period, indicating missed opportunities for diagnosis.

Optic Fiber Microsensor Reveals Specific Spatiotemporal Oxygen Uptake Profiles at the Mammalian Ocular Surface.

Oxygen (O2) uptake by cells and tissues is a critical indicator of metabolic demand, changes in microenvironment, and pathophysiology. O2 uptake from the atmosphere accounts for virtually all the O2 consumption in the avascular cornea; however, a detailed spatiotemporal profile of corneal O2 uptake (COU) remains undetermined. Here, we used a non-invasive self-referencing optical fiber O2 sensor-the scanning micro-optrode technique (SMOT)-to report the O2 partial pressure and flux variations at the ocular surface of rodents and non-human primates. In vivo spatial mapping in mice revealed a distinct COU, characterized by a centripetal gradient with a significantly higher O2 influx at the limbus and conjunctiva regions than at the center of the cornea. This regional COU profile was reproduced ex vivo in freshly enucleated eyes. The centripetal gradient was conserved across the following species analyzed: mice, rats, and rhesus monkeys. In vivo temporal mapping in mice showed a significant increase in the O2 flux in the limbus in the evening compared to other times. Altogether, the data unveiled a conserved centripetal COU profile, which may be associated with the limbal epithelial stem cells residing at the intersection of the limbus and conjunctiva. These physiological observations will serve as a useful baseline for comparative studies with contact lens wear, ocular disease, diabetes, etc. Moreover, the sensor may be applied to understand the responses of the cornea and other tissues to various insults, drugs, or changes in the environment.

Supramolecular organization and dynamics of mannosylated phosphatidylinositol lipids in the mycobacterial plasma membrane.

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis (TB), a disease that claims ~1.6 million lives annually. The current treatment regime is long and expensive, and missed doses contribute to drug resistance. Therefore, development of new anti-TB drugs remains one of the highest public health priorities. Mtb has evolved a complex cell envelope that represents a formidable barrier to antibiotics. The Mtb cell envelop consists of four distinct layers enriched for Mtb specific lipids and glycans. Although the outer membrane, comprised of mycolic acid esters, has been extensively studied, less is known about the plasma membrane, which also plays a critical role in impacting antibiotic efficacy. The Mtb plasma membrane has a unique lipid composition, with mannosylated phosphatidylinositol lipids (phosphatidyl-myoinositol mannosides, PIMs) comprising more than 50% of the lipids. However, the role of PIMs in the structure and function of the membrane remains elusive. Here, we used multiscale molecular dynamics (MD) simulations to understand the structure-function relationship of the PIM lipid family and decipher how they self-organize to shape the biophysical properties of mycobacterial plasma membranes. We assess both symmetric and asymmetric assemblies of the Mtb plasma membrane and compare this with residue distributions of Mtb integral membrane protein structures. To further validate the model, we tested known anti-TB drugs and demonstrated that our models agree with experimental results. Thus, our work sheds new light on the organization of the mycobacterial plasma membrane. This paves the way for future studies on antibiotic development and understanding Mtb membrane protein function.

Considerations for Hand Surgery in Patients With Scleroderma.

Systemic sclerosis (scleroderma, SSc) is an autoimmune disease that causes significant dysfunction to multiple organ systems, including the musculoskeletal system. It poses significant challenges to the hand surgeon, including calcinosis, ischemic changes, Raynaud phenomenon, tendinopathies, synovitis, and joint contractures. Patients with SSc also suffer from multiorgan dysfunction, which makes them high-risk surgical patients. The hand surgeon must understand the pathophysiology, treatment strategies, and special operative considerations required in this population to avoid complications and help maintain or improve hand function.

Interrogation of the Pathogen Box reveals small molecule ligands against the mycobacterial trehalose transporter LpqY-SugABC.

Tuberculosis, caused by Mycobacterium tuberculosis, claims ∼1.5 million lives annually. Effective chemotherapy is essential to control TB, however the emergence of drug-resistant strains of TB have seriously threatened global attempts to control and eradicate this deadly pathogen. Trehalose recycling via the LpqY-SugABC importer is essential for the virulence and survival of Mtb and inhibiting or hijacking this transport system is an attractive approach for the development of novel anti-tubercular and diagnostic agents. Therefore, we interrogated the drug-like compounds in the open-source Medicines for Malaria Pathogen Box and successfully identified seven compounds from the TB, kinetoplastids and reference compound disease sets that recognise LpqY. The molecules have diverse chemical scaffolds, are not specific trehalose analogues, and may be used as novel templates to facilitate the development of therapeutics that kill Mtb with a novel mechanism of action via the mycobacterial trehalose LpqY-SugABC transport system.

What is the role of leukopenia in the assessment of septic arthritis?

Background: It is not well-understood how leukopenia affects the synovial white blood cell (WBC) and percent neutrophils (%PMNs) in the setting of septic arthritis. We sought to determine 1. Do synovial WBC and %PMNs differ between patients with culture positive septic arthritis with or without leukopenia? And 2. Are traditional thresholds of synovial fluid studies for accurately diagnosing septic arthritis still applicable in the leukopenic patient population? Methods: A retrospective cohort study was performed at a single institution of 79 non-leukopenic and 11 leukopenic patients diagnosed with culture-positive septic arthritis. Demographic data, serum laboratory values, synovial laboratory values, and culture results were recorded. Significant differences in synovial laboratory values were evaluated using the Wilcoxon-Mann-Whitney test. Results are reported as median, interquartile range, and p values. Results: There was a significant difference in synovial WBC in leukopenic patients compared to non-leukopenic patients with culture positive septic arthritis (p = 0.01). No significant difference was found in the synovial %PMNs between two cohorts (p = 0.33). Conclusion: Leukopenic patients with culture positive septic arthritis have significantly lower synovial WBCs compared to non-leukopenic patients. Traditional thresholds for synovial WBC are not reliable for excluding diagnosis of septic arthritis in leukopenic patients.

Peptidoglycan biosynthesis is driven by lipid transfer along enzyme-substrate affinity gradients.

Maintenance of bacterial cell shape and resistance to osmotic stress by the peptidoglycan (PG) renders PG biosynthetic enzymes and precursors attractive targets for combating bacterial infections. Here, by applying native mass spectrometry, we elucidate the effects of lipid substrates on the PG membrane enzymes MraY, MurG, and MurJ. We show that dimerization of MraY is coupled with binding of the carrier lipid substrate undecaprenyl phosphate (C55-P). Further, we demonstrate the use of native MS for biosynthetic reaction monitoring and find that the passage of substrates and products is controlled by the relative binding affinities of the different membrane enzymes. Overall, we provide a molecular view of how PG membrane enzymes convey lipid precursors through favourable binding events and highlight possible opportunities for intervention.

Splice-switching of the insulin receptor pre-mRNA alleviates tumorigenic hallmarks in rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is an aggressive pediatric tumor with a poor prognosis for metastasis and recurrent disease. Large-scale sequencing endeavors demonstrate that Rhabdomyosarcomas have a dearth of precisely targetable driver mutations. However, IGF-2 signaling is known to be grossly altered in RMS. The insulin receptor (IR) exists in two alternatively spliced isoforms, IR-A and IR-B. The IGF-2 signaling molecule binds both its innate IGF-1 receptor as well as the insulin receptor variant A (IR-A) with high affinity. Mitogenic and proliferative signaling via the canonical IGF-2 pathway is, therefore, augmented by IR-A. This study shows that RMS patients express increased IR-A levels compared to control tissues that predominantly express the IR-B isoform. We also found that Hif-1α is significantly increased in RMS tumors, portraying their hypoxic phenotype. Concordantly, the alternative splicing of IR adapts to produce more IR-A in response to hypoxic stress. Upon examining the pre-mRNA structure of the gene, we identified a potential hypoxia-responsive element, which is also the binding site for the RNA-binding protein CUG-BP1 (CELF1). We designed Splice Switching Oligonucleotides (SSO) against this binding site to decrease IR-A levels in RMS cell lines and, consequently, rescue the IR-B expression levels. SSO treatment resulted in a significant reduction in cell proliferation, migration, and angiogenesis. Our data shows promising insight into how impeding the IGF-2 pathway by reducing IR-A expression mitigates tumor growth. It is evident that Rhabdomyosarcomas use IR alternative splicing as yet another survival strategy that can be exploited as a therapeutic intervention in conjunction with already established anti-IGF-1 receptor therapies.

Assessment for Septic Arthritis in Immunocompetent and Immunocompromised Patients: A Single-Institution Study.

INTRODUCTION: Prompt diagnosis of septic arthritis is imperative to prevent irreversible joint damage. Immunocompromised patients are at an increased risk of septic arthritis as well as secondary systemic infection. Our aims were to identify features predictive of septic arthritis and to determine whether these features differed between immunocompetent and immunocompromised patients. METHODS: A single institution retrospective cohort study was performed of 173 immunocompetent and 70 immunocompromised patients who underwent aspiration or arthrotomy for suspected septic arthritis from 2010 to 2018. Demographic data, symptoms, laboratory values, and imaging findings were recorded. Multiple variable logistic regression models were used to assess for predictive factors for septic arthritis in both cohorts. Results were reported as odds ratios, 95% confidence intervals, and P values. RESULTS: In the regression analysis, independent predictive factors for septic arthritis in immunocompetent patients were younger age (P = 0.004), presence of radiographic abnormalities (P = 0.006), and C-reactive protein (CRP) (P < 0.001). For immunocompromised patients, only CRP was an independent continuous predictive factor (P = 0.008) for septic arthritis. A risk stratification tool for predicting septic arthritis in immunocompetent patients using age <55 years, CRP >100 mg/dL, and presence of radiographic abnormalities was developed. A similar tool was created using CRP >180 mg/dL and radiographic abnormalities in immunocompromised patients. DISCUSSION: Differences in predictive factors for septic arthritis between immunocompromised and immunocompetent patients suggest dissimilar clinical presentations. The developed risk stratification tools allow one to predict the likelihood of septic arthritis in both groups. This may permit more accurate selection of patients for surgical intervention in the setting of insufficient data from synovial aspiration.

Sequencing and Integration of Cervical Manual Therapy and Vestibulo-oculomotor Therapy for Concussion Symptoms: Retrospective Analysis.

BACKGROUND: After concussion many people have cervicogenic headache, visual dysfunction, and vestibular deficits that can be attributed to brain injury, cervical injury, or both. While clinical practice guidelines outline treatments to address the symptoms that arise from the multiple involved systems, no preferred treatment sequence for post-concussion syndrome has emerged. PURPOSE: This study sought to describe the clinical and patient-reported outcomes for people with post-concussion symptoms after a protocol sequenced to address cervical dysfunction and benign paroxysmal positional vertigo within the first three weeks of injury, followed by integrated vision and vestibular therapy. STUDY DESIGN: Retrospective longitudinal cohort analysis. METHODS: Records from a concussion clinic for 38 patients (25 male 13 female, aged 26.9±19.7 years) with post-concussion symptoms due to sports, falls, assaults, and motor vehicle accident injuries were analyzed. Musculoskeletal, vision, and vestibular system functions were assessed after pragmatic treatment including early cervical manual therapy and canalith repositioning treatment-when indicated-integrated with advanced vision and vestibular rehabilitation. Patient-reported outcomes included the Post-Concussion Symptom Scale (PCSS) for general symptoms; and for specific symptoms, the Dizziness Handicap Index (DHI), Convergence Insufficiency Symptom Scale (CISS), Activities-specific Balance Confidence scale (ABC), and the Brain Injury Vision Symptom Survey (BIVSS). Paired t-tests with Bonferroni correction to minimize familywise error (p<0.05) were used to analyze the clinical and patient-reported outcomes. RESULTS: After 10.4±4.8 sessions over 57.6±34.0 days, general symptoms improved on the PCSS (p=0.001, 95%CI=12.4-30.6); and specific symptoms on the DHI (p<0.001, 95%CI=14.5-33.2), CISS (p<0.002, 95%CI=7.1-18.3), ABC (p<0.024, 95%CI=-.3 - -.1), and BIVSS (p<0.001, 95%CI=13.4-28.0). Clinical measures improved including cervical range-of-motion (55.6% fully restored), benign paroxysmal positional vertigo symptoms (28/28, fully resolved), Brock string visual convergence (p<0.001, 95%CI=3.3-6.3), and score on the Balance Error Scoring System (p<0.001, 95%CI=5.5-11.6). CONCLUSION: A rehabilitation approach for post-concussion syndrome that sequenced cervical dysfunction and benign paroxysmal positional vertigo treatment within the first three weeks of injury followed by integrated vision and vestibular therapy improved clinical and patient-reported outcomes. Level of Evidence: 2b.