Native ß-barrel substrates pass through two shared intermediates during folding on the BAM complex.

The assembly of ß-barrel proteins into membranes is mediated by the evolutionarily conserved ß-barrel assembly machine (BAM) complex. In Escherichia coli, BAM folds numerous substrates which vary considerably in size and shape. How BAM is able to efficiently fold such a diverse array of ß-barrel substrates is not clear. Here, we develop a disulfide crosslinking method to trap native substrates in vivo as they fold on BAM. By placing a cysteine within the luminal wall of the BamA barrel as well as in the substrate ß-strands, we can compare the residence time of each substrate strand within the BamA lumen. We validated this method using two defective, slow-folding substrates. We used this method to characterize stable intermediates which occur during folding of two structurally different native substrates. Strikingly, these intermediates occur during identical stages of folding for both substrates: soon after folding has begun and just before folding is completed. We suggest that these intermediates arise due to barriers to folding that are common between ß-barrel substrates, and that the BAM catalyst is able to fold so many different substrates because it addresses these common challenges.

Progression to type 1 diabetes in the DPT-1 and TN07 clinical trials is critically associated with specific residues in HLA-DQA1-B1 heterodimers.

AIMS/HYPOTHESIS: The aim of this work was to explore molecular amino acids (AAs) and related structures of HLA-DQA1-DQB1 that underlie its contribution to the progression from stages 1 or 2 to stage 3 type 1 diabetes. METHODS: Using high-resolution DQA1 and DQB1 genotypes from 1216 participants in the Diabetes Prevention Trial-Type 1 and the Diabetes Prevention Trial, we applied hierarchically organised haplotype association analysis (HOH) to decipher which AAs contributed to the associations of DQ with disease and their structural properties. HOH relied on the Cox regression to quantify the association of DQ with time-to-onset of type 1 diabetes. RESULTS: By numerating all possible DQ heterodimers of α- and ß-chains, we showed that the heterodimerisation increases genetic diversity at the cellular level from 43 empirically observed haplotypes to 186 possible heterodimers. Heterodimerisation turned several neutral haplotypes (DQ2.2, DQ2.3 and DQ4.4) to risk haplotypes (DQ2.2/2.3-DQ4.4 and DQ4.4-DQ2.2). HOH uncovered eight AAs on the α-chain (-16α, -13α, -6α, α22, α23, α44, α72, α157) and six AAs on the ß-chain (-18ß, ß9, ß13, ß26, ß57, ß135) that contributed to the association of DQ with progression of type 1 diabetes. The specific AAs concerned the signal peptide (minus sign, possible linkage to expression levels), pockets 1, 4 and 9 in the antigen-binding groove of the α1ß1 domain, and the putative homodimerisation of the αß heterodimers. CONCLUSIONS/INTERPRETATION: These results unveil the contribution made by DQ to type 1 diabetes progression at individual residues and related protein structures, shedding light on its immunological mechanisms and providing new leads for developing treatment strategies. DATA AVAILABILITY: Clinical trial data and biospecimen samples are available through the National Institute of Diabetes and Digestive and Kidney Diseases Central Repository portal ( https://repository.niddk.nih.gov/studies ).

Multiscale topological analysis of kinetoplast DNA via high-resolution AFM.

Kinetoplast DNA is a complex nanoscale network, naturally assembled from thousands of interconnected DNA circles within the mitochondrion of certain parasites. Despite the relevance of this molecule to parasitology and the recent discovery of tuneable mechanics, its topology remains highly contested. Here we present a multiscale analysis into the structure of kDNA using a combination of high-resolution atomic force microscopy and custom-designed image analysis protocols. By capturing a notably large set of high-resolution images, we are able to look beyond individual kDNA variations and quantify population properties throughout several length scales. Within the sample, geometric fluctuations of area and mean curvature are observed, corresponding with previous in vitro measurements. These translate to localised variations in density, with a sample-wide decrease in DNA density from the outer rim of the molecule to the centre and an increase in pore size. Nodes were investigated in a single molecule study, and their estimated connectivity significantly exceeded mean valence, with a high dependence on their position in the network. While node separation was approximately half the minicircle circumference, it followed a strong bimodal distribution, suggesting more complex underlying behaviour. Finally, upon selective digestion of the network, breakdown of the fibril-cap heterogeneity was observed, with molecules expanding less upon immobilisation on the mica surface. Additionally, preferential digestion was seen in localised areas of the network, increasing pore size disproportionately. Overall, the combination of high-resolution AFM and single molecule image analysis provides a promising method to the continued investigation of complex nanoscale structures. These findings support the ongoing characterisation of kDNA topology to aid understanding of its biological and mechanical phenomena.

Indoor air concentrations of PM2.5 quartz fiber filter-collected ionic PFAS and emissions to outdoor air: findings from the IPA campaign.

Per- and polyfluoroalkyl substances (PFAS) are prevalent in consumer products used indoors. However, few measurements of ionic PFAS exist for indoor air. We analyzed samples collected on PM2.5 quartz fiber filters (QFFs) in 11 North Carolina homes 1-3 times in living rooms (two QFFs in series), and immediately outside each home (single QFF), for 26 ionic PFAS as part of the 9 months Indoor PFAS Assessment (IPA) Campaign. All targeted PFAS, except for PFDS and 8:2 monoPAP, were detected indoors. PFBA, PFHpA, PFHxA, PFOA, PFOS, and 6:2 diPAP were detected in >50% of indoor samples. PFHxA, PFOA, and PFOS had the highest detection frequency (DF = 80%; medians = 0.5-0.7 pg m-3), while median PFBA concentrations (3.6 pg m-3; DF = 67%) were highest indoors. Residential indoor air concentrations (sum of measured PFAS) were, on average, 3.4 times higher than residential outdoor air concentrations, and an order of magnitude higher than regional background concentrations. Indoor-to-outdoor emission rate estimates suggest that emissions from single unit homes could be a meaningful contributor to PFBA, PFOA, and PFOS emissions in populated areas far from major point sources. Backup QFFs were observed to adsorb some targeted PFAS from the gas-phase, making reported values upper-bounds for particle-phase and lower-bounds for total air (gas plus particle) concentrations. We found that higher concentrations of carbonaceous aerosol were associated with a shift in partitioning of short chain PFCAs and long chain PFSAs toward the particle phase.

Osteosarcoma and Langerhans Cell Histiocytosis in a Pediatric Patient with Lynch Syndrome: A Case Report.

CASE: Lynch syndrome (hereditary nonpolyposis colorectal cancer) is associated with extracolonic manifestations, but skeletal tumors are rare. Our patient, a 12-year-old boy with Lynch syndrome, developed osteosarcoma of the left femur. Treatment included cytotoxic chemotherapy, wide resection, and pembrolizumab. Two years later, he developed an aggressive lesion in the contralateral femur that was thought to be metastatic osteosarcoma but which histology revealed to be Langerhans cell histiocytosis. CONCLUSION: This case underscores the importance of advanced testing in patients with osteosarcoma and poor response to chemotherapy, and of tissue sampling when patients with a primary malignancy develop new bone lesions. LEVEL OF EVIDENCE: IV.

Hierarchically Vascularized and Suturable Tissue Constructs created through Angiogenesis from Tissue-Engineered Vascular Grafts.

A major roadblock in implementing engineered tissues clinically lies in their limited vascularization. After implantation, such tissues do not integrate with the host's circulation as quickly as needed, commonly resulting in loss of viability and functionality. This study presents a solution to the vascularization problem that could enable the survival and function of large, transplantable, and vascularized engineered tissues. The technique allows vascularization of a cell laden hydrogel through angiogenesis from a suturable tissue-engineered vascular graft (TEVG) constructed from electrospun polycaprolactone with macropores. The graft is surrounded by a layer of cell-laden gelatin-methacryloyl hydrogel. The constructs are suturable and possess mechanical properties like native vessels. Angiogenesis occurs through the pores in the graft, resulting in a hydrogel containing an extensive vascular network that is connected to an implantable TEVG. The size of the engineered tissue and the degree of vascularization can be increased by adding multiple TEVGs into a single construct. The engineered tissue has the potential to be immediately perfused by the patient's blood upon surgical anastomosis to host vessels, enabling survival of implanted cells. These findings provide a meaningful step to address the longstanding problem of fabricating suturable pre-vascularized tissues which could survive upon implantation in vivo. STATEMENT OF SIGNIFICANCE: Creating vascularized engineered tissues that can be transplanted and rapidly perfused by the host blood supply is a major challenge which has limited the clinical impact of tissue engineering. In this study we demonstrate a technique to fabricate vascularized tissue constructs via angiogenesis from a suturable tissue-engineered vascular graft. The macroporous graft is surrounded with hydrogel, allowing endothelial cells to migrate from the lumen and vascularize the hydrogel layer with capillary-like structures connected to the macrovessel. The graft has comparable mechanical properties to native blood vessels and larger constructs can be fabricated by incorporating multiple grafts. These constructs could potentially be connected surgically to the circulation at an implantation site to support their immediate perfusion and survival.

Positive affect longitudinally buffers the negative effect of health anxiety on gastrointestinal symptoms.

People with significant health anxiety may experience brain-gut dysregulation, leading to increased visceral sensitivity and greater gastrointestinal (GI) symptoms. Positive affect (PA), on the other hand, may serve as a protective characteristic, buffering the negative impact of health anxiety on GI symptoms. This study investigated interrelationships between health anxiety, PA, and GI symptoms. Longitudinal data were collected on health anxiety, PA, and GI symptoms via an online survey from 861 adults at two timepoints, 4 weeks apart. Regression models were conducted to examine the moderating effect of PA at baseline on future GI symptoms. Greater health anxiety at baseline predicted GI symptoms at follow-up, whereas higher levels of PA predicted reduced GI symptoms at follow-up. Post hoc testing revealed that PA significantly moderated the relationship between health anxiety and GI symptoms over time, such that higher levels of PA attenuated the effect of health anxiety on belly pain, nausea and vomiting, and reflux at follow-up. This study provides preliminary evidence PA may buffer the negative impact of health anxiety on GI symptoms. Future studies should explore whether the promotion of PA through interventions similarly attenuates health related anxiety's impact on GI symptoms.

Evaluating Personalized Add-On Ayurveda Therapy in Oxygen-Dependent Diabetic COVID-19 Patients: A 60-Day Study of Symptoms, Inflammation, and Radiological Changes.

Background Effective management of both acute and post-acute sequelae of SARS-CoV-2 is essential, particularly for type 2 diabetes mellitus (T2DM) patients, who are at increased risk of severe pro-inflammatory responses and complications. Persistent symptoms and residual lung and cardiovascular damage in post-coronavirus disease (COVID-19) individuals highlight the need for comprehensive long-term treatment strategies. Conventional treatments, including Remdesivir and glucocorticoids, have limitations, suggesting that further investigation into Ayurvedic therapies could be beneficial, though controlled trials are currently limited. Objectives Evaluate the effectiveness and safety of Ayurveda with the standard of care (SOC) versus SOC in improving symptoms, moderating immune responses (interleukin-6 (IL-6), C-reactive protein (CRP), neutrophil-lymphocyte ratio (NLR), and radiological outcomes in oxygen-dependent, high-risk, non-vaccinated type 2 diabetes COVID-19 patients over 60 days, and thus addressing their heightened vulnerability to severe infections. Methods A controlled trial with 50 diabetic COVID-19 patients, aged 18-80, with an NLR of >= 4, primarily on Remdesivir, was assigned to Group 1 (Add-on Ayurveda+SOC, n=30) or Group 2 (SOC, n=20) based on their voluntary choice with follow-up on days 14, 28, and 60. Parametric outcomes in group analysis were assessed with robust regression and non-parametric outcomes with Cochran-Mantel-Haenszel, log-rank test, and chi-square tests at 95% confidence interval (CI). Results Group 1 exhibited statistically significant improvements in fever, cough, diarrhea, as well as NLR, IL-6, and CRP by 14 days, and in anosmia, loss of taste, shortness of breath, general weakness, and headache by 60 days. Though the sample size is small, notable improvements can be seen in troponin levels in Group 1 at 28 and 60 days. High-resolution computer tomography COVID-19 reporting and data system (HRCT CO-RADS) scores improved more slowly in Group 2 than in Group 1. Survival rates were 96.4% for Group 1 and 90% for Group 2. Numbers were too small for reliable comparisons at 60 days.  Conclusion The add-on Ayurveda group showed a better symptomatic response, and faster normalization in inflammatory markers, including IL-6 and NLR by 14 days, and cardiac markers by 28 days. Minimal clinical and no laboratory adverse events were observed. This study supports the need for a randomized, double-blind trial.

Embracing multiple infection models to tackle Q fever: A review of in vitro, in vivo, and lung ex vivo models.

Multiple animal and cell culture models are employed to study pathogenesis of Coxiella burnetii, the causative agent of acute and chronic human Q fever. C. burnetii is a lung pathogen that is aerosolized in contaminated products and inhaled by humans to cause acute disease that can disseminate to other organs and establish chronic infection. Cellular models of Q fever include a variety of tissue-derived cell lines from mice and humans such as lung alveolar ex vivo cells. These models have the advantage of being cost-effective and reproducible. Similarly, animal models including mice and guinea pigs are cost-effective, although only immunocompromised SCID mice display a severe disease phenotype in response to Nine Mile I and Nine Mile II isolates of C. burnetii while immunocompetent guinea pigs display human-like symptoms and robust immune responses. Non-human primates such as macaques and marmosets are the closest model of human disease but are costly and largely used for adaptive immune response studies. All animal models are used for vaccine development but many differences exist in the pathogen's ability to establish lung infection when considering infection routes, bacterial isolates, and host genetic background. Similarly, while cellular models are useful for characterization of host-pathogen mechanisms, future developments should include use of a lung infection platform to draw appropriate conclusions. Here, we summarize the current state of the C. burnetii lung pathogenesis field by discussing the contribution of different animal and cell culture models and include suggestions for continuing to move the field forward.

CD4 downregulation precedes Env expression and protects HIV-1-infected cells from ADCC mediated by non-neutralizing antibodies.

HIV-1 envelope glycoprotein (Env) conformation substantially impacts antibody-dependent cellular cytotoxicity (ADCC). Envs from primary HIV-1 isolates adopt a prefusion "closed" conformation, which is targeted by broadly neutralizing antibodies (bnAbs). CD4 binding drives Env into more "open" conformations, which are recognized by non-neutralizing Abs (nnAbs). To better understand Env-Ab and Env-CD4 interaction in CD4+ T cells infected with HIV-1, we simultaneously measured antibody binding and HIV-1 mRNA expression using multiparametric flow cytometry and RNA flow fluorescent in situ hybridization (FISH) techniques. We observed that env mRNA is almost exclusively expressed by HIV-1 productively infected cells that already downmodulated CD4. This suggests that CD4 downmodulation precedes env mRNA expression. Consequently, productively infected cells express "closed" Envs on their surface, which renders them resistant to nnAbs. Cells recognized by nnAbs were all env mRNA negative, indicating Ab binding through shed gp120 or virions attached to their surface. Consistent with these findings, treatment of HIV-1-infected humanized mice with the ADCC-mediating nnAb A32 failed to lower viral replication or reduce the size of the viral reservoir. These findings confirm the resistance of productively infected CD4+ T cells to nnAbs-mediated ADCC and question the rationale of immunotherapy approaches using this strategy. IMPORTANCE: Antibody-dependent cellular cytotoxicity (ADCC) represents an effective immune response for clearing virally infected cells, making ADCC-mediating antibodies promising therapeutic candidates for HIV-1 cure strategies. Broadly neutralizing antibodies (bNAbs) target epitopes present on the native "closed" envelope glycoprotein (Env), while non-neutralizing antibodies (nnAbs) recognize epitopes exposed upon Env-CD4 interaction. Here, we provide evidence that env mRNA is predominantly expressed by productively infected cells that have already downmodulated cell-surface CD4. This indicates that CD4 downmodulation by HIV-1 precedes Env expression, making productively infected cells resistant to ADCC mediated by nnAbs but sensitive to those mediated by bnAbs. These findings offer critical insights for the development of immunotherapy-based strategies aimed at targeting and eliminating productively infected cells in people living with HIV.

Risk prediction modelling in idiopathic inflammatory myositis-associated interstitial lung disease based on seven factors including serum KL-6 and lung ultrasound B-lines.

OBJECTIVES: To develop a user-friendly nomogram-based predictive model for interstitial lung disease (ILD) in patients with idiopathic inflammatory myositis (IIM). METHODS: A retrospective study was conducted at Shantou Central Hospital, encompassing 205 IIM patients diagnosed between January 2013 and December 2022. We used the LASSO regression method in the discovery set to select features for model construction, followed by efficacy verification through AUC of ROC. Afterwards, KL-6 values and LUS B-lines number were added into this model to evaluate whether these 2 factors added to the model efficiency. Finally, a web version was constructed to make it more available. RESULTS: Among the 205 IIM patients, 115 (56.1%) patients were diagnosed with ILD, and 90 (43.9%) did not. The predictive model, derived from the training set, comprised four independent risk factors, including age, presence of respiratory symptoms, anti-melanoma differentiation-associated gene 5 (MDA-5) antibody positivity, and anti-aminoacyl transfer RNA synthetase (anti-ARS) antibodies positivity. Notably, anti-TIF1-γ antibody positivity emerged as a protective factor. The AUC of the ROC based on these 5 factors was 0.876 in the training set and 0.861 in the validation set. The AUC of the ROC based on the 5 factors plus KL-6 was 0.922, 5 factors plus B-line number was 0.949 and 5 factors plus both KL-6 and B-line number was 0.951. Accordingly, a nomogram and a web version were developed. CONCLUSIONS: This predictive model demonstrates robust capability to assess ILD risk in IIM patients, particularly when augmented with serum KL-6 level or/and LUS B-line number.

Infection length and host environment influence on Plasmodium falciparum dry season reservoir.

Persistence of malaria parasites in asymptomatic hosts is crucial in areas of seasonally-interrupted transmission, where P. falciparum bridges wet seasons months apart. During the dry season, infected erythrocytes exhibit extended circulation with reduced cytoadherence, increasing the risk of splenic clearance of infected cells and hindering parasitaemia increase. However, what determines parasite persistence for long periods of time remains unknown. Here, we investigated whether seasonality affects plasma composition so that P. falciparum can detect and adjust to changing serological cues; or if alternatively, parasite infection length dictates clinical presentation and persistency. Data from Malian children exposed to alternating ~6-month wet and dry seasons show that plasma composition is unrelated to time of year in non-infected children, and that carrying P. falciparum only minimally affects plasma constitution in asymptomatic hosts. Parasites persisting in the blood of asymptomatic children from the dry into the ensuing wet season rarely if ever appeared to cause malaria in their hosts as seasons changed. In vitro culture in the presence of plasma collected in the dry or the wet seasons did not affect parasite development, replication or host-cell remodelling. The absence of a parasite-encoded sensing mechanism was further supported by the observation of similar features in P. falciparum persisting asymptomatically in the dry season and parasites in age- and sex-matched asymptomatic children in the wet season. Conversely, we show that P. falciparum clones transmitted early in the wet season had lower chance of surviving until the end of the following dry season, contrasting with a higher likelihood of survival of clones transmitted towards the end of the wet season, allowing for the re-initiation of transmission. We propose that the decreased virulence observed in persisting parasites during the dry season is not due to the parasites sensing ability, nor is it linked to a decreased capacity for parasite replication but rather a consequence decreased cytoadhesion associated with infection length.

A Longitudinal Treatment Effect Analysis of Antipsychotics on Behavior of Residents in Long-Term Care.

OBJECTIVE: The proportion of long-term care (LTC) residents being treated with antipsychotic medication is high, and these medications may exacerbate behavioral symptoms. We used propensity scores to investigate the effect of antipsychotic use on the worsening of behavioral symptoms among residents in LTC facilities. DESIGN: A retrospective study. SETTING AND PARTICIPANTS: Residents in LTC in 8 provinces and 1 territory in Canada, without severe aggressive behavior at baseline and reassessed at follow-up, between March 2000 and March 2022. METHODS: We used propensity score matching and weighting to balance baseline covariates and logistic regression to estimate the effect of antipsychotics on the worsening of behavioral symptoms in the original, matched, and weighted cohorts. The treatment variable was use of antipsychotic medication at baseline and the outcome was worsening of behavior at follow-up. RESULTS: A total of 494,215 participants were included (318,234 women and 175,981 men; mean age 82.8 years [SD 10.1; range 18-112]).130 558 (26.4%) used antipsychotics at baseline and 88,632 (17.9%) had worsening behavior in follow-up. In the matched cohort, there were 249,698 participants, and 124,849 were matched (1:1) in each treatment group. There was a significant association between antipsychotic use at baseline and worsening in behavior at follow-up in the adjusted regression models (OR 1.27 [95% CI 1.25-1.29], <0.0001) as well as in matched (OR 1.20 [95% CI 1.17-1.21], <0.0001) and weighted (OR 1.26 [95% CI 1.24-1.28], <0.0001) cohorts. CONCLUSIONS AND IMPLICATIONS: This study further evidence to support the cautious use of antipsychotics in LTC facilities. Future research in LTC facilities could include a more granular analyses of behavior change, including bidirectional analyses between different symptom severity classifications.

DOTATATE PET Imaging in Olfactory Neuroblastoma and Association with SSTR Expression.

Introduction Olfactory neuroblastoma (ONB), or esthesioneuroblastoma, is a rare neuroectodermal tumor of the nasal cavity and paranasal sinuses. Most of these tumors express somatostatin receptors (SSTRs), providing a potential target for radionuclide imaging with Ga-68 DOTATATE. However, this imaging modality has not been extensively studied in ONB. Methods We conducted a retrospective chart review of 96 endoscopic endonasal skull base surgery cases for ONB performed at our institution between 2000 and 2021. Histo (H) scores were assigned to each tumor and normalized DOTATATE standardized uptake values (nSUVs) were measured as well. Results Nine patients (5 males and 4 females) with ONB were ultimately included in the study. The average age of the patients was 50 years. All ONBs had a positive SSTR2 expression (H-score > 105; mean: 180). All ONBs showed DOTATATE avidity (mean nSUV for ONB: 6.7). However, there was no correlation between H-score and nSUV, with an r 2 of 0.24 ( p = 0.18). Conclusion Our study shows that SSTR2 expression is found in all ONBs with associated DOTATATE avidity, which may serve as a valuable imaging modality to monitor for recurrent and metastatic disease in ONB.

"Rehabbed to Death" in Oncology: Where Do We Go From Here?

To break the cycle of "rehabbed to death" in oncology, we must focus on improving communication and care coordination.