A Rapid Screening Platform for Simultaneous Evaluation of Biodegradation and Therapeutic Release of an Ocular Hydrogel.

This study attempts to address the challenge of accurately measuring the degradation of biodegradable hydrogels, which are frequently employed in drug delivery for controlled and sustained release. The traditional method utilizes a mass-loss approach, which is cumbersome and time consuming. The aim of this study was to develop an innovative screening platform using a millifluidic device coupled with automated image analysis to measure the degradation of Gelatin methacrylate (GelMA) and the subsequent release of an entrapped wetting agent, polyvinyl alcohol (PVA). Gel samples were placed within circular wells on a custom millifluidic chip and stained with a red dye for enhanced visualization. A camera module captured time-lapse images of the gels throughout their degradation. An image-analysis algorithm was used to translate the image data into degradation rates. Simultaneously, the eluate from the chip was collected to quantify the amount of GelMA degraded and PVA released at various time points. The visual method was validated by comparing it with the mass-loss approach (R = 0.91), as well as the amount of GelMA eluted (R = 0.97). The degradation of the GelMA gels was also facilitated with matrix metalloproteinases 9. Notably, as the gels degraded, there was an increase in the amount of PVA released. Overall, these results support the use of the screening platform to assess hydrogel degradation and the subsequent release of entrapped therapeutic compounds.

SEMPro: A Data-Driven Pipeline To Learn Structure-Property Insights from Scanning Electron Microscopy Images.

Analyzing hydrogel microstructure through scanning electron microscopy (SEM) images is crucial in understanding hydrogel properties. However, the analysis of SEM images in hydrogel research heavily relies on the intuition of individual researchers and is constrained by the limited size of the dataset. To address this, we propose SEMPro, a data-driven solution using web-scraping and deep learning (DL) to compile and analyze the structure-property relationships of hydrogels through SEM images. It accurately predicts the elastic modulus from SEM images within the same order of magnitude and displays a learned extraction of modulus-relevant features in SEM images as seen through the nontrivial activation mapping and transfer learning. By employing Explainable AI through activation map exposure, SEMPro validates the model predictions. SEMPro represents a closed-loop data collection and analysis pipeline, providing critical insights into hydrogels and soft materials. This innovative approach has the potential to revolutionize hydrogel research, offering high-dimensional insights for further advancements.

A cross-species proteomic map reveals neoteny of human synapse development.

The molecular mechanisms and evolutionary changes accompanying synapse development are still poorly understood1,2. Here we generate a cross-species proteomic map of synapse development in the human, macaque and mouse neocortex. By tracking the changes of more than 1,000 postsynaptic density (PSD) proteins from midgestation to young adulthood, we find that PSD maturation in humans separates into three major phases that are dominated by distinct pathways. Cross-species comparisons reveal that human PSDs mature about two to three times slower than those of other species and contain higher levels of Rho guanine nucleotide exchange factors (RhoGEFs) in the perinatal period. Enhancement of RhoGEF signalling in human neurons delays morphological maturation of dendritic spines and functional maturation of synapses, potentially contributing to the neotenic traits of human brain development. In addition, PSD proteins can be divided into four modules that exert stage- and cell-type-specific functions, possibly explaining their differential associations with cognitive functions and diseases. Our proteomic map of synapse development provides a blueprint for studying the molecular basis and evolutionary changes of synapse maturation.

RNF8 ubiquitylation of XRN2 facilitates R-loop resolution and restrains genomic instability in BRCA1 mutant cells.

Breast cancer linked with BRCA1/2 mutations commonly recur and resist current therapies, including PARP inhibitors. Given the lack of effective targeted therapies for BRCA1-mutant cancers, we sought to identify novel targets to selectively kill these cancers. Here, we report that loss of RNF8 significantly protects Brca1-mutant mice against mammary tumorigenesis. RNF8 deficiency in human BRCA1-mutant breast cancer cells was found to promote R-loop accumulation and replication fork instability, leading to increased DNA damage, senescence, and synthetic lethality. Mechanistically, RNF8 interacts with XRN2, which is crucial for transcription termination and R-loop resolution. We report that RNF8 ubiquitylates XRN2 to facilitate its recruitment to R-loop-prone genomic loci and that RNF8 deficiency in BRCA1-mutant breast cancer cells decreases XRN2 occupancy at R-loop-prone sites, thereby promoting R-loop accumulation, transcription-replication collisions, excessive genomic instability, and cancer cell death. Collectively, our work identifies a synthetic lethal interaction between RNF8 and BRCA1, which is mediated by a pathological accumulation of R-loops.

Identification of a drug binding pocket in TMEM16F calcium-activated ion channel and lipid scramblase.

The dual functions of TMEM16F as Ca2+-activated ion channel and lipid scramblase raise intriguing questions regarding their molecular basis. Intrigued by the ability of the FDA-approved drug niclosamide to inhibit TMEM16F-dependent syncytia formation induced by SARS-CoV-2, we examined cryo-EM structures of TMEM16F with or without bound niclosamide or 1PBC, a known blocker of TMEM16A Ca2+-activated Cl- channel. Here, we report evidence for a lipid scrambling pathway along a groove harboring a lipid trail outside the ion permeation pore. This groove contains the binding pocket for niclosamide and 1PBC. Mutations of two residues in this groove specifically affect lipid scrambling. Whereas mutations of some residues in the binding pocket of niclosamide and 1PBC reduce their inhibition of TMEM16F-mediated Ca2+ influx and PS exposure, other mutations preferentially affect the ability of niclosamide and/or 1PBC to inhibit TMEM16F-mediated PS exposure, providing further support for separate pathways for ion permeation and lipid scrambling.

Mec1-independent activation of the Rad53 checkpoint kinase revealed by quantitative analysis of protein localization dynamics.

The replication checkpoint is essential for accurate DNA replication and repair, and maintenance of genomic integrity when a cell is challenged with genotoxic stress. Several studies have defined the complement of proteins that change subcellular location in the budding yeast Saccharomyces cerevisiae following chemically induced DNA replication stress using methyl methanesulfonate (MMS) or hydroxyurea (HU). How these protein movements are regulated remains largely unexplored. We find that the essential checkpoint kinases Mec1 and Rad53 are responsible for regulating the subcellular localization of 159 proteins during MMS-induced replication stress. Unexpectedly, Rad53 regulation of the localization of 52 proteins is independent of its known kinase activator Mec1, and in some scenarios independent of Tel1 or the mediator proteins Rad9 and Mrc1. We demonstrate that Rad53 is phosphorylated and active following MMS exposure in cells lacking Mec1 and Tel1. This noncanonical mode of Rad53 activation depends partly on the retrograde signaling transcription factor Rtg3, which also facilitates proper DNA replication dynamics. We conclude that there are biologically important modes of Rad53 protein kinase activation that respond to replication stress and operate in parallel to Mec1 and Tel1.

A length-adjustable vacuum-powered artificial muscle for wearable physiotherapy assistance in infants.

Soft pneumatic artificial muscles are increasingly popular in the field of soft robotics due to their light-weight, complex motions, and safe interfacing with humans. In this paper, we present a Vacuum-Powered Artificial Muscle (VPAM) with an adjustable operating length that offers adaptability throughout its use, particularly in settings with variable workspaces. To achieve the adjustable operating length, we designed the VPAM with a modular structure consisting of cells that can be clipped in a collapsed state and unclipped as desired. We then conducted a case study in infant physical therapy to demonstrate the capabilities of our actuator. We developed a dynamic model of the device and a model-informed open-loop control system, and validated their accuracy in a simulated patient setup. Our results showed that the VPAM maintains its performance as it grows. This is crucial in applications such as infant physical therapy where the device must adapt to the growth of the patient during a 6-month treatment regime without actuator replacement. The ability to adjust the length of the VPAM on demand offers a significant advantage over traditional fixed-length actuators, making it a promising solution for soft robotics. This actuator has potential for various applications that can leverage on demand expansion and shrinking, including exoskeletons, wearable devices, medical robots, and exploration robots.

Vaccine coverage and factors associated with vaccine adherence in persons with HIV at an urban infectious disease clinic.

Information on vaccination rates and factors associated with adherence in persons with HIV (PWH) is limited. We report vaccine adherence in 653 adult PWH attending an urban Infectious Disease Clinic from January 2015 to December 2021. Vaccines evaluated included influenza, pneumococcal, tetanus, hepatitis A virus (HAV) and hepatitis B virus (HBV), human papillomavirus (HPV), and zoster vaccines. Vaccine reminders were triggered at every visit, and all vaccines were accessible in the clinic. The mean age was 50 y (±SD 13), male gender was 78.6%, and black race was 74.3%. The overall adherence to all recommended vaccines was 63.6%. Vaccine adherence was >90% for influenza, pneumococcal, and tetanus, >80% for HAV and HBV, and ≥60% for HPV and zoster vaccines. The main predictor of adherence to all vaccines was ≥2 annual clinic visits (odds ratio [OR] 3.45; 95% confidence interval [CI] 2.36-5.05; p < .001). Other predictors included an assigned primary care provider within the system (OR 2.89 [95% CI 1.71-5.00, p < .001]) and CD4 >200 cell/mm3 at entry into care (OR 1.91 [95% CI 1.24-2.94, p = .0003]). Retention in care combined with vaccine reminders and accessibility of vaccines in the clinic can achieve high vaccine uptake in PWH.

Development of a yeast whole-cell biocatalyst for MHET conversion into terephthalic acid and ethylene glycol.

BACKGROUND: Over the 70 years since the introduction of plastic into everyday items, plastic waste has become an increasing problem. With over 360 million tonnes of plastics produced every year, solutions for plastic recycling and plastic waste reduction are sorely needed. Recently, multiple enzymes capable of degrading PET (polyethylene terephthalate) plastic have been identified and engineered. In particular, the enzymes PETase and MHETase from Ideonella sakaiensis depolymerize PET into the two building blocks used for its synthesis, ethylene glycol (EG) and terephthalic acid (TPA). Importantly, EG and TPA can be re-used for PET synthesis allowing complete and sustainable PET recycling. RESULTS: In this study we used Saccharomyces cerevisiae, a species utilized widely in bioindustrial fermentation processes, as a platform to develop a whole-cell catalyst expressing the MHETase enzyme, which converts monohydroxyethyl terephthalate (MHET) into TPA and EG. We assessed six expression architectures and identified those resulting in efficient MHETase expression on the yeast cell surface. We show that the MHETase whole-cell catalyst has activity comparable to recombinant MHETase purified from Escherichia coli. Finally, we demonstrate that surface displayed MHETase is active across a range of pHs, temperatures, and for at least 12 days at room temperature. CONCLUSIONS: We demonstrate the feasibility of using S. cerevisiae as a platform for the expression and surface display of PET degrading enzymes and predict that the whole-cell catalyst will be a viable alternative to protein purification-based approaches for plastic degradation.

A flexible adhesive surface electrode array capable of cervical electroneurography during a sequential autonomic stress challenge.

This study introduces a flexible, adhesive-integrated electrode array that was developed to enable non-invasive monitoring of cervical nerve activity. The device uses silver-silver chloride as the electrode material of choice and combines it with an electrode array consisting of a customized biopotential data acquisition unit and integrated graphical user interface (GUI) for visualization of real-time monitoring. Preliminary testing demonstrated this electrode design can achieve a high signal to noise ratio during cervical neural recordings. To demonstrate the capability of the surface electrodes to detect changes in cervical neuronal activity, the cold-pressor test (CPT) and a timed respiratory challenge were employed as stressors to the autonomic nervous system. This sensor system recording, a new technique, was termed Cervical Electroneurography (CEN). By applying a custom spike sorting algorithm to the electrode measurements, neural activity was classified in two ways: (1) pre-to-post CPT, and (2) during a timed respiratory challenge. Unique to this work: (1) rostral to caudal channel position-specific (cephalad to caudal) firing patterns and (2) cross challenge biotype-specific change in average CEN firing, were observed with both CPT and the timed respiratory challenge. Future work is planned to develop an ambulatory CEN recording device that could provide immediate notification of autonomic nervous system activity changes that might indicate autonomic dysregulation in healthy subjects and clinical disease states.

Yeast goes viral: probing SARS-CoV-2 biology using S. cerevisiae.

The budding yeast Saccharomyces cerevisiae has long been an outstanding platform for understanding the biology of eukaryotic cells. Robust genetics, cell biology, molecular biology, and biochemistry complement deep and detailed genome annotation, a multitude of genome-scale strain collections for functional genomics, and substantial gene conservation with Metazoa to comprise a powerful model for modern biological research. Recently, the yeast model has demonstrated its utility in a perhaps unexpected area, that of eukaryotic virology. Here we discuss three innovative applications of the yeast model system to reveal functions and investigate variants of proteins encoded by the SARS-CoV-2 virus.

Relationship Between Food Insecurity and Neighborhood Child Opportunity Index.

OBJECTIVES: To evaluate the association between the Child Opportunity Index (COI) and food insecurity. STUDY DESIGN: This was a secondary analysis of a comprehensive screening instrument for social determinants of health and behavioral health risks. It was administered in 2 urban pediatric emergency departments to adolescents aged 13-21 years and caregivers of children aged 0-17 years. Food insecurity was assessed using the 2-item Hunger Vital Sign. Residential addresses were geocoded and linked with COI data. Bivariable and multivariable logistic regression models were developed to measure the relationship between COI and food insecurity. RESULTS: Of the 954 participants (384 adolescents, 570 caregivers) who underwent screening, 15.7% identified food insecurity (14.3% of adolescent and 16.7% of caregiver participants). The majority of participants were non-Hispanic Black (overall, 62.3%; food secure, 60.9%; food insecure, 72.0%), were publicly insured (overall, 56.6%; food secure, 53.1%; food insecure, 73.3%), and lived in neighborhoods of low/very low opportunity (overall, 76.9%; food secure, 74.7%; food insecure, 88.3%). In adjusted analyses, participants living in neighborhoods of low/very low child opportunity had 3-fold greater odds of being food insecure compared with children living in neighborhoods of high child opportunity (aOR, 3.0; 95% CI, 1.4-6.3). CONCLUSION: We demonstrate that food insecurity is associated with lower neighborhood opportunity. Our results could inform future screening initiatives and support the development of novel, place-based interventions to tackle the complex issue of food insecurity.

Identification of a conserved drug binding pocket in TMEM16 proteins.

The TMEM16 family of calcium-activated membrane proteins includes ten mammalian paralogs (TMEM16A-K) playing distinct physiological roles with some implicated in cancer and airway diseases. Their modulators with therapeutic potential include 1PBC, a potent inhibitor with anti-tumoral properties, and the FDA-approved drug niclosamide that targets TMEM16F to inhibit syncytia formation induced by SARS-CoV-2 infection. Here, we report cryo-EM structures of TMEM16F associated with 1PBC and niclosamide, revealing that both molecules bind the same drug binding pocket. We functionally and computationally validate this binding pocket in TMEM16A as well as TMEM16F, thereby showing that drug modulation also involves residues that are not conserved between TMEM16A and TMEM16F. This study establishes a much-needed structural framework for the development of more potent and more specific drug molecules targeting TMEM16 proteins.

A Vacuum-Powered Artificial Muscle Designed for Infant Rehabilitation.

The majority of soft pneumatic actuators for rehabilitation exercises have been designed for adult users. Specifically, there is a paucity of soft rehabilitative devices designed for infants with upper and lower limb motor disabilities. We present a low-profile vacuum-powered artificial muscle (LP-VPAM) with dimensions suitable for infants. The actuator produced a maximum force of 26 N at vacuum pressures of -40 kPa. When implemented in an experimental model of an infant leg in an antagonistic-agonist configuration to measure resultant knee flexion, the actuator generated knee flexion angles of 43° and 61° in the prone and side-lying position, respectively.

A Modular Geometrical Framework for Modelling the Force-Contraction Profile of Vacuum-Powered Soft Actuators.

In this paper, we present a generalized modeling tool for predicting the output force profile of vacuum-powered soft actuators using a simplified geometrical approach and the principle of virtual work. Previous work has derived analytical formulas to model the force-contraction profile of specific actuators. To enhance the versatility and the efficiency of the modelling process we propose a generalized numerical algorithm based purely on geometrical inputs, which can be tailored to the desired actuator, to estimate its force-contraction profile quickly and for any combination of varying geometrical parameters. We identify a class of linearly contracting vacuum actuators that consists of a polymeric skin guided by a rigid skeleton and apply our model to two such actuators-vacuum bellows and Fluid-driven Origami-inspired Artificial Muscles-to demonstrate the versatility of our model. We perform experiments to validate that our model can predict the force profile of the actuators using its geometric principles, modularly combined with design-specific external adjustment factors. Our framework can be used as a versatile design tool that allows users to perform parametric studies and rapidly and efficiently tune actuator dimensions to produce a force-contraction profile to meet their needs, and as a pre-screening tool to obviate the need for multiple rounds of time-intensive actuator fabrication and testing.

RNF168 regulates R-loop resolution and genomic stability in BRCA1/2-deficient tumors.

Germline mutations in BRCA1 and BRCA2 (BRCA1/2) genes considerably increase breast and ovarian cancer risk. Given that tumors with these mutations have elevated genomic instability, they exhibit relative vulnerability to certain chemotherapies and targeted treatments based on poly (ADP-ribose) polymerase (PARP) inhibition. However, the molecular mechanisms that influence cancer risk and therapeutic benefit or resistance remain only partially understood. BRCA1 and BRCA2 have also been implicated in the suppression of R-loops, triple-stranded nucleic acid structures composed of a DNA:RNA hybrid and a displaced ssDNA strand. Here, we report that loss of RNF168, an E3 ubiquitin ligase and DNA double-strand break (DSB) responder, remarkably protected Brca1-mutant mice against mammary tumorigenesis. We demonstrate that RNF168 deficiency resulted in accumulation of R-loops in BRCA1/2-mutant breast and ovarian cancer cells, leading to DSBs, senescence, and subsequent cell death. Using interactome assays, we identified RNF168 interaction with DHX9, a helicase involved in the resolution and removal of R-loops. Mechanistically, RNF168 directly ubiquitylated DHX9 to facilitate its recruitment to R-loop-prone genomic loci. Consequently, loss of RNF168 impaired DHX9 recruitment to R-loops, thereby abrogating its ability to resolve R-loops. The data presented in this study highlight a dependence of BRCA1/2-defective tumors on factors that suppress R-loops and reveal a fundamental RNF168-mediated molecular mechanism that governs cancer development and vulnerability.

Efficacy of quality improvement and patient safety workshops for students: a pilot study.

BACKGROUND: While the Association of American Medical Colleges encourages medical schools to incorporate quality improvement and patient safety (QI/PS) into their curriculum, medical students continue to have limited QI/PS exposure. To prepare medical students for careers that involve QI/PS, the Institute for Healthcare Improvement chapter at an allopathic medical school and school of allied health professions initiated self-directed learning by offering student-led workshops to equip learners with skills to improve the quality and safety of healthcare processes. METHODS: In this prospective cohort study, workshops were hosted for medical students between 2015 and 2018 on five QI/PS topics: Process Mapping, Root-Cause Analysis (RCA), Plan-Do-Study-Act (PDSA) Cycles, Evidence Based Medicine (EBM), and Patient Handoffs. Each workshop included a hands-on component to engage learners in practical applications of QI/PS skills in their careers. Change in knowledge, attitudes, and behaviors was assessed via pre- and post-surveys using 5-point Likert scales, and analyzed using either the McNemar test or non-parametric Wilcoxon signed-rank test. Surveys also gathered qualitative feedback regarding strengths, future areas for improvement, and reasons for attending the workshops. RESULTS: Data was collected from 88.5% of learners (n = 185/209); 19.5% of learners reported prior formal instruction in these topics. Statistically significant improvements in learners' confidence were observed for each workshop. Additionally, after attending workshops, learners felt comfortable teaching the learned QI/PS skill to colleagues (mean pre/post difference 1.96, p < 0.0001, n = 139) and were more likely to pursue QI/PS projects in their careers (mean pre/post difference 0.45, p < 0.0001, n = 139). Lastly, learners demonstrated a statistically significant increase in knowledge in four out of five skills workshop topics. CONCLUSION: Few medical students have formal instruction in QI/PS tools. This pilot study highlights advantages of incorporating an innovative, student-directed modified 'flipped classroom' methodology, with a focus on active experiential learning and minimal didactic instruction.

Correlation of clinical and chest radiograph findings in pediatric submersion cases.

BACKGROUND: Submersion injuries are a leading cause of injury death in children in the United States. The clinical course of a submersion patient varies depending on the presence of anoxic brain injury and acute respiratory failure. OBJECTIVE: We studied changes in clinical findings and chest radiograph findings and determined the sensitivity/specificity of the presenting chest radiograph in predicting clinical improvement within the first 24 h in pediatric submersion cases. MATERIALS AND METHODS: We conducted a cross-sectional study of pediatric submersion patients through age 18 years treated at a children's hospital from 2010 to 2013. We reviewed demographics, comorbidities, prehospital/hospital course and chest radiographic findings. Clinical improvement occurred when a child demonstrated normal vital signs and mentation. We compared radiographic findings among children based on clinical improvement up to 24 h post submersion. Using odds ratios, we calculated associations between radiographic findings and clinical improvement. We studied the sensitivity/specificity of the presenting chest radiograph in predicting clinical improvement within 24 h. RESULTS: One hundred forty-two of 262 (54%) patients had initial chest radiographs; 41% had follow-up radiographs. The odds of an abnormal initial chest radiograph were 4 times higher in children with respiratory distress or abnormal mentation at emergency department (ED) presentation compared to children without these findings (odds ratio [OR]=4.83; 95% confidence interval [CI]=2.1-10.85; P<0.001). Improvement in radiographic findings occurred in 85% of children within 24 h. Children with an abnormal initial chest radiograph were 87% less likely to improve clinically by 24 h (P<0.001). A presenting chest radiograph that was normal or with mild pulmonary edema/atelectasis predicted clinical improvement within 24 h (sensitivity 95%, specificity 57%). CONCLUSION: Most chest radiographic findings improve in pediatric submersion patients who recover within the first 24 h. An initial chest radiograph that is normal or with mild pulmonary edema/atelectasis satisfactorily predicts clinical improvement by 24 h post submersion.

Sonic hedgehog accelerates DNA replication to cause replication stress promoting cancer initiation in medulloblastoma.

The mechanisms generating cancer-initiating mutations are not well understood. Sonic hedgehog (SHH) pathway activation is frequent in medulloblastoma (MB), with PTCH1 mutations being a common initiating event. Here we investigated the role of the developmental mitogen SHH in initiating carcinogenesis in the cells of origin: granule cell progenitors (GCPs). We delineate a molecular mechanism for tumor initiation in MB. Exposure of GCPs to Shh causes a distinct form of DNA replication stress, increasing both origin firing and fork velocity. Shh promotes DNA helicase loading and activation, with increased Cdc7-dependent origin firing. The S-phase duration is reduced and hyper-recombination occurs, causing copy number neutral loss of heterozygosity-a frequent event at the PTCH1/ptch1 locus. Moreover, Cdc7 inhibition to attenuate origin firing reduces recombination and preneoplastic tumor formation in mice. Therefore, tissue-specific replication stress induced by Shh promotes loss of heterozygosity, which in tumor-prone Ptch1+/- GCPs results in loss of this tumor suppressor-an early cancer-initiating event.