A minimal RNA substrate with dual fluorescent probes enables rapid kinetics and provides insight into bacterial RNase P active site interactions.

Bacterial ribonuclease P (RNase P) is a tRNA processing endonuclease that occurs primarily as a ribonucleoprotein with a catalytic RNA subunit (P RNA). As one of the first ribozymes discovered, P RNA is a well-studied model system for understanding RNA catalysis and substrate recognition. Extensive structural and biochemical studies have revealed the structure of RNase P bound to precursor tRNA (ptRNA) and product tRNA. These studies also helped to define active site residues and propose the molecular interactions that are involved in substrate binding and catalysis. However, a detailed quantitative model of the reaction cycle that includes the structures of intermediates and the process of positioning active site metal ions for catalysis is lacking. To further this goal, we used a chemically modified minimal RNA duplex substrate (MD1) to establish a kinetic framework for measuring the functional effects of P RNA active site mutations. Substitution of U69, a critical nucleotide involved in active site Mg2+ binding, was found to reduce catalysis >500-fold as expected, but had no measurable effect on ptRNA binding kinetics. In contrast, the same U69 mutations had little effect on catalysis in Ca2+ compared to reactions containing native Mg2+ ions. CryoEM structures and SHAPE mapping suggested increased flexibility of U69 and adjacent nucleotides in Ca2+ compared to Mg2+. These results support a model in which slow catalysis in Ca2+ is due to inability to engage U69. These studies establish a set of experimental tools to analyze RNase P kinetics and mechanism and can be expanded to gain new insights into the assembly of the active RNase P-ptRNA complex.

Towards targeting the breast cancer immune microenvironment.

The tumour immune microenvironment is shaped by the crosstalk between cancer cells, immune cells, fibroblasts, endothelial cells and other stromal components. Although the immune tumour microenvironment (TME) serves as a source of therapeutic targets, it is also considered a friend or foe to tumour-directed therapies. This is readily illustrated by the importance of T cells in triple-negative breast cancer (TNBC), culminating in the advent of immune checkpoint therapy in combination with cytotoxic chemotherapy as standard of care for both early and advanced-stage TNBC, as well as recent promising signs of efficacy in a subset of hormone receptor-positive disease. In this Review, we discuss the various components of the immune TME in breast cancer and therapies that target or impact the immune TME, as well as the complexity of host physiology.

Effect of Non-ablative Medical Therapy on Progression of Hearing Loss in Menière's Disease: A Systematic Review and meta-Analysis.

OBJECTIVE: To systematically review how audiometric data change over time in patients with Menière's disease (MD) undergoing non-ablative medical therapy. DATABASES REVIEWED: Medline (via PubMed), Scopus, Web of Science, Cumulated Index to Nursing and Allied Health Literature (CINAHL), Google Scholar. METHODS: A systematic review and meta-analysis of the literature was performed. Adult patients undergoing non-ablative medical therapy and reported duration of disease or follow-up were included and pooled estimates of pure-tone average (PTA) were tabulated. Studies were excluded if they did not use established MD, did not have pure-tone average (PTA) audiometric data, underwent ear surgery or ablative therapies, and were systematic reviews or case reports. RESULTS: Out of 198 articles meeting full eligibility, 13 studies, involving 950 patients with MD, were included in the review and further analyzed. No effect on progression of PTA from initial diagnosis was seen between the different medical therapies within 2 years of non-ablative medical treatment. There was a significant worsening of PTA after 2 year, regardless of treatment used. High levels of heterogeneity among studies were noted up to 6 months from diagnosis (I2 = 79%), likely reflecting differences in patient characteristics, treatment regimens, and study design. Overall, the risk of bias was low for the majority of included studies. CONCLUSIONS: Patients diagnosed with MD who are undergoing non-ablative medical therapy should be counseled on the likelihood of worsening of hearing loss over the course of the disease despite elected treatment.

Tumor biomechanical stiffness by magnetic resonance elastography predicts surgical outcomes and identifies biomarkers in vestibular schwannoma and meningioma.

Variations in the biomechanical stiffness of brain tumors can not only influence the difficulty of surgical resection but also impact postoperative outcomes. In a prospective, single-blinded study, we utilize pre-operative magnetic resonance elastography (MRE) to predict the stiffness of intracranial tumors intraoperatively and assess the impact of increased tumor stiffness on clinical outcomes following microsurgical resection of vestibular schwannomas (VS) and meningiomas. MRE measurements significantly correlated with intraoperative tumor stiffness and baseline hearing status of VS patients. Additionally, MRE stiffness was elevated in patients that underwent sub-total tumor resection compared to gross total resection and those with worse postoperative facial nerve function. Furthermore, we identify tumor microenvironment biomarkers of increased stiffness, including αSMA + myogenic fibroblasts, CD163 + macrophages, and HABP (hyaluronic acid binding protein). In a human VS cell line, a dose-dependent upregulation of HAS1-3, enzymes responsible for hyaluronan synthesis, was observed following stimulation with TNFα, a proinflammatory cytokine present in VS. Taken together, MRE is an accurate, non-invasive predictor of tumor stiffness in VS and meningiomas. VS with increased stiffness portends worse preoperative hearing and poorer postoperative outcomes. Moreover, inflammation-mediated hyaluronan deposition may lead to increased stiffness.

Failure in HiRes Ultra Series Recall Devices Does Not Necessarily Lead to Decrement in Performance.

OBJECTIVE: To understand the impact on speech perception for patients experiencing Advanced Bionics V1 series Ultra and Ultra 3D cochlear implant failure. STUDY DESIGN: Retrospective cohort study. SETTING: Tertiary academic center. PATIENTS: Adult patients implanted with V1 series devices. INTERVENTIONS: Device integrity and speech perception testing. MAIN OUTCOME MEASURES: consonant-nucleus-consonant and AzBio in quiet speech recognition scores. RESULTS: At our institution, 116 V1 series cochlear implants were placed in 114 patients. Thirteen devices in prelingual patients were excluded, leaving 103 (89%) for final analysis. Forty-eight (46.6%) devices were considered as failed using the company provided EFI analysis tool. There were 36 (65.5%) of the remaining 55 devices that consistently tested within normal range; the remainder lost to follow-up with unknown status. Among the 48 device failures, 29 were revised and 19 patients were not revised. Among those not revised, 11 self-opted for observation (57.9%). Observed patients, despite impedance changes meeting failure criteria, had no subjective or objective changes in speech perception. Sentence testing scores for failure patients who elected observation (82.9 ± 11.4%) were significantly higher at failure compared with those opting for revision (55 ± 22.8%, p = 0.006). For those undergoing revision surgery, significant improvement in post-activation scores was noted as compared with time of failure with a mean improvement of 12.9% (p = 0.002, n = 24) for consonant-nucleus-consonant word scores and 17.2% (p = 0.001, n = 19) for AzBio in quiet scores. CONCLUSIONS: Proactive monitoring using EFI identifies a higher rate of Ultra Series V1 device failure than previously reported. However, about 20% of these patients may not have subjective change in hearing or objective decline in test scores and could be observed. Should performance worsen, reimplantation provides significant improvement in speech recognition.

Deleting the ribosomal prolyl hydroxylase OGFOD1 protects mice against diet-induced obesity and insulin resistance.

Type 2 diabetes predisposes patients to heart disease, which is the primary cause of death across the globe. Type 2 diabetes often accompanies obesity and is defined by insulin resistance and abnormal glucose handling. Insulin resistance impairs glucose uptake and results in hyperglycemia, which damages tissues such as kidneys, liver, and heart. 2-oxoglutarate (2-OG)- and iron-dependent oxygenases (2-OGDOs), a family of enzymes regulating various aspects of cellular physiology, have been studied for their role in obesity and diet-induced insulin resistance. However, nothing is known of the 2-OGDO family member 2-oxoglutarate and iron-dependent prolyl hydroxylase domain containing protein 1 (OGFOD1) in this setting. OGFOD1 deletion leads to protection in cardiac ischemia-reperfusion injury and cardiac hypertrophy, which are two cardiac events that can lead to heart failure. Considering the remarkable correlation between heart disease and diabetes, the cardioprotection observed in OGFOD1-knockout mice led us to challenge these knockouts with high-fat diet. Wildtype mice fed a high-fat diet developed diet-induced obesity, insulin resistance, and glucose intolerance, but OGFOD1 knockout mice fed this same diet were resistant to diet-induced obesity and insulin resistance. These results support OGFOD1 down-regulation as a strategy for preventing obesity and insulin handling defects.

Rapid Ribonuclease P Kinetics Measured by Stopped-Flow Fluorescence and Fluorescence Anisotropy.

Stopped-flow fluorescence spectroscopy is a highly sensitive method for measuring rapid enzyme kinetics. A wide range of fluorophores can be employed, and fluorescence and fluorescence polarization can be measured. Thus, binding, conformational changes, and catalysis can, in principle, be measured, making it helpful in probing the entire kinetic landscape of a reaction. In this chapter, we use the bacterial RNA processing enzyme ribonuclease P (RNase P) as a model system to illustrate the determination of the kinetic constants for substrate binding and cleavage, thus allowing mechanistic questions regarding the effects of reaction conditions, mutations, or drug binding to be answered.

Why are medical students so motivated to learn ultrasound skills? A qualitative study.

BACKGROUND: The introduction of ultrasound (US) courses into medical undergraduate courses is usually met with a particularly high level of student motivation. The reasons for this are unclear. The aim of this study was to investigate the factors that contribute to undergraduate medical students' motivation to learn US skills. Understanding what motivates students to learn US will inform the efforts of faculty to foster students' motivation to learn. METHODS: We carried out in-depth semi-structured one-to-one interviews with medical students participating in an optional US course at two Swiss universities. The interview guide consisted of 10 main questions. The content was informed by experts in the field of medical education and US, as well as by a literature review of motivation theories for learning, in particular by self-determination theory (SDT). SDT was used to guide the development of the interview guide and to reflect on the resulting themes in the discussion section. The interview guide was piloted with two medical students. The interviews lasted an average of 45 min and were audio recorded and transcribed. Thematic analysis was used to analyse the data. RESULTS: Fourteen undergraduate medical students in their preclinical (year 3) and clinical studies (years 4 and 5) elaborated on a wide range of reasons for their high motivation to learn US. They were motivated for US training because of the positive nimbus of the US modality, emphasising the advantages of visualisation. Students acknowledged the potential professional benefits of learning US and described it as a fun, exciting group activity. CONCLUSIONS: The four themes we found in our analysis can all be related to the three universal needs described in SDT. The strong focus on the visual aspect and the positive nimbus of the modality goes beyond that and reflects the visuo-centric Zeitgeist, which claims the superiority of visual information over other data. Educators should be aware that motivation to learn is affected by the Zeitgeist and ensuing preconceptions, such as the perception of the positive nimbus surrounding a topic. Other key elements that can be implemented to motivate students are just-in-time feedback, enabling group experiences and creating awareness of the clinical relevance of learning content.

Analysis of the limited M. tuberculosis accessory genome reveals potential pitfalls of pan-genome analysis approaches.

Pan-genome analysis is a fundamental tool for studying bacterial genome evolution; however, the variety of methods used to define and measure the pan-genome poses challenges to the interpretation and reliability of results. To quantify sources of bias and error related to common pan-genome analysis approaches, we evaluated different approaches applied to curated collection of 151 Mycobacterium tuberculosis ( Mtb ) isolates. Mtb is characterized by its clonal evolution, absence of horizontal gene transfer, and limited accessory genome, making it an ideal test case for this study. Using a state-of-the-art graph-genome approach, we found that a majority of the structural variation observed in Mtb originates from rearrangement, deletion, and duplication of redundant nucleotide sequences. In contrast, we found that pan-genome analyses that focus on comparison of coding sequences (at the amino acid level) can yield surprisingly variable results, driven by differences in assembly quality and the softwares used. Upon closer inspection, we found that coding sequence annotation discrepancies were a major contributor to inflated Mtb accessory genome estimates. To address this, we developed panqc, a software that detects annotation discrepancies and collapses nucleotide redundancy in pan-genome estimates. When applied to Mtb and E. coli pan-genomes, panqc exposed distinct biases influenced by the genomic diversity of the population studied. Our findings underscore the need for careful methodological selection and quality control to accurately map the evolutionary dynamics of a bacterial species.

Congenitally Corrected Transposition of the Great Arteries in Utero: Morphological Spectrum, Outcomes and Pitfalls in Fetal Diagnosis.

Congenitally corrected transposition of the great arteries (ccTGA) is a rare malformation with diverse morphology. We assessed features of fetuses with ccTGA and evaluated neonatal and pediatric outcomes. This was a retrospective review of fetuses with ccTGA at Birmingham Women's and Children's Hospital born from 2005 to 2019. Of thirty-six fetuses identified, six had unavailable prenatal data, one was postnatally diagnosed with isomerism and 29 fetuses were evaluated. ccTGA without associated cardiac lesions was found in 28% (8/29), ccTGA with significant VSD in 31% (9/29), ccTGA with pulmonary obstruction in 24% (7/29) and ccTGA with complex anomalies in 17% (5/29). Tricuspid regurgitation (TR) was observed in 17% (5/29) and heart block (HB) in 10% (3/29) prenatally. Six, that is 21% underwent genetic testing of which one was abnormal. Five extra-cardiac anomalies were reported prenatally and postnatally. Pregnancy was discontinued in five, of which two had moderate TR. There were thirty-one liveborn. Coarctation of the aorta was found in five postnatally but not suspected prenatally. In one, pulmonary stenosis was underestimated; otherwise, prenatal morphology was confirmed. Cardiac interventions were performed in 77% (24/31) liveborn with 39% (12/31) undergoing neonatal intervention. Overall, 6/31 liveborn died including all three with prenatal heart block and one with TR. Estimated survival for all liveborn at 1, 5 and 10 years was 87% (95% CI 76-100%), 83% (95% CI 72-98%) and 80% (95% CI 66-96%) respectively. Accurate prenatal diagnosis of ccTGA is critical for counseling. Early outcomes are favorable with 77% of liveborn undergoing surgery. Fetuses with prenatal diagnosis of complex associated abnormalities, HB and TR appear to do less well.

Digital variance angiography in patients undergoing lower limb arterial recanalization: cost-effectiveness analysis within the English healthcare setting.

Aim: Digital variance angiography (DVA) is a recently developed image processing method capable of improving image quality compared with the traditionally used digital subtraction angiography (DSA), among patients undergoing lower limb x-ray angiography. This study aims to explore the potential cost-effectiveness of DVA from an English National Health Service perspective. Materials & methods: A two-part economic model, consisting of a decision tree and a Markov model, was developed to consider the costs and health outcomes associated with the use of DVA as part of current practice imaging, compared with x-ray angiography using standard DSA. The model explored the impact of DVA on the development of acute kidney injury (AKI), chronic kidney disease and radiation-induced cancer over a lifetime horizon. Both deterministic and probabilistic analyses were performed to assess the cost per quality-adjusted life-year (QALY). Results: Base-case results indicate that DVA results in cost savings of £309 per patient, with QALYs also improving (+0.025) over a lifetime. As shown in sensitivity analysis, a key driver of model results is the relative risk (RR) reduction of contrast-associated acute kidney injury associated with use of DVA. The intervention also decreases the risk of carcinoma over a lifetime. Scenario analyses show that cost savings range from £310 to £553, with QALY gains ranging from 0.048 to 0.109 per patient. Conclusion: The use of DVA could result in a decrease in costs and an increase in QALYs over a lifetime, compared with existing imaging practice. The potential for this technology to offer an economically viable alternative to existing image processing methods, through a reduction in contrast media volume and radiation exposure, has been demonstrated.

The anti-cancer immune response in breast cancer: current and emerging biomarkers and treatments.

Triple-negative breast cancers (TNBCs) exhibit heightened T cell infiltration, contributing to an enhanced response to immune checkpoint blockade (ICB) compared with other subtypes. An immune-rich immune microenvironment correlates with improved prognosis in early and advanced TNBC. Combination chemotherapy and ICB is now the standard of care in early- and late-stage TNBC. Although programmed death ligand-1 (PD-L1) positivity predicts ICB response in advanced stages, its role in early-stage disease remains uncertain. Despite neoadjuvant ICB becoming common in early-stage TNBC, the necessity of adjuvant ICB after surgery remains unclear. Understanding the molecular basis of the immune response in breast cancer is vital for precise biomarkers for ICB and effective combination therapy strategies.

Optimal timing of faculty teaching when combined with near-peer teaching: A mixed methods analysis.

BACKGROUND: Near-peer teaching is increasingly used in medical education, supporting or replacing faculty teaching. It has positive aspects for learners and tutors, some of which are explained by higher social and cognitive congruence between learners and near-peer tutors (NPTs). This study investigates the optimal combination of faculty tutors (FTs) and NPTs in an abdominal ultrasound course. METHODS: Sixty-four third-year medical students underwent a basic ultrasound course, with 75% of lessons taught by NPTs and 25% by FTs. Each of four groups had a different faculty teaching timing. A mixed methods approach used a survey and semi-structured interviews at the course end to elicit learners' preferences, and end-of-course examination scores to look for differences in outcomes. RESULTS: Most learners preferred having faculty teaching in the second half of the course, saying it would be overwhelming to start with FTs. Learners preferred between a quarter and a third of the teaching to be from FTs, with NPTs rated better at teaching basics, and FTs contributing unique, helpful clinical knowledge. There was no significant between-group difference in examination scores. CONCLUSIONS: Medical students preferred most of their teaching to be from NPTs, with some faculty input in the second half of the course.

Social Determinants of Health Screening in Type 1 Diabetes Management.

Type 1 diabetes management is intricately influenced by social determinants of health. Economic status impacts access to vital resources like insulin and diabetes technology. Racism, social injustice, and implicit biases affect equitable delivery of care. Education levels affect understanding of self-care, leading to disparities in glycemic outcomes. Geographic location can limit access to health care facilities. Stressors from discrimination or financial strain can disrupt disease management. Addressing these social factors is crucial for equitable diabetes care, emphasizing the need for comprehensive strategies that go beyond medical interventions to ensure optimal health outcomes for all individuals with type 1 diabetes.

Comparison Between Movement Pattern Training and Strengthening on Kinematics and Kinetics in Patients With Chronic Hip-Related Groin Pain.

The purpose of this study was to compare the preliminary effects of movement pattern training (MoveTrain) versus strengthening/flexibility (standard) treatment on hip and pelvic biomechanics in patients with chronic hip-related groin pain. This is a secondary analysis of data collected during a pilot randomized clinical trial. Thirty patients with hip pain, between the ages of 15 and 40 years, were randomized to MoveTrain or standard. Both groups completed 10 treatment sessions over 12 weeks along with a daily home exercise program. Three-dimensional motion analysis was used to collect kinematic and kinetic data of the pelvis and hip during a single-leg squat task at pretreatment and immediately posttreatment. Compared with the standard group, the MoveTrain group demonstrated smaller hip adduction angles (P = .006) and smaller hip external adduction moments (P = .008) at posttreatment. The desired changes to hip joint biomechanics, as found in this study, may require specificity in training that could allow health care professionals to better customize the rehabilitation of patients with hip pain. These findings can also be applied to the design and implementation of future clinical trials to strengthen our understanding of the long-term implications of different rehabilitation techniques for patients with hip pain.

Novel Presentation of Coccidioidomycosis with Myriad Free-Floating Proteinaceous Spheres in the Pericardial Sac of a Southern Sea Otter (Enhydra lutris nereis).

A southern sea otter (Enhydra lutris nereis) stranded dead in central California, USA, with a distended pericardial sac containing thousands of free-floating proteinaceous masses. Serology, fungal culture, PCR, and sequencing confirmed the etiology of this novel lesion as Coccidioides immitis. Range expansion of this zoonotic pathogen is predicted with climate change.

The specificity landscape of bacterial ribonuclease P.

Developing quantitative models of substrate specificity for RNA processing enzymes is a key step toward understanding their biology and guiding applications in biotechnology and biomedicine. Optimally, models to predict relative rate constants for alternative substrates should integrate an understanding of structures of the enzyme bound to "fast" and "slow" substrates, large datasets of rate constants for alternative substrates, and transcriptomic data identifying in vivo processing sites. Such data are either available or emerging for bacterial ribonucleoprotein RNase P a widespread and essential tRNA 5' processing endonuclease, thus making it a valuable model system for investigating principles of biological specificity. Indeed, the well-established structure and kinetics of bacterial RNase P enabled the development of high throughput measurements of rate constants for tRNA variants and provided the necessary framework for quantitative specificity modeling. Several studies document the importance of conformational changes in the precursor tRNA substrate as well as the RNA and protein subunits of bacterial RNase P during binding, although the functional roles and dynamics are still being resolved. Recently, results from cryo-EM studies of E. coli RNase P with alternative precursor tRNAs are revealing prospective mechanistic relationships between conformational changes and substrate specificity. Yet, extensive uncharted territory remains, including leveraging these advances for drug discovery, achieving a complete accounting of RNase P substrates, and understanding how the cellular context contributes to RNA processing specificity in vivo.

What would primary care practitioners do differently after a delayed cancer diagnosis? Learning lessons from their experiences.

OBJECTIVE: Diagnosis of cancer is challenging in primary care due to the low incidence of cancer cases in primary care practice. A prolonged diagnostic interval may be due to doctor, patient or system factors, or may be due to the characteristics of the cancer itself. The objective of this study was to learn from Primary Care Physicians' (PCP) experiences of incidents when they had failed to think of, or act on, a cancer diagnosis. DESIGN: A qualitative, online survey eliciting PCP narratives. Thematic analysis was used to analyse the data. SETTING AND SUBJECTS: A primary care study, with narratives from 159 PCPs in 23 European countries. MAIN OUTCOME MEASURES: PCPs' narratives on the question 'If you saw this patient with cancer presenting in the same way today, what would you do differently? RESULTS: The main themes identified were: thinking broadly; improvement in communication and clinical management; use of other available resources and 'I wouldn't do anything differently'. CONCLUSION (IMPLICATIONS): To achieve more timely cancer diagnosis, PCPs need to provide a long-term, holistic and active approach with effective communication, and to ensure shared decision-making, follow-up and continuing re-assessment of the patients' clinical conditions.

Diagnosing cancer in primary care is challenging due to the low incidence of cancer in practice and the multiple confounding factors that are involved in the diagnostic process.The need to think broadly, make improvements in communication and clinical management, and use other available resources were the main themes from Primary Care Physicians' (PCPs') narratives about their learning experiences from missed or late cancer diagnoses.A long-term, holistic and active approach with effective communication, follow-up and continuing re-assessment of the patients' clinical conditions was another theme for making improvements.Some PCPs, on reflection, would not have done anything differently.

In Vitro Glycosylation of the Membrane Protein γ-Sarcoglycan in Nanodiscs.

Membrane glycoproteins are proteins that reside in the membranes of cells and are post-translationally modified to have sugars attached to their amino acid side chains. Studies of this subset of proteins in their native states are becoming more important since they have been linked to numerous human diseases. However, these proteins are difficult to study due to their hydrophobic nature and their propensity to aggregate. Using membrane mimetics allows us to solubilize these proteins, which, in turn, allows us to perform glycosylation in vitro to study the effects of the modification on protein structure, dynamics, and interactions. Here, the membrane glycoprotein γ-sarcoglycan was incorporated into nanodiscs composed of long-chain lipids and membrane scaffold proteins to perform N-linked glycosylation in which an enzyme attaches a sugar to the asparagine side chain within the glycosylation site. We previously performed glycosylation of membrane proteins in vitro when the protein had been solubilized using different detergents and short-chain lipids. This work demonstrates successful glycosylation of a full-length membrane protein in nanodiscs providing a more biologically relevant sample to study the effects of the modification.

Arthritis Foundation/HSS Workshop on Hip Osteoarthritis, Part 2: Detecting Hips at Risk: Early Biomechanical and Structural Mechanisms.

Far more publications are available for osteoarthritis of the knee than of the hip. Recognizing this research gap, the Arthritis Foundation (AF), in partnership with the Hospital for Special Surgery (HSS), convened an in-person meeting of thought leaders to review the state of the science of and clinical approaches to hip osteoarthritis. This article summarizes the recommendations gleaned from 5 presentations given in the "early hip osteoarthritis" session of the 2023 Hip Osteoarthritis Clinical Studies Conference, which took place on February 17 and 18, 2023, in New York City. It also summarizes the workgroup recommendations from a small-group discussion on clinical research gaps.