Retrotransposable elements (RTEs) are common mobile genetic elements comprising ~42% of the human genome. RTEs play critical roles in gene regulation and function, but how they are specifically involved in complex diseases is largely unknown. Here, we investigate the cellular heterogeneity of RTEs using 12 single-cell transcriptome profiles covering three neurodegenerative diseases, Alzheimer's disease (AD), Parkinson's disease, and multiple sclerosis. We identify cell type marker RTEs in neurons, astrocytes, oligodendrocytes, and oligodendrocyte precursor cells that are related to these diseases. The differential expression analysis reveals the landscape of dysregulated RTE expression, especially L1s, in excitatory neurons of multiple neurodegenerative diseases. Machine learning algorithms for predicting cell disease stage using a combination of RTE and gene expression features suggests dynamic regulation of RTEs in AD. Furthermore, we construct a single-cell atlas of retrotransposable elements in neurodegenerative disease (scARE) using these data sets and features. scARE has six feature analysis modules to explore RTE dynamics in a user-defined condition. To our knowledge, scARE represents the first systematic investigation of RTE dynamics at the single-cell level within the context of neurodegenerative diseases.

PWAS (proteome-wide association study) is an innovative genetic association approach that complements widely used methods like GWAS (genome-wide association study). The PWAS approach involves consecutive phases. Initially, machine learning modeling and probabilistic considerations quantify the impact of genetic variants on protein-coding genes’ biochemical functions. Secondly, for each individual, aggregating the variants per gene determines a gene-damaging score. Finally, standard statistical tests are activated in the case-control setting to yield statistically significant genes per phenotype. The PWAS Hub offers a user-friendly interface for an in-depth exploration of gene–disease associations from the UK Biobank (UKB). Results from PWAS cover 99 common diseases and conditions, each with over 10,000 diagnosed individuals per phenotype. Users can explore genes associated with these diseases, with separate analyses conducted for males and females. For each phenotype, the analyses account for sex-based genetic effects, inheritance modes (dominant and recessive), and the pleiotropic nature of associated genes. The PWAS Hub showcases its usefulness for asthma by navigating through proteomic-genetic analyses. Inspecting PWAS asthma-listed genes (a total of 27) provide insights into the underlying cellular and molecular mechanisms. Comparison of PWAS-statistically significant genes for common diseases to the Open Targets benchmark shows partial but significant overlap in gene associations for most phenotypes. Graphical tools facilitate comparing genetic effects between PWAS and coding GWAS results, aiding in understanding the sex-specific genetic impact on common diseases. This adaptable platform is attractive to clinicians, researchers, and individuals interested in delving into gene–disease associations and sex-specific genetic effects.

Sequence variation observed in populations of pathogens can be used for important public health and evolutionary genomic analyses, especially outbreak analysis and transmission reconstruction. Identifying this variation is typically achieved by aligning sequence reads to a reference genome, but this approach is susceptible to reference biases and requires careful filtering of called genotypes. There is a need for tools that can process this growing volume of bacterial genome data, providing rapid results, but that remain simple so they can be used without highly trained bioinformaticians, expensive data analysis, and long-term storage and processing of large files. Here we describe split k-mer analysis (SKA2), a method that supports both reference-free and reference-based mapping to quickly and accurately genotype populations of bacteria using sequencing reads or genome assemblies. SKA2 is highly accurate for closely related samples, and in outbreak simulations, we show superior variant recall compared with reference-based methods, with no false positives. SKA2 can also accurately map variants to a reference and be used with recombination detection methods to rapidly reconstruct vertical evolutionary history. SKA2 is many times faster than comparable methods and can be used to add new genomes to an existing call set, allowing sequential use without the need to reanalyze entire collections. With an inherent absence of reference bias, high accuracy, and a robust implementation, SKA2 has the potential to become the tool of choice for genotyping bacteria. SKA2 is implemented in Rust and is freely available as open-source software.

High-throughput sequencing (HTS) technologies have been instrumental in investigating biological questions at the bulk and single-cell levels. Comparative analysis of two HTS data sets often relies on testing the statistical significance for the difference of two negative binomial distributions (DOTNB). Although negative binomial distributions are well studied, the theoretical results for DOTNB remain largely unexplored. Here, we derive basic analytical results for DOTNB and examine its asymptotic properties. As a state-of-the-art application of DOTNB, we introduce DEGage, a computational method for detecting differentially expressed genes (DEGs) in scRNA-seq data. DEGage calculates the mean of the sample-wise differences of gene expression levels as the test statistic and determines significant differential expression by computing the P-value with DOTNB. Extensive validation using simulated and real scRNA-seq data sets demonstrates that DEGage outperforms five popular DEG analysis tools: DEGseq2, DEsingle, edgeR, Monocle3, and scDD. DEGage is robust against high dropout levels and exhibits superior sensitivity when applied to balanced and imbalanced data sets, even with small sample sizes. We utilize DEGage to analyze prostate cancer scRNA-seq data sets and identify marker genes for 17 cell types. Furthermore, we apply DEGage to scRNA-seq data sets of mouse neurons with and without fear memory and reveal eight potential memory-related genes overlooked in previous analyses. The theoretical results and supporting software for DOTNB can be widely applied to comparative analyses of dispersed count data in HTS and broad research questions.

Most studies of genome organization have focused on intrachromosomal (cis) contacts because they harbor key features such as DNA loops and topologically associating domains. Interchromosomal (trans) contacts have received much less attention, and tools for interrogating potential biologically relevant trans structures are lacking. Here, we develop a computational framework that uses Hi-C data to identify sets of loci that jointly interact in trans. This method, trans-C, initiates probabilistic random walks with restarts from a set of seed loci to traverse an input Hi-C contact network, thereby identifying sets of trans-contacting loci. We validate trans-C in three increasingly complex models of established trans contacts: the Plasmodium falciparum var genes, the mouse olfactory receptor "Greek islands," and the human RBM20 cardiac splicing factory. We then apply trans-C to systematically test the hypothesis that genes coregulated by the same trans-acting element (i.e., a transcription or splicing factor) colocalize in three dimensions to form "RNA factories" that maximize the efficiency and accuracy of RNA biogenesis. We find that many loci with multiple binding sites of the same DNA-binding proteins interact with one another in trans, especially those bound by factors with intrinsically disordered domains. Similarly, clustered binding of a subset of RNA-binding proteins correlates with trans interaction of the encoding loci. We observe that these trans-interacting loci are close to nuclear speckles. These findings support the existence of trans-interacting chromatin domains (TIDs) driven by RNA biogenesis. Trans-C provides an efficient computational framework for studying these and other types of trans interactions, empowering studies of a poorly understood aspect of genome architecture.

The poly(A) signal, together with auxiliary elements, directs cleavage of a pre-mRNA and thus determines the 3' end of the mature transcript. In many species, including humans, the poly(A) signal is an AAUAAA hexamer, but we recently found that the deeply branching eukaryote Giardia lamblia uses a distinct hexamer (AGURAA) and lacks any known auxiliary elements. Our discovery prompted us to explore the evolutionary dynamics of poly(A) signals and auxiliary elements in the eukaryotic kingdom. We use direct RNA sequencing to determine poly(A) signals for four protists within the Metamonada clade (which also contains G. lamblia) and two outgroup protists. These experiments reveal that the AAUAAA hexamer serves as the poly(A) signal in at least four different eukaryotic clades, indicating that it is likely the ancestral signal, whereas the unusual Giardia version is derived. We find that the use and relative strengths of auxiliary elements are also plastic; in fact, within Metamonada, species like G. lamblia make use of a previously unrecognized auxiliary element where nucleotides flanking the poly(A) signal itself specify genuine cleavage sites. Thus, despite the fundamental nature of pre-mRNA cleavage for the expression of all protein-coding genes, the motifs controlling this process are dynamic on evolutionary timescales, providing motivation for future biochemical and structural studies as well as new therapeutic angles to target eukaryotic pathogens.

The human major histocompatibility complex (MHC) is a ~4 Mb genomic segment on Chromosome 6 that plays a pivotal role in the immune response. Despite its importance in various traits and diseases, its complex nature makes it challenging to accurately characterize on a routine basis. We present a novel approach allowing targeted sequencing and de novo haplotypic assembly of the MHC region in heterozygous samples, using long-read sequencing technologies. Our approach is validated using two reference samples, two family trios, and an African-American sample. We achieved excellent coverage (96.6%–99.9% with at least 30x depth) and high accuracy (99.89%–99.99%) for the different haplotypes. This methodology offers a reliable and cost-effective method for sequencing and fully characterizing the MHC without the need for whole-genome sequencing, facilitating broader studies on this important genomic segment and having significant implications in immunology, genetics, and medicine.

Two decades into the era of Electronic Health Records (EHRs), the promise of streamlining clinical care, reducing burden, and improving patient outcomes has yet to be realized. A cross-sectional family physician census conducted by the American Board of Family Medicine in 2022 and 2023 included self-reported physician EHR satisfaction. Of the nearly 10,000 responding family physicians, only one-in-four (26.2%) report being very satisfied and one-in-three (33.8%) were not satisfied. These low levels of satisfaction point to the need for greater transparency in the marketplace and pressure to increase user-centric EHR design.

Family physicians are fielding questions about cannabis --particularly for the use of cannabis for treatment of pain. Like about every substance ingested to treat medical conditions, cannabis has risks and benefits. But regarding evidence-based practice and practice-based recommendations for patients about cannabis use, the cart is in front of the horse. Cannabis use is still illegal at a federal level and a Schedule 1 drug, but most states have challenged federal law by decriminalizing or legalizing cannabis for a variety of uses. Research is difficult due to this federal status as a Schedule 1 drug since federal funding is not readily available to support research. As a result, physicians have little to no guidance about the clinical usefulness of the product. This article explores what we know and what we are learning about cannabis, and the authors provide clinical guidance for patient care based on this evidence.

The rising obesity epidemic is a phenomenon that has gained increasing attention from health providers and health policy makers. This led to recognition of nonalcoholic fatty liver disease (MASLD). The standard for its assessment has been histologic, which is neither practical nor acceptable by patients. Subsequently, a number of noninvasive assessment methods have been developed. However, despite ease of implementation, their confounding variables do hinder their accuracy. Nonetheless, the development of the liver stiffness measurement (LSM) and incorporation of other biological parameters has minimized but not eliminated the need for liver biopsy. Imaging methods are useful in evaluation, estimation, and following the progression of steatosis and fibrosis with particular attention to controlled attenuation parameter (CAP) and MRI–Proton Density Fat Fraction (MRI-PDFF). The choices for the family physician are broad and rely on tests’ availability, cost, and patient acceptance. Great efforts have been undertaken to produce more robust and novel noninvasive markers that indicate fibrinogenesis directly in an implementable and cost-effective way.

Background:

Discharge communication between hospitalists and primary care clinicians is essential to improve care coordination, minimize adverse events, and decrease unplanned health services use. Health-related social needs are key drivers of health, and hospitalists and primary care clinicians value communicating social needs at discharge.

Purpose:

Sexual misconduct by physicians is a consequential violation of patient trust. The purpose of this study was to determine the frequency and patterns of sexual misconduct by physicians certified by the American Board of Family Medicine (ABFM).

Background:

Certain health-related risk factors require legal interventions. Medical-legal partnerships (MLPs) are collaborations between clinics and lawyers that address these health-harming legal needs (HHLNs) and have been shown to improve health and reduce utilization.

Background:

Higher trust in healthcare providers has been linked to better health outcomes and satisfaction. Lower trust has been associated with healthcare-based discrimination.

Introduction:

High-quality primary care can reduce avoidable emergency department visits and emergency hospitalizations. The availability of electronic medical record (EMR) data and capacities for data storage and processing have created opportunities for predictive analytics. This systematic review examines studies which predict emergency department visits, hospitalizations, and mortality using EMR data from primary care.

COPD and asthma are two of the most common chronic lung diseases, affecting over 545 million people globally and 34 million in the United States. Annual health care costs related to chronic lung disease are estimated at €380 billion in the European Union, and $24–$50 billion in the United States averaging to $4,000 in out-of-pocket costs per person in the U.S. A full-text literature search was conducted for English publications between January 1, 2005–March 18, 2024. It returned over 5,000 publications that were further narrowed using key search words, resulting in 172 peer-reviewed articles. Using their experience and subject expertise, the authors further narrowed the peer-reviewed articles to 55 that were in their opinion relevant. Also, 38 recently published industry reports and news articles specific to downstream effects of inhaler market changes and the future impact were included. The literature suggests that individuals with chronic lung disease face increased challenges with access to inhaled medication due to rising medication costs, discontinuation of branded medications, introduction of generic medications not covered by insurance, exclusionary preferred drug list tactics that force health care providers into non-medical switching of medication or devices, and ongoing medication shortages. Providers experience ongoing hurdles in prescribing appropriate inhaled medications for individuals with chronic lung disease, including increased time and costs spent on administrative tasks due to inhaler denials, a loss of patient trust, and limits on their ability to prescribe appropriate inhaled medication for individuals with chronic lung disease.

BACKGROUND:This study sought to estimate the overall cumulative incidence and odds of Hospital-acquired venous thromboembolism (VTE) among critically ill children with and without exposure to invasive ventilation. In doing so, we also aimed to describe the temporal relationship between invasive ventilation and hospital-acquired VTE development.METHODS:We performed a retrospective cohort study using Virtual Pediatric Systems (VPS) data from 142 North American pediatric ICUs among children < 18 y of age from January 1, 2016–December 31, 2022. After exclusion criteria were applied, cohorts were identified by presence of invasive ventilation exposure. The primary outcome was cumulative incidence of hospital-acquired VTE, defined as limb/neck deep venous thrombosis or pulmonary embolism. Multivariate logistic regression was used to determine whether invasive ventilation was an independent risk factor for hospital-acquired VTE development.RESULTS:Of 691,118 children studied, 86,922 (12.4%) underwent invasive ventilation. The cumulative incidence of hospital-acquired VTE for those who received invasive ventilation was 1.9% and 0.12% for those who did not (P < .001). The median time to hospital-acquired VTE after endotracheal intubation was 6 (interquartile range 3–14) d. In multivariate models, invasive ventilation exposure and duration were each independently associated with development of hospital-acquired VTE (adjusted odds ratio 1.64 [95% CI 1.42–1.86], P < .001; and adjusted odds ratio 1.03 [95% CI 1.02–1.03], P < .001, respectively).CONCLUSIONS:In this multi-center retrospective review from the VPS registry, invasive ventilation exposure and duration were independent risk factors for hospital-acquired VTE among critically ill children. Children undergoing invasive ventilation represent an important target population for risk-stratified thromboprophylaxis trials.

BACKGROUND:Post–COVID-19 syndrome has affected millions of people, with rehabilitation being at the center of non-pharmacologic care. However, numerous published studies show conflicting results due to, among other factors, considerable variation in subject characteristics. Currently, the effects of age, sex, time of implementation, and prior disease severity on the outcomes of a supervised rehabilitation program after COVID-19 remain unknown.METHODS:This was a non-randomized case-control study. Subjects with post–COVID-19 sequelae were enrolled. Among study participants, those who could attend an 8-week, supervised rehabilitation program composed the intervention group, whereas those who couldn’t the control group. Measurements were collected at baseline and 8 weeks thereafter.RESULTS:Study groups (N = 119) had similar baseline measurements. Participation in rehabilitation (n = 47) was associated with clinically important improvements in the 6-min walk test (6MWT) distance, adjusted (for potential confounders) odds ratio (AOR) 4.56 (95% CI 1.95–10.66); 1-min sit-to-stand test, AOR 4.64 (1.88-11.48); Short Physical Performance Battery, AOR 7.93 (2.82–22.26); health-related quality of life (HRQOL) 5-level EuroQol-5D (Visual Analog Scale), AOR 3.12 (1.37–7.08); Montreal Cognitive Assessment, AOR 6.25 (2.16–18.04); International Physical Activity Questionnaire, AOR 3.63 (1.53–8.59); Fatigue Severity Scale, AOR 4.07 (1.51–10.98); Chalder Fatigue Scale (bimodal score), AOR 3.33 (1.45–7.67); Modified Medical Research Council dyspnea scale (mMRC), AOR 4.43 (1.83–10.74); Post–COVID-19 Functional Scale (PCFS), AOR 3.46 (1.51–7.95); and COPD Assessment Test, AOR 7.40 (2.92–18.75). Time from disease onset was marginally associated only with 6MWT distance, AOR 0.99 (0.99–1.00). Prior hospitalization was associated with clinically important improvements in the mMRC dyspnea scale, AOR 3.50 (1.06–11.51); and PCFS, AOR 3.42 (1.16–10.06). Age, sex, and ICU admission were not associated with the results of any of the aforementioned tests/grading scales.CONCLUSIONS:In this non-randomized, case-control study, post–COVID-19 rehabilitation was associated with improvements in physical function, activity, HRQOL, respiratory symptoms, fatigue, and cognitive impairment. These associations were observed independently of timing of rehabilitation, age, sex, prior hospitalization, and ICU admission.

BACKGROUND:Training in mechanical ventilation is a key goal in critical care fellowship education. Web-based simulators offer a cost-effective and readily available alternative to traditional on-site simulators. However, it is unclear how effective they are as teaching tools. In this study, we evaluated the test scores of fellows who underwent mechanical ventilation training by using a web-based simulator compared with fellows who used an on-site simulator during a mechanical ventilation course.METHODS:This was a nonrandomized controlled trial conducted as part of a mechanical ventilation course that involved 70 first-year critical care fellows. The course was identical except for the simulation technology used. One group of instructors used a traditional on-site simulator, the ASL 5000 Lung Solution (n = 39). The second group was instructed in using a web-based simulator, VentSim (n = 31). Each fellow completed a pre-course test and a post-course test by using a validated, case-based ventilator waveform examination that consisted of 5 questions with a total possible score of 100. The primary outcome was a comparison of the mean scores on the posttest between the 2 groups. The study was designed as a non-inferiority trial with a predetermined margin of 10 points.RESULTS:There was no significant difference in the mean ± SD pretest scores between the web-based and the on-site groups (21.1 ± 12.6 and 26.9 ± 13.6 respectively; P = .11). The mean ± SD posttest scores were 45.6 ± 25.0 for the web-based simulator and 43.4 ± 16.5 for on-site simulator (mean difference 2.2; one-sided 95% CI –7.0 to ∞; Pnon-inferiority = .02 [non-inferiority confirmed]). Changes in mean ± SD scores (posttest – pretest) were 25.9 ± 20.9 for the web-based simulator and 16.5 ± 15.9 for the on-site simulator (mean difference 9.4, one-sided 95% CI 0.9 to ∞; Pnon-inferiority < .001 [non-inferiority confirmed]).CONCLUSIONS:In the education of first-year critical care fellows on mechanical ventilation waveform analysis, a web-based mechanical ventilation simulator was non-inferior to a traditional on-site mechanical ventilation simulator.

Descriptive statistics (DS) play a crucial role in establishing a solid foundation for study analysis and are important for understanding the results of a study or data set. If the data from DS is used incorrectly, the study may be misinterpreted. Descriptive statistics summarizes and organizes data, making analysis easier and providing an overview of the characteristics of sampled data. This analysis is comprised of measures of central tendency, which includes the mean, median, and mode of a particular data set. Understanding how to use each metric is essential for basic statistical analysis. The purpose of this short report is to review descriptive statistics and describe how to best utilize them during data analysis. The authors aim to provide this short report as an educational resource to assist the dental hygiene research community in understanding statistical analysis through descriptive statistics.

Purpose Health care professionals (HCPs) working collaboratively can improve patient outcomes and also increase their understanding of each other’s professional roles. This descriptive study aimed to explore dental hygienists’ perceptions of collaboration with dentists and intraprofessional educational (IntraPE) experiences.Methods A convenience sampling method was used to assess DHs perceptions of collaboration with dentists using the Interprofessional Collaboration Scale (ICS), a validated scale that measures perceptions of communication, accommodation, and isolation among HCPs. One open-ended question was added to explore IntraPE. Demographics, work characteristics and responses from the ICS were analyzed using frequency, mean, standard deviation, Pearson’s correlation, t-test, ANOVA, and multivariable regression. Responses from the open-ended question were transcribed, organized, and coded. Themes were identified using the Delve Qualitative Analysis Tool.Results Of the 264 participants, the average age was 38.9, and most identified as female (98.9%). Data analysis revealed that DHs had positive perceptions of collaboration with dentists. Significant relationships were found between ICS factor accommodation and the average number of patients treated per day (rs = −0.242, p<0.001), dentists’ age (rs = −.145, p<0.05). Isolation showed a significant negative correlation with the average number of patients treated per day (rs = −0.156, p<0.05). Most reported having no opportunities for IntraPE education experiences with dentists. Five categories of themes were identified from the open-ended question: shared academic setting, clinic dentist, externships, desire for more shared learning, and shared patient experiences.Conclusion Dental hygienists in this study had an overall more positive than negative perception of collaboration with dentists. Dental and dental hygiene programs should focus on intraprofessional education experiences to continue to enhance collaboration.

Purpose The posterior superior alveolar (PSA) block injection is one of many techniques used to provide profound anesthesia for invasive dental procedures. This technique has a high success rate but is not without complication risks. The purpose of this study was to determine if pulpal anesthesia of the maxillary second molar could be achieved using a reduced needle depth of 10mm or 5mm compared to the traditional needle depth of 16mm.Methods Sixty participants were asked to participate in three sessions. Each session started with a pre neural response test, followed by one randomized needle depth PSA injection, and ending with a post neural response test. The neural response test consisted of two parts, a cold refrigerant and a dental probe, on the buccal and interproximal surface of the maxillary second molar. After receiving a positive neural response, each participant received a posterior superior alveolar block injection using a short (21mm), 27-gauge dental needle with a randomized needle penetration depth of 16mm, 10mm, or 5mm. A post neural response test consisting of the same two parts as the pre-test was conducted on the maxillary second molar to evaluate for profound anesthesia.Results Positive neural responses were obtained from 100% of the participants (n=167) during the pre-tests. Study results demonstrated an 85% success rate at the traditional 16mm needle depth and a 93% and 92% success rates for the reduced needle depths of 10mm and 5mm, respectively. Pulpal anesthesia of the maxillary second molar had been achieved at all three needle depths with no statistically significant difference in the rate of success. Furthermore, there were no adverse events observed.Conclusion The reduced needle depth technique showed promise in achieving desired results of pulpal anesthesia with a reduced risk for complications associated with the PSA block injection. Additional studies are recommended to achieve evidence-based support for this reduced needle depth technique.

Purpose Dental professionals are exposed to hazardous noise levels on a daily basis in clinical practice. The purpose of this study was to compare the hearing status of dental hygienists who utilize ultrasonic scalers in the workplace compared to age-matched control participants (non-dental hygienists) who were not exposed to ultrasonic noise.Methods A convenience sample of nineteen dental hygienists (experimental) and nineteen non-dental hygienists (control) was recruited for this study. A matched pairs design was utilized; participants in each group were matched based on age and gender to eliminate confounding variables. The testing procedure consisted of an audiologist performing a series of auditory tests including otoacoustic emissions test, pure-tone audiometry, and tympanometry on the experimental and control groups.Results In the right ear, there were notable differences from 1000 Hz – 10,000 Hz and in the left ear from 6000 Hz – 10,000 Hz, with higher hearing thresholds in the experimental group of dental hygienists. While 56% of the univariate tests conducted on how many days were worked per week showed statistical significance, the regression line slope indicated those that worked more days had better hearing statuses. The variables for years in practice for dental hygienists, how many of those years were full-time employment, and how many years the dental hygienist had used an ultrasonic scaling device, also had many significant univariate tests for the experimental group only. These variables were more likely to serve as proxies representing true noise exposure. The paired t-test between the groups demonstrated statistically significant differences between the experimental and control group at 9000 Hz in both ears.Conclusion While results from this study demonstrated various qualitative differences in hearing status of the control group (non-dental hygienists) and experimental group (dental hygienists), age was found to be the most critical variable. Furthermore, this data demonstrated differences in hearing status based on various frequencies between dental hygienists and age-matched controls that should be further explored with a larger population.

The Coalition for Epidemic Preparedness Innovations (CEPI) has developed a robust CMC (Chemistry, Manufacturing, and Controls) Framework to enhance the likelihood of successful vaccine development. This Framework serves as a comprehensive guide, aiding developers in building effective strategies to overcome the challenges posed by the different phases of vaccine development, including the ones often referred to as the "valleys of death". The Framework lists stage-appropriate deliverables, categorized and refined, spanning five key areas: manufacturing process, formulation and stability, analytics, supply chain, and compliance. By emphasizing the critical aspects of CMC development, CEPI's objective is to expedite the progression of vaccine candidates from research to deployment, reducing delays, mitigating risks, and optimizing the overall development process, all while upholding uncompromising quality standards, ultimately increasing the probability of success.

Visible particle is an important issue in the biopharmaceutical industry, and it may occur across all the stages in the life cycle of biologics. Upon the occurrence of visible particles, it is often necessary to conduct chemical identification and root cause analysis to safeguard the safety and efficacy of the biotherapeutic products. In this article, we present a number of typical particles and relevant root cause analysis in the categories of extrinsic, intrinsic, and inherent particles that are commonly encountered in the biopharma industry. In particular, the optical images of particles obtained both in situ and after isolation are provided, along with spectral and elemental information. The particle identification was carried out with multiple microscopic and microspectroscopic techniques, including stereo optical microscopy, Fourier-transform infrared microscopy, confocal Raman microscopy, scanning electron microscopy, and energy dispersive X-ray spectroscopy. Both commercial and in-house spectral databases were used for comparison and identification. In addition to particle identification, we placed significant efforts on the root cause analysis of the addressed particles with the intention to provide a relatively whole picture of the particle-related issues and practical references to particle mitigation for our peers in the biopharmaceutical industry.

The analysis of extractables and leachables and subsequent risk assessment is an important aspect of the determination of biocompatibility for many medical devices. Leachable chemicals have the potential to pose a toxicological risk to patients, and therefore it is required that they be adequately characterized and assessed for potential safety concerns. One important consideration in the assessment of leachables is the choice of a suitable simulating solvent intended to replicate the use condition for the device and its biological environment. This aspect of study design is especially difficult for blood-contacting medical devices due to the complexity of simulating the biological matrix. This publication reports a comparison of the extracting power of different binary solvent mixtures and saline in comparison with whole blood for a bloodline tubing set connected to a hemodialyzer. Ten different known extractables, spanning a range of physicochemical properties and molecular weights, were quantified. The results indicated that for low-molecular-weight analytes, a suitable exaggeration for whole blood can be obtained using a low-concentration ethanol/water mixture (20%), and in general, extracted quantity increases with the concentration of alcohol cosolvent. For polyvinylpyrrolidone, the opposite trend was observed, as solubility of the polymer was found to decrease with increasing alcohol concentration, resulting in lower extracted quantities at high alcohol concentrations. Analysis of ethanol/water concentrations in the extract solutions post extraction indicated no change in solvent composition.

Cellular plasticity in adult multicellular organisms is a protective mechanism that allows certain tissues to regenerate in response to injury. Considering that aging involves exposure to repeated injuries over a lifetime, it is conceivable that cell identity itself is more malleable—and potentially erroneous—with age. In this review, we summarize and critically discuss the available evidence that cells undergo age-related shifts in identity, with an emphasis on those that contribute to age-associated pathologies, including neurodegeneration and cancer. Specifically, we focus on reported instances of programs associated with dedifferentiation, biased differentiation, acquisition of features from alternative lineages, and entry into a preneoplastic state. As some of the most promising approaches to rejuvenate cells reportedly also elicit transient changes to cell identity, we further discuss whether cell state change and rejuvenation can be uncoupled to yield more tractable therapeutic strategies.

Thermoregulation, responsible for maintaining a stable core temperature during wide fluctuations in external and internal thermal environments, is an iconic homeostatic process. However, we suggest that despite its fundamental physiological significance, the potential for required cool housing temperatures and thermoregulatory mechanisms to influence the interpretation of experimental data is not sufficiently appreciated. Moreover, although it is generally assumed that the major thermoregulatory pathways are well understood, here we discuss new research that suggests otherwise and reveals the emergence of a new wave of exciting ideas for this "old" field of research.

The brain's capacity to predict and anticipate changes in internal and external environments is fundamental to initiating efficient adaptive responses, behaviors, and reflexes that minimize disruptions to physiology. In the context of feeding control, the brain predicts and anticipates responses to the consumption of dietary substances, thus driving adaptive behaviors in the form of food choices, physiological preparation for meals, and engagement of defensive mechanisms. Here, we provide an integrative perspective on the multisensory computation between exteroceptive and interoceptive cues that guides feeding strategy and may result in food-related disorders.

The study of biological mechanisms, while crucial, cannot fully explain complex phenomena like the instinct to eat. The mind–body connection, as exemplified by the concept of "voodoo death," highlights the profound influence of belief and cultural context on physiology. Indigenous knowledge systems further emphasize the interconnectedness of humans with their environment. Recent discoveries in gut–brain communication reveal the intricate neural circuits that drive our visceral desires, but a holistic approach that integrates both physiological mechanisms and the subjective experience of life, informed by diverse cultural perspectives, will be essential to truly understand what it means to be alive.

Caenorhabditis elegans is an important model organism for human health and disease, with foundational contributions to the understanding of gene expression and tissue patterning in animals. An invaluable tool in modern gene expression research is the presence of a high-resolution ribosome structure, though no such structure exists for C. elegans. Here, we present a high-resolution single-particle cryogenic electron microscopy (cryo-EM) reconstruction and molecular model of a C. elegans ribosome, revealing a significantly streamlined animal ribosome. Many facets of ribosome structure are conserved in C. elegans, including overall ribosomal architecture and the mechanism of cycloheximide, whereas other facets, such as expansion segments and eL28, are rapidly evolving. We identify uL5 and uL23 as two instances of tissue-specific ribosomal protein paralog expression conserved in Caenorhabditis, suggesting that C. elegans ribosomes vary across tissues. The C. elegans ribosome structure will provide a basis for future structural, biochemical, and genetic studies of translation in this important animal system.

The noncoding RNA BC200 is elevated in human cancers and is implicated in translation regulation as well as cell survival and proliferation. Upon BC200 overexpression, we observed correlated expression of a second, smaller RNA species. This RNA is expressed endogenously and exhibits cell-type-dependent variability relative to BC200. Aptamer-tagged expression constructs confirmed that the RNA is a truncated form of BC200, and sequencing revealed a modal length of 120 nt; thus, we refer to the RNA fragment as BC120. We present a methodology for accurate and specific detection of BC120 and establish that BC120 is expressed in several normal human tissues and is also elevated in ovarian cancer. BC120 exhibits remarkable stability relative to BC200 and is resistant to knockdown strategies that target the 3' unique sequence of BC200. Combined knockdown of BC200 and BC120 exhibits greater phenotypic impacts than knockdown of BC200 alone, and overexpression of BC120 negatively impacts translation of a GFP reporter, providing insight into a potential translational regulatory role for this RNA. The presence of a novel, truncated, and stable form of BC200 adds complexity to the investigation of this noncoding RNA that must be considered in future studies of BC200 and other related Alu RNAs.

Vault RNAs (vtRNAs) are evolutionarily conserved small noncoding RNAs transcribed by RNA polymerase III. Vault RNAs were initially described as components of the vault particle, but have since been assigned multiple vault-independent functions, including regulation of PKR activity, apoptosis, autophagy, lysosome biogenesis, and viral particle trafficking. The full-length transcript has also been described as a noncanonical source of miRNAs, which are processed in a DICER-dependent manner. As central molecules in vault-dependent and independent processes, vtRNAs have been attributed numerous biological roles, including regulation of cell proliferation and survival, response to viral infections, drug resistance, and animal development. Yet, their impact to mammalian physiology remains largely unexplored. To study vault RNAs in vivo, we generated a mouse line with a conditional Vaultrc5 loss-of-function allele. Because Vaultrc5 is the sole murine vtRNA, this allele enables the characterization of the physiological requirements of this conserved class of small regulatory RNAs in mammals. Using this strain, we show that mice constitutively null for Vaultrc5 are viable and histologically normal but have a slight reduction in platelet counts, pointing to a potential role for vtRNAs in hematopoiesis. This work paves the way for further in vivo characterizations of this abundant but mysterious RNA molecule. Specifically, it enables the study of the biological consequences of constitutive or lineage-specific Vaultrc5 deletion and of the physiological requirements for an intact Vaultrc5 during normal hematopoiesis or in response to cellular stresses such as oncogene expression, viral infection, or drug treatment.

The SARS-CoV-2 frameshifting element (FSE) has been intensely studied and explored as a therapeutic target for coronavirus diseases, including COVID-19. Besides the intriguing virology, this small RNA is known to adopt many length-dependent conformations, as verified by multiple experimental and computational approaches. However, the role these alternative conformations play in the frameshifting mechanism and how to quantify this structural abundance has been an ongoing challenge. Here, we show by DMS and dual-luciferase functional assays that previously predicted FSE mutants (using the RAG graph theory approach) suppress structural transitions and abolish frameshifting. Furthermore, correlated mutation analysis of DMS data by three programs (DREEM, DRACO, and DANCE-MaP) reveals important differences in their estimation of specific RNA conformations, suggesting caution in the interpretation of such complex conformational landscapes. Overall, the abolished frameshifting in three different mutants confirms that all alternative conformations play a role in the pathways of ribosomal transition.

SEquence Evaluation through k-mer Representation (SEEKR) is a method of sequence comparison that uses sequence substrings called k-mers to quantify the nonlinear similarity between nucleic acid species. We describe the development of new functions within SEEKR that enable end-users to estimate P-values that ascribe statistical significance to SEEKR-derived similarities, as well as visualize different aspects of k-mer similarity. We apply the new functions to identify chromatin-enriched lncRNAs that contain XIST-like sequence features, and we demonstrate the utility of applying SEEKR on lncRNA fragments to identify potential RNA-protein interaction domains. We also highlight ways in which SEEKR can be applied to augment studies of lncRNA conservation, and we outline the best practice of visualizing RNA-seq read density to evaluate support for lncRNA annotations before their in-depth study in cell types of interest.

Background

Exacerbations of noncystic fibrosis bronchiectasis (bronchiectasis) are associated with reduced health-related quality of life and increased mortality, likelihood of hospitalisation and lung function decline. This study investigated patient clinical characteristics associated with exacerbation frequency.

Background

Quality of life is impaired in patients with sarcoidosis. The King's Sarcoidosis Questionnaire (KSQ) is a brief questionnaire assessing health-related quality of life in patients with sarcoidosis, comprising subdomains of General Health Status (GHS), Lung, Medication, Skin and Eyes. The aim of this study was to enhance the validation of the KSQ, incorporating longitudinal validation and known-groups validity in a cohort with mild sarcoidosis.

Extract

There is an expanding literature on the association between obstructive sleep apnoea (OSA) and cancer risk [1, 2]. Evidence is growing from population- and clinic-based cohort studies that the severity of OSA and sleep-related hypoxaemia may adversely affect both overall cancer risk and incidence of certain cancers [3–7]. These clinical findings are supported by the identification of the intermediate mechanisms by which intermittent hypoxia and sleep fragmentation, the hallmark features of OSA, might promote oncogenesis, tumour growth and metastasis [8]. Although studies have shown a relationship between OSA and cancer, few have evaluated whether the risk of cancer development or progression in patients with OSA is modified by continuous positive airway pressure (CPAP) therapy (the primary treatment for OSA) [1, 2].

Extract

With a disproportionate burden of tuberculosis (TB) amongst migrants in Europe [1], Burman et al. [2] have highlighted the pressing need for alternative approaches to make TB infection (TBI) screening comprehensive and accessible. Across high-income Organisation for Economic Co-operation and development countries, a median of 52% of TB cases occur in foreign-born individuals, who are at their highest risk of developing TB disease within the first 5 years of migration [3]. Molecular epidemiological studies indicate that the majority of these cases occur as a result of TBI reactivation, often acquired overseas [4]. Within the UK, overseas-born migrants have a 14-fold higher TB incidence than UK-born individuals [5]. The World Health Organization therefore recommends that migrants from countries with a high TB burden may be prioritised for TBI screening [6, 7].

Extract

In cystic fibrosis (CF), Pseudomonas aeruginosa acquisition represents a turning point in disease progression. The presence of chronic P. aeruginosa infection is associated with worsening lung function and increased risk of earlier death, whereas treatment substantially improves lung function and survival [1, 2]. Efforts to diagnose and eradicate early P. aeruginosa provide lasting benefits for children with CF [3, 4]. However, the timing of infection varies considerably between individuals with CF, treatment centres [5, 6], and different birth cohorts of people with the disease [7, 8].

Background

Computer-aided detection (CAD) systems hold promise for improving tuberculosis (TB) detection on digital chest radiographs. However, data on their performance in exclusively paediatric populations are scarce.

The management of chylothorax remains challenging given the limited evidence and significant heterogeneity in practice. In addition, there are no practical guidelines on the optimal approach to manage this complex condition. We convened an international group of 27 experts from 20 institutions across five countries and four specialties (pulmonary, interventional radiology, thoracic surgery and nutrition) with experience and expertise in managing adult patients with chylothorax. We performed a literature and internet search for reports addressing seven clinically relevant PICO (Patient, Intervention, Comparison and Outcome) questions pertaining to the management of adult patients with chylothorax. This consensus statement, consisting of best practice statements based on expert consensus addressing these seven PICO questions, was formulated by a systematic and rigorous process involving the evaluation of published evidence, augmented with provider experience. Panel members participated in the development of the final best practice statements using the modified Delphi technique. Our consensus statement aims to offer guidance in clinical decision making when managing patients with chylothorax while also identifying gaps in knowledge and informing future research.

Background

Pseudomonas aeruginosa is a common pathogen that contributes to progressive lung disease in cystic fibrosis (CF). Genetic factors other than CF-causing CFTR (CF transmembrane conductance regulator) variations contribute ~85% of the variation in chronic P. aeruginosa infection age in CF according to twin studies, but the susceptibility loci remain unknown. Our objective is to advance understanding of the genetic basis of host susceptibility to P. aeruginosa infection.

Background

Control of latent tuberculosis infection (LTBI) is a priority in the World Health Organization strategy to eliminate TB. Many high-income, low TB incidence countries have prioritised LTBI screening and treatment in recent migrants. We tested whether a novel model of care, based entirely within primary care, was effective and safe compared to secondary care.

Hepatocyte nuclear factor 4 alpha antisense 1 (HNF4A-AS1) is a long noncoding RNA (lncRNA) gene physically located next to the transcription factor HNF4A gene in the human genome. Its transcription products have been reported to inhibit the progression of hepatocellular carcinoma (HCC) and negatively regulate the expression of cytochrome P450s (CYPs), including CYP1A2, 2B6, 2C9, 2C19, 2E1, and 3A4. By altering CYP expression, lncRNA HNF4A-AS1 also contributes to the susceptibility of drug-induced liver injury. Thus, HNF4A-AS1 lncRNA is a promising target for controlling HCC and modulating drug metabolism. However, HNF4A-AS1 has four annotated alternative transcripts in the human genome browsers, and it is unclear which transcripts the small interfering RNAs or small hairpin RNAs used in the previous studies are silenced and which transcripts should be used as the target. In this study, four annotated and two newly identified transcripts were confirmed. These six transcripts showed different expression levels in different liver disease conditions, including metabolic dysfunction-associated steatotic liver disease, alcohol-associated liver disease, and obesity. The expression patterns of all HNF4A-AS1 transcripts were further investigated in liver cell growth from human embryonic stem cells to matured hepatocyte-like cells, HepaRG differentiation, and exposure to rifampicin treatment. Several HNF4A-AS1 transcripts highly displayed correlations with these situations. In addition, some of the HNF4A-AS1 transcripts also showed a strong correlation with CYP3A4 during HepaRG maturation and rifampicin exposure. Our findings provide valuable insights into the specific roles of HNF4A-AS1 transcripts, paving the way for more targeted therapeutic strategies for liver diseases and drug metabolism.

Human organic anion transporting polypeptide (OATP) 1B1 and 1B3 are two highly homologous liver-specific uptake transporters. However, 2’,7’-dichlorofluorescein (DCF) is preferably transported by OATP1B1. In the present study, the molecular mechanisms for the selective transport of DCF by OATP1B1 were investigated by constructing and characterizing an array of OATP1B1/1B3 chimeras and site-directed mutagenesis. Our results show that transmembrane domain (TM) 10 is crucial for the surface expression and function of OATP1B1, in which Q541 and L545 play the most important roles in DCF transport. Replacement of TM10 in OATP1B1 with its OATP1B3 counterpart led to OATP1B1’s complete intracellular retention. Q541 and L545 may interact with DCF directly via hydrogen bonding and hydrophobic interactions. The decrease of DCF uptake by Q541A and L545S was due to their reduced binding affinity for DCF as compared with OATP1B1. In addition, Q541 and L545 are also crucial for the transport of estradiol-17β-glucuronide (E17βG) but not for the transport of estrone-3-sulfate (E3S), indicating different interaction modes between DCF/E17βG and E3S in OATP1B1. Taken together, Q541 and L545 in TM10 are critical for OATP1B1-mediated DCF uptake, but their effect is substrate-dependent.

Solute carrier family 6 member 19 (SLC6A19) inhibitors are being studied as therapeutic agents for phenylketonuria. In this work, a potent SLC6A19 inhibitor (RA836) elevated rat kidney uremic toxin indoxyl sulfate (IDS) levels by intensity (arbitrary unit) of 13.7 ± 7.7 compared with vehicle 0.3 ± 0.1 (P = 0.01) as determined by tissue mass spectrometry imaging analysis. We hypothesized that increased plasma and kidney levels of IDS could be caused by the simultaneous inhibition of both Slc6a19 and a kidney IDS transporter responsible for excretion of IDS into urine. To test this, we first confirmed the formation of IDS through tryptophan metabolism by feeding rats a Trp-free diet. Inhibiting Slc6a19 with RA836 led to increased IDS in these rats. Next, RA836 and its key metabolites were evaluated in vitro for inhibiting kidney transporters such as organic anion transporter (OAT)1, OAT3, and breast cancer resistance protein (BCRP). RA836 inhibits BCRP with an IC₅₀ of 0.045 μM but shows no significant inhibition of OAT1 or OAT3. Finally, RA836 analogs with either potent or no inhibition of SLC6A19 and/or BCRP were synthesized and administered to rats fed a normal diet. Plasma and kidney samples were collected to quantify IDS using liquid chromatography–mass spectrometry. Neither a SLC6A19 inactive but potent BCRP inhibitor nor a SLC6A19 active but weak BCRP inhibitor raised IDS levels, whereas compounds inhibiting both transporters caused IDS accumulation in rat plasma and kidney, supporting the hypothesis that rat Bcrp contributes to the excretion of IDS. In summary, we identified that inhibiting Slc6a19 increases IDS formation, while simultaneously inhibiting Bcrp results in IDS accumulation in the kidney and plasma.

Exercise significantly alters human physiological functions, such as increasing cardiac output and muscle blood flow and decreasing glomerular filtration rate (GFR) and liver blood flow, thereby altering the absorption, distribution, metabolism, and excretion of drugs. In this study, we aimed to establish a database of human physiological parameters during exercise and to construct equations for the relationship between changes in each physiological parameter and exercise intensity, including cardiac output, organ blood flow (e.g., muscle blood flow and kidney blood flow), oxygen uptake, plasma pH and GFR, etc. The polynomial equation P = a_iHRⁱ was used for illustrating the relationship between the physiological parameters (P) and heart rate (HR), which served as an index of exercise intensity. The pharmacokinetics of midazolam, quinidine, digoxin, and lidocaine during exercise were predicted by a whole-body physiologically based pharmacokinetic (WB-PBPK) model and the developed database of physiological parameters following administration to 100 virtual subjects. The WB-PBPK model simulation results showed that most of the observed plasma drug concentrations fell within the 5^th–95^th percentiles of the simulations, and the estimated peak concentrations (C_max) and area under the curve (AUC) of drugs were also within 0.5–2.0 folds of observations. Sensitivity analysis showed that exercise intensity, exercise duration, medication time, and alterations in physiological parameters significantly affected drug pharmacokinetics and the net effect depending on drug characteristics and exercise conditions. In conclusion, the pharmacokinetics of drugs during exercise could be quantitatively predicted using the developed WB-PBPK model and database of physiological parameters.

Organic anion transporting polypeptides (OATP, gene symbol SLCO) are well-recognized key determinants for the absorption, distribution, and excretion of a wide spectrum of endogenous and exogenous compounds including many antineoplastic agents. It was therefore proposed as a potential drug target for cancer therapy. In our previous study, it was found that low-dose X-ray and carbon ion irradiation both upregulated the expression of OATP family member OATP1A2 and in turn, led to a more dramatic killing effect when cancer cells were cotreated with antitumor drugs such as methotrexate. In the present study, the underlying mechanism of the phenomenon was explored in breast cancer cell line MCF-7. It was found that the nonreceptor tyrosine kinase v-YES-1 Yamaguchi sarcoma viral oncogene homolog 1 (YES-1) was temporally coordinated with the change of OATP1A2 after irradiation. The overexpression of YES-1 significantly increased OATP1A2 both at the mRNA and protein level. The signal transducer and activator of transcription 3 (STAT3) pathway is likely the downstream target of YES-1 because phosphorylation and nuclear accumulation of STAT3 were both enhanced after overexpressing YES-1 in MCF-7 cells. Further investigation revealed that there are two possible binding sites of STAT3 localized at the upstream sequence of SLCO1A2, the encoding gene of OATP1A2. Electrophoretic mobility shift assay and chromatin immunoprecipitation analysis suggested that these two sites bound to STAT3 specifically and the overexpression of YES-1 significantly increased the association of the transcription factor with the putative binding sites. Finally, inhibition or knockdown of YES-1 attenuated the induction effect of radiation on the expression of OATP1A2.