bluuebunny/crossref_metadata_2025_split huggingface dataset:
Single-molecule visualization of sequence-specific RNA binding by a designer PPR protein
Authors: Marzano Nicholas , Johnston Brady , Paudel Bishnu P. , Schmidberger Jason , Jergic Slobodan , Böcking Till , Agostino Mark , Small Ian , van Oijen Antoine M. , Bond Charles S. 
Published: April 2024

AbstractPentatricopeptide repeat (PPR) proteins are a large family of modular RNA-binding proteins that recognize specific ssRNA target sequences. There is significant interest in developing ‘designer’ PPRs for use in diagnostics or as tools to detect and localize target RNA sequences. However, it is unclear how PPRs search for target sequences within complex transcriptomes and current models to predict PPR binding sites struggle to reconcile the effects that RNA mismatches and secondary structure have on PPR binding. To address this, we determined the structure of a designer PPR (dPPR10) bound to its target sequence and used two- and three-colour single-molecule FRET to interrogate the mechanism of ssRNA binding to individual PPR proteins in real time. We demonstrate that longer RNA sequences were significantly slower to bind (or could not bind at all) and that this is, in part, due to their propensity to form stable secondary structures that sequester the target sequence from dPPR10. Importantly, dPPR10 does not associate with non-target flanking sequences, binding specifically to its target sequence within longer ssRNA species. This data provides evidence that PPRs have limited to no capacity to ‘scan’ RNA transcripts for target sequences and instead rely on diffusion for cognate searching. The kinetic constraints imposed by random three-dimensional diffusion may explain the long-standing conundrum of why PPR proteins are abundant in organelles, but almost unknown outside them (i.e. in the cytosol and nucleus). These findings will inform improved prediction of PPR binding sites for the development of designer PPRs.SummaryPentatricopeptide repeat proteins (PPR) are a large family of modular RNA-binding proteins, whereby each module can be ‘designed’ to bind to a specific ssRNA nucleobase and thus any RNA sequence of interest. As such, there is substantial interest in developing ‘designer’ PPRs for a range of biotechnology applications, including diagnostics orin vivolocalisation of RNA species; however, the mechanistic details regarding how PPRs search for and bind to target sequences is unclear. As such, we combined structure-based and single- molecule approaches and determined that PPRs bind only to their target sequences (i.e., they do not associate with non-target RNA sequences) and do not ‘scan’ longer RNA oligonucleotides for the target sequence. Instead, target searching appears kinetically-constrained by random three-dimensional diffusion, providing an explanation as to why PPRs are found almost exclusively in organelle compartments that typically have smaller transcriptomes. Collectively, this work identifies several key considerations for future ‘designer’ PPR developments.
Dose-Efficient Cryo-Electron Microscopy for Thick Samples using Tilt-Corrected Scanning Transmission Electron Microscopy, Demonstrated on Cells and Single Particles
Authors: Yu Yue , Spoth Katherine A. , Colletta Michael , Nguyen Kayla X. , Zeltmann Steven E. , Zhang Xiyue S. , Paraan Mohammadreza , Kopylov Mykhailo , Dubbeldam Charlie , Serwas Daniel , Siems Hannah , Muller David A. , Kourkoutis Lena F. 
Published: April 2024

AbstractCryo-EM is a powerful tool in structural biology, providing insights through techniques like single-particle analysis (SPA) and cryogenic electron tomography (cryo-ET). In thick specimens, challenges arise as an exponentially larger fraction of the transmitted electrons lose energy from inelastic scattering and can no longer be properly focused as a result of chromatic aberrations in the post-specimen optics. Rather than filtering out the inelastic scattering at the price of reducing potential signal, as is done in energy-filtered transmission electron microscopy (EFTEM), we show how a dose-efficient and unfiltered image can be rapidly obtained using tilt-corrected bright-field scanning-TEM (tcBF-STEM) data collected on a pixelated detector. Enhanced contrast and a 3-5x improvement in collection efficiency are observed for 2D images of intact bacterial cells and large organelles using tcBF-STEM compared to EFTEM for thicknesses beyond 500 nm. As a proof of concept for the technique’s performance in structural determination, we present an SPA map at subnanometer resolution for a highly symmetric virus-like particle (VLP) with 789 particles. These findings suggest applications for tcBF-STEM in cryo-EM of thicker cellular volumes where current approaches struggle.
Improved specificity and efficacy of base-editing therapies with hybrid guide RNAs
Authors: Whittaker Madelynn N. , Brooks Dominique L. , Quigley Aidan , Jindal Ishaan , Said Hooda , Qu Ping , Wang Josh Zhiyong , Ahrens-Nicklas Rebecca C. , Musunuru Kiran , Alameh Mohamad-Gabriel , Peranteau William H. , Wang Xiao 
Published: April 2024

AbstractPhenylketonuria (PKU), hereditary tyrosinemia type 1 (HT1), and mucopolysaccharidosis type 1 (MPSI) are autosomal recessive disorders linked to the phenylalanine hydroxylase (PAH) gene, fumarylacetoacetate hydrolase (FAH) gene, and alpha-L-iduronidase (IDUA) gene, respectively. Potential therapeutic strategies to ameliorate disease include corrective editing of pathogenic variants in thePAHandIDUAgenes and, as a variant-agnostic approach, inactivation of the 4-hydroxyphenylpyruvate dioxygenase (HPD) gene, a modifier of HT1, via adenine base editing. Here we evaluated the off-target editing profiles of therapeutic lead guide RNAs (gRNAs) that, when combined with adenine base editors correct the recurrentPAHP281L variant,PAHR408W variant, orIDUAW402X variant or disrupt theHPDgene in human hepatocytes. To mitigate off-target mutagenesis, we systematically screened hybrid gRNAs with DNA nucleotide substitutions. Comprehensive and variant-aware specificity profiling of these hybrid gRNAs reveal dramatically reduced off-target editing and reduced bystander editing. Lastly, in a humanizedPAHP281L mouse model, we showed that when formulated in lipid nanoparticles (LNPs) with adenine base editor mRNA, selected hybrid gRNAs revert the PKU phenotype, substantially enhance on-target editing, and reduce bystander editingin vivo. These studies highlight the utility of hybrid gRNAs to improve the safety and efficacy of base-editing therapies.
Role of m6A RNA methylation in dosage compensation
Authors: Naik Hemant C , Baro Runumi , Sarkar Amritesh , M Muralidhar Nayak , Sunagar Kartik , Gayen Srimonta 
Published: April 2024

AbstractIn therian mammals, inactivation of one of the X-chromosomes in female cells (XX) balances the dosage of X-linked gene expression between the sexes, resulting in monoallelic expression of X-linked genes in females similar to males (XY). Therefore, it creates a dosage imbalance for X-linked genes with respect to the biallelically expressing autosomal genes (AA). X-to-autosome dosage compensation is thought to be achieved by the transcriptional upregulation of many genes from the active X-chromosome in both sexes. Here, we have delineated the role of m6A RNA methylation in the maintenance of dosage compensation in a variety of cell types, including embryonic, extra-embryonic and somatic cells. We show that m6A marks tend to be less enriched on X-linked transcripts compared to the autosomal transcripts in most of the studied cell types. However, our analysis demonstrates that m6A RNA methylation plays a minor role in dosage compensation. We show that the loss of m6A RNA methylation does not affect the maintenance of X-linked gene silencing on the inactive-X in early embryonic lineages, suggesting m6A is dispensable for the maintenance of X-chromosome inactivation. On the contrary, we show that the depletion of m6A affects X-to-autosome dosage compensation, however, the effect is minor and it occurs in a lineage-specific manner. Taken together, our study demonstrates that, although m6A marks are lowly enriched on X-linked transcripts compared to autosomal ones, it is dispensable for the maintenance of X-inactivation and has a minor contribution to the maintenance of X-to-autosome dosage compensation.
Intersectin1 promotes clathrin-mediated endocytosis by organizing and stabilizing endocytic protein interaction networks
Authors: Jin Meiyan , Iwamoto Yuichiro , Shirazinejad Cyna , Drubin David G. 
Published: April 2024

SummaryDuring clathrin-mediated endocytosis (CME), dozens of proteins are recruited to nascent CME sites on the plasma membrane. Coordination of endocytic protein recruitment in time and space is important for efficient CME. Here, we show that the multivalent scaffold protein intersectin1 (ITSN1) promotes CME by organizing and stabilizing endocytic protein interaction networks. By live-cell imaging of genome-edited cells, we observed that endogenously labeled ITSN1 is recruited to CME sites shortly after they begin to assemble. Knocking down ITSN1 impaired endocytic protein recruitment during the stabilization stage of CME site assembly. Artificially locating ITSN1 to the mitochondria surface was sufficient to assemble puncta consisting of CME initiation proteins, including EPS15, FCHO, adaptor proteins, the AP2 complex and epsin1 (EPN1), and the vesicle scission GTPase dynamin2 (DNM2). ITSN1 can form puncta and recruit DNM2 independently of EPS15/FCHO or EPN1. Our work redefines ITSN1’s primary endocytic role as organizing and stabilizing the CME protein interaction networks rather than a previously suggested role in initiation and provides new insights into the multi-step and multi-zone organization of CME site assembly.
Beyond membrane permeability: A role for the small RNA MicF in regulation of chromosome replication and partitioning
Authors: Stibelman Aaron Y. , Sariles Amy Y. , Takahashi Melissa K. 
Published: April 2024

AbstractSmall regulatory RNAs (sRNA) have been shown to play a large role in the management of stress responses inEscherichia coliand other bacteria. sRNAs act post-transcriptionally on target mRNA through an imperfect base pairing mechanism to regulate downstream protein expression. The imperfect base pairing allows a single sRNA to bind and regulate a variety mRNA targets which can form intricate regulatory networks that connect different physiological processes for the cell’s response. Upon exposure to antimicrobials and superoxide generating agents, the MicF sRNA inE. colihas been shown to regulate a small set of genes involved in the management of membrane permeability. Currently, it is unknown whether MicF acts on other processes to mediate the response to these agents. Using an sRNA interaction prediction tool, we identified genes inE. colithat are potentially regulated by MicF. Through subsequent analysis using a sfGFP-based reporter-gene fusion, we have validated two novel targets of MicF regulation: SeqA, a negative modulator of DNA replication, and ObgE, a GTPase crucial for chromosome partitioning. Importantly, the interaction between MicF and these target mRNAs is contingent upon the presence of the RNA chaperone protein, Hfq. Furthermore, our findings affirm the role of MicF’s conserved 5’ seed pairing region in initiating these regulatory interactions. Our study suggests that, beyond its established role in membrane permeability management, MicF exerts control over chromosome dynamics in response to distinct environmental cues, implicating a more multifaceted regulatory function in bacterial stress adaptation.
Estimating heterogeneity of treatment effect in psychiatric clinical trials
Authors: Siegel Joshua S. , Zhong Jinglin , Tomioka Sasagu , Ogirala Ajay , Faraone Stephen V. , Szabo Steven T. , Koblan Kenneth S. , Hopkins Seth C. 
Published: April 2024

AbstractCurrently, placebo-controlled clinical trials report mean change and effect sizes, which masks information about heterogeneity of treatment effects (HTE). Here, we present a method to estimate HTE and evaluate the null hypothesis (H0) that a drug has equal benefit for all participants (HTE=0). We developed measure termed ‘estimated heterogeneity of treatment effect’ oreHTE, which estimates variability in drug response by comparing distributions between study arms. This approach was tested across numerous large placebo-controlled clinical trials. In contrast with variance-based methods which have not identified heterogeneity in psychiatric trials, reproducible instances of treatment heterogeneity were found. For example, heterogeneous response was found in a trial of venlafaxine for depression (peHTE=0.034), and two trials of dasotraline for binge eating disorder (Phase 2, peHTE=0.002; Phase 3, 4mg peHTE=0.011; Phase 3, 6mg peHTE=0.003). Significant response heterogeneity was detected in other datasets as well, often despite no difference in variance between placebo and drug arms. The implications of eHTE as a clinical trial outcomes independent from central tendency of the group is considered and the important of the eHTE method and results for drug developers, providers, and patients is discussed.
Cholesterol distribution among adults in Nigeria: A descriptive cross-sectional study in a rural tertiary hospital
Authors: Asogun Danny , Akyea Ralph K , Osuji Kingsley C , Imafidon Nathaniel O , Qureshi Nadeem , Iyen Barbara 
Published: April 2024

AbstractBackgroundThere is an increasing burden of cardiovascular disease in the Nigerian population, but very little known about the cholesterol distribution within subgroups of the general population.MethodsA cross-sectional study of adults was done in a tertiary hospital in the Southern region of Nigeria. Using results of blood tests over an 8-month period, the cholesterol distribution and prevalence of dyslipidaemias were determined.ResultsA total of 46.8% of patients had raised total cholesterol (mean total cholesterol 198.93mg/dl (SD 57.81)), 14.6% had raised low-density lipoprotein cholesterol (LDL-C) (mean LDL-C 120.83mg/dl (SD 51.62)), and 27.9% of patients had raised triglycerides (mean triglycerides 128.83mg/dl (SD 75.26). Women had higher mean values than men, of total cholesterol (203.3mg/dl vs 193.4mg/dl), LDL-C (125.1mg/dl vs 115.5mg/dl), and non-HDL cholesterol (148.4mg/dl vs 141.1mg/dl). The 95thpercentile values of total cholesterol and LDL-C in the general population were 294mg/dl (7.6mmol/L) and 205.7mg/dl (5.3mmol/L) respectively.ConclusionHypercholesterolaemia is highly prevalent in the Nigerian general population, with a higher prevalence in women compared to men. The 95thpercentile identified is likely to represent the thresholds associated with elevated risk of atherosclerotic cardiovascular disease risk within this population.What is already known on this topicThere is a rising prevalence of cardiovascular disease (CVD) and CVD-related deaths in Nigeria, and a pressing need for targeted strategies and interventions to reduce this burden. However, there is lack of robust evidence on cholesterol distribution within subgroups of the Nigerian population.What this study addsThis study described the distribution of cholesterol concentration in Nigerian adults by sex and age groups, and also determined the cholesterol 95thpercentile thresholds which are likely to be associated with elevated risks of atherosclerotic cardiovascular disease risk within this populationHow this study might affect research, practice or policyEvidence from this study will aid appropriate targeting of interventions to reduce the burden of hypercholesterolaemia and consequent risk of atherosclerotic cardiovascular disease.
Performance of low-cost non-invasive blood markers of liver cirrhosis in adults with chronic hepatitis B infection with and without comorbid alcohol use in Zambia
Authors: Mpisa Sydney , Kahere Morris , Kanunga Annie , Vinikoor Michael , Sinkala Edford 
Published: April 2024

AbstractBackgroundDiagnosis of liver cirrhosis in patients with chronic hepatitis B is challenging given rare use of biopsy. In low and middle-income countries, transient elastography (TE), a recommended non-invasive imaging test for cirrhosis is rarely accessible. We therefore investigated the performance of multiple low-cost and more accessible blood-based liver fibrosis markers in patients with chronic hepatitis B infection in Zambia. As alcohol use complicates the assessment and outcomes of hepatitis B, we also considered alcohol use patterns in our evaluation.MethodsWe performed a hospital-based cross-sectional study, in Lusaka, Zambia, among consecutive treatment-naive adults with chronic hepatitis B mono-infection (i.e., HIV-negative) presenting to our hospital. The reference test for cirrhosis was TE of >/=9.6 kPa. Low-cost markers were the AST-to-platelet ratio index (APRI) at recommended threshold >2, as well as lower proposed alternative thresholds for Africa, >0.5 and >0.65, AST/ALT ratio and FIB-4 index >3.25. We evaluated the performance of each marker versus TE. In a secondary analysis, we evaluated marker performance in participants with current alcohol use versus lifetime abstinence.ResultsAmong 239 adults with HBV mono-infection analyzed, the mean age was 34.7 years and 53 (22.2%) reported current alcohol use. The prevalence of cirrhosis by TE was 16.3% (95% CI: 11.87-21.63). The area under the receiver operating characteristic curve was 0.83, 0.80, 0.79 and 0.73 for FIB-4, APRI >0.5, APRI >0.65 and APRI >2 respectively. Virtually all indices performed less well in people with current alcohol use.ConclusionThese data support the adoption of a lower APRI threshold in Africa, and the use of the FIB-4 index, for diagnosis of cirrhosis among patients with chronic hepatitis B infection. The currently-recommended APRI threshold may exclude people with cirrhosis who need antiviral therapy. Clinicians adopting these markers should screen for alcohol use and consider re-assessment of cirrhosis after alcohol reduction.
Essential conserved neuronal motors kinesin-1 and kinesin-3 regulate Aβ42 toxicity in vivo
Authors: Francis Deepthy , Paonessa Francesco , Fabre Caroline C.G. , Hewitt Victoria L. , Giannakou Maria E. , Peset Isabel , Whitworth Alexander J. , Livesey Frederick J. , Palacios Isabel M. 
Published: April 2024

AbstractAlzheimer’s Disease is the leading cause of dementia and the most common neurodegenerative disorder. Understanding the molecular pathology of Alzheimer’s Disease may help identify new ways to reduce neuronal damage. In the past decadesDrosophilahas become a powerful tool in modelling mechanisms underlying human diseases. Here we investigate how the expression of the human 42-residue β-amyloid (Aβ) carrying the E22G pathogenic “Arctic” mutation (Aβ42Arc) affects axonal health and behaviour ofDrosophila. We find that Aβ42Arcflies present aberrant neurons, with altered axonal transport of mitochondrial and an increased number of terminal boutons at neuromuscular junctions. We demonstrate that the major axonal motor proteins kinesin-1 and kinesin-3 are essential for the correct development of neurons inDrosophilalarvae and similar findings are replicated in human iPSC-derived cortical neurons. We then show that the over-expression of kinesin-1 or kinesin-3 restores the correct number of terminal boutons in Aβ42Arcexpressing neurons and that this is associated with a rescue of the overall neuronal function, measured by negative geotaxis locomotor behavioural assay. We therefore provide new evidence in understanding the mechanisms of axonal transport defects in Alzheimer’s Disease, and our results indicate that kinesins should be considered as potential drug targets to help reduce dementia-associated disorders.
Silversol®(a colloidal nanosilver formulation) inhibits growth of antibiotic-resistantStaphylococcus aureusby disrupting its physiology in multiple ways
Authors: Thakkar Nidhi , Gajera Gemini , Godse Chhaya , DeSouza Anselm , Mehta Dilip , Kothari Vijay 
Published: April 2024

AbstractAntibacterial effect of a colloidal nanosilver formulation Silversol®was investigated against an antibiotic-resistant strain ofStaphylococcus aureus. Lower concentrations of the test formulation exerted bacteriostatic and, its higher concentrations exerted bactericidal effect against this pathogen. Silversol®at sub-lethal concentration was found to disturb multiple physiological traits ofS. aureussuch as growth, antibiotic susceptibility, membrane permeability, efflux, protein synthesis and export, biofilm and exopolysaccharide production, etc. Transcriptome data revealed the genes coding for transcriptional regulators, efflux machinery, transferases, β-lactam resistance, oxidoreductases, metal homeostasis, virulence factors, and arginine biosynthesis to get expressed differently under influence of the test formulation. Genes (argGandargH) involved in arginine biosynthesis emerged among the major targets of Silversol®inS. aureus.
Safety in context: routines and the effect of a balanced safety and operations focus on worker perceptions and performance
Authors: Roberts Matthew D. , Douglas Matthew A. , Overstreet Robert E. 
Published: April 2024

PurposeTo investigate the influence of logistics and transportation workers’ perceptions of their management’s simultaneous safety and operations focus (or lack thereof) on related worker safety and operational perceptions and behaviors.Design/methodology/approachThis multi-method research consisted of two studies. Study 1 aimed to establish correlational relationships by evaluating the impact of individual-level worker perceptions of operationally focused routines (as a moderator) on the relationship between worker perceptions of safety-related routines and workers’ self-reported safety and in-role operational behaviors using a survey. Study 2 aimed to establish causal relationships by evaluating the same conceptual relationships in a behavioral-type experiment utilizing vehicle simulators. After receiving one of four pre-task briefings, participants completed a driving task scenario in a driving simulator.FindingsIn Study 1, the relationship between perceived safety focus and safety behavior/in-role operational behavior was strengthened at higher levels of perceived operations focus. In Study 2, participants who received the balanced pre-task briefing committed significantly fewer safety violations than the other 3 treatment groups. However, in-role driving deviations were not impacted as hypothesized.Originality/valueThis research is conducted at the individual (worker) level of analysis to capture the little-known perspectives of logistics and transportation workers and explore the influence of balanced safety and operational routines from a more micro perspective, thus contributing to a deeper understanding of how balanced routines might influence worker behavior when conducting dynamic tasks to ensure safe, effective outcomes.