Live imaging of excitable axonal microdomains in ankyrin-G-GFP mice.

The axon initial segment (AIS) constitutes not only the site of action potential initiation, but also a hub for activity-dependent modulation of output generation. Recent studies shedding light on AIS function used predominantly post-hoc approaches since no robust murine in vivo live reporters exist. Here, we introduce a reporter line in which the AIS is intrinsically labeled by an ankyrin-G-GFP fusion protein activated by Cre recombinase, tagging the native Ank3 gene. Using confocal, superresolution, and two-photon microscopy as well as whole-cell patch-clamp recordings in vitro, ex vivo, and in vivo, we confirm that the subcellular scaffold of the AIS and electrophysiological parameters of labeled cells remain unchanged. We further uncover rapid AIS remodeling following increased network activity in this model system, as well as highly reproducible in vivo labeling of AIS over weeks. This novel reporter line allows longitudinal studies of AIS modulation and plasticity in vivo in real-time and thus provides a unique approach to study subcellular plasticity in a broad range of applications.

Experimentally validated modeling of dynamic drug-hERG channel interactions reproducing the binding mechanisms and its importance in action potential duration.

BACKGROUND AND OBJECTIVE: Assessment of drug cardiotoxicity is critical in the development of new compounds and modeling of drug-binding dynamics to hERG can improve early cardiotoxicity assessment. We previously developed a methodology to generate Markovian models reproducing preferential state-dependent binding properties, trapping dynamics and the onset of IKr block using simple voltage clamp protocols. Here, we test this methodology with real IKr blockers and investigate the impact of drug dynamics on action potential prolongation. METHODS: Experiments were performed on HEK cells stably transfected with hERG and using the Nanion SyncroPatch 384i. Three protocols, P-80, P0 and P 40, were applied to obtain the experimental data from the drugs and the Markovian models were generated using our pipeline. The corresponding static models were also generated and a modified version of the O´Hara-Rudy action potential model was used to simulate the action potential duration. RESULTS: The experimental Hill plots and the onset of IKr block of ten compounds were obtained using our voltage clamp protocols and the models generated successfully mimicked these experimental data, unlike the CiPA dynamic models. Marked differences in APD prolongation were observed when drug effects were simulated using the dynamic models and the static models. CONCLUSIONS: These new dynamic models of ten well-known IKr blockers constitute a validation of our methodology to model dynamic drug-hERG channel interactions and highlight the importance of state-dependent binding, trapping dynamics and the time-course of IKr block to assess drug effects even at the steady-state.

Global impacts of marine heatwaves on coastal foundation species.

With increasingly intense marine heatwaves affecting nearshore regions, foundation species are coming under increasing stress. To better understand their impacts, we examine responses of critical, habitat-forming foundation species (macroalgae, seagrass, corals) to marine heatwaves in 1322 shallow coastal areas located across 85 marine ecoregions. We find compelling evidence that intense, summer marine heatwaves play a significant role in the decline of foundation species globally. Critically, detrimental effects increase towards species warm-range edges and over time. We also identify several ecoregions where foundation species don't respond to marine heatwaves, suggestive of some resilience to warming events. Cumulative marine heatwave intensity, absolute temperature, and location within a species' range are key factors mediating impacts. Our results suggest many coastal ecosystems are losing foundation species, potentially impacting associated biodiversity, ecological function, and ecosystem services provision. Understanding relationships between marine heatwaves and foundation species offers the potential to predict impacts that are critical for developing management and adaptation approaches.

Genotype-to-phenotype mapping of somatic clonal mosaicism via single-cell co-capture of DNA mutations and mRNA transcripts.

Somatic mosaicism is a hallmark of malignancy that is also pervasively observed in human physiological aging, with clonal expansions of cells harboring mutations in recurrently mutated driver genes. Bulk sequencing of tissue microdissection captures mutation frequencies, but cannot distinguish which mutations co-occur in the same clones to reconstruct clonal architectures, nor phenotypically profile clonal populations to delineate how driver mutations impact cellular behavior. To address these challenges, we developed single-cell Genotype-to-Phenotype sequencing (scG2P) for high-throughput, highly-multiplexed, single-cell joint capture of recurrently mutated genomic regions and mRNA phenotypic markers in cells or nuclei isolated from solid tissues. We applied scG2P to aged esophagus samples from five individuals with high alcohol and tobacco exposure and observed a clonal landscape dominated by a large number of clones with a single driver event, but only rare clones with two driver mutations. NOTCH1 mutants dominate the clonal landscape and are linked to stunted epithelial differentiation, while TP53 mutants and double-driver mutants promote clonal expansion through both differentiation biases and increased cell cycling. Thus, joint single-cell highly multiplexed capture of somatic mutations and mRNA transcripts enables high resolution reconstruction of clonal architecture and associated phenotypes in solid tissue somatic mosaicism.

A RAB7A phosphoswitch coordinates Rubicon Homology protein regulation of Parkin-dependent mitophagy.

Activation of PINK1 and Parkin in response to mitochondrial damage initiates a response that includes phosphorylation of RAB7A at Ser72. Rubicon is a RAB7A binding negative regulator of autophagy. The structure of the Rubicon:RAB7A complex suggests that phosphorylation of RAB7A at Ser72 would block Rubicon binding. Indeed, in vitro phosphorylation of RAB7A by TBK1 abrogates Rubicon:RAB7A binding. Pacer, a positive regulator of autophagy, has an RH domain with a basic triad predicted to bind an introduced phosphate. Consistent with this, Pacer-RH binds to phosho-RAB7A but not to unphosphorylated RAB7A. In cells, mitochondrial depolarization reduces Rubicon:RAB7A colocalization whilst recruiting Pacer to phospho-RAB7A-positive puncta. Pacer knockout reduces Parkin mitophagy with little effect on bulk autophagy or Parkin-independent mitophagy. Rescue of Parkin-dependent mitophagy requires the intact pRAB7A phosphate-binding basic triad of Pacer. Together these structural and functional data support a model in which the TBK1-dependent phosphorylation of RAB7A serves as a switch, promoting mitophagy by relieving Rubicon inhibition and favoring Pacer activation.

Variation in farming damselfish behaviour creates a competitive landscape of risk on coral reefs.

Interspecific interactions are fundamental drivers of animal space use. Yet while non-consumptive effects of predation risk on prey space use are well-known, the risk of aggressive interactions on space use of competitors is largely unknown. We apply the landscape of risk framework to competition-driven space use for the first time, with the hypothesis that less aggressive competitors may alter their behaviour to avoid areas of high competitor density. Specifically, we test how aggressive risk from territorial algal-farming damselfishes can shape the spatial distribution of herbivore fish competitors. We found that only the most aggressive damselfish had fewer competitors in their surrounding area, demonstrating that individual-level behavioural variation can shape spatial distributions. In contradiction to the landscape of risk framework, abundances of farming damselfish and other fishes were positively associated. Our results suggest that reef fishes do not simply avoid areas of high damselfish abundance, but that spatial variation in aggressive behaviour, rather than of individuals, created a competitive landscape of risk. We emphasize the importance of individual-level behaviour in identifying patterns of space use and propose expanding the landscape of risk framework to non-predatory interactions to explore cascading behavioural responses to aggressive risk.

Mapping genotypes to chromatin accessibility profiles in single cells.

In somatic tissue differentiation, chromatin accessibility changes govern priming and precursor commitment towards cellular fates1-3. Therefore, somatic mutations are likely to alter chromatin accessibility patterns, as they disrupt differentiation topologies leading to abnormal clonal outgrowth. However, defining the impact of somatic mutations on the epigenome in human samples is challenging due to admixed mutated and wild-type cells. Here, to chart how somatic mutations disrupt epigenetic landscapes in human clonal outgrowths, we developed genotyping of targeted loci with single-cell chromatin accessibility (GoT-ChA). This high-throughput platform links genotypes to chromatin accessibility at single-cell resolution across thousands of cells within a single assay. We applied GoT-ChA to CD34+ cells from patients with myeloproliferative neoplasms with JAK2V617F-mutated haematopoiesis. Differential accessibility analysis between wild-type and JAK2V617F-mutant progenitors revealed both cell-intrinsic and cell-state-specific shifts within mutant haematopoietic precursors, including cell-intrinsic pro-inflammatory signatures in haematopoietic stem cells, and a distinct profibrotic inflammatory chromatin landscape in megakaryocytic progenitors. Integration of mitochondrial genome profiling and cell-surface protein expression measurement allowed expansion of genotyping onto DOGMA-seq through imputation, enabling single-cell capture of genotypes, chromatin accessibility, RNA expression and cell-surface protein expression. Collectively, we show that the JAK2V617F mutation leads to epigenetic rewiring in a cell-intrinsic and cell type-specific manner, influencing inflammation states and differentiation trajectories. We envision that GoT-ChA will empower broad future investigations of the critical link between somatic mutations and epigenetic alterations across clonal populations in malignant and non-malignant contexts.

Temporal and spatial drivers of the structure of macroinvertebrate assemblages associated with Laminaria hyperborea detritus in the northeast Atlantic.

Kelp forests occur on more than a quarter of the world's coastlines, serving as foundation species supporting high levels of biodiversity. They are also a major source of organic matter in coastal ecosystems, with the majority of primary production released and exported as detritus. Kelp detritus also provides food and shelter for macroinvertebrates, which comprise important components of inshore food-webs. Hitherto, research on kelp detritus-associated macroinvertebrate assemblages remains relatively limited. We quantified spatiotemporal variability in the structure of detritus-associated macroinvertebrate assemblages within Laminaria hyperborea forests and evaluated the influence of putative drivers of the observed variability in assemblages across eight study sites within four regions of the United Kingdom in May and September 2015. We documented 5167 individuals from 106 taxa with Malacostraca, Gastropoda, Isopoda and Bivalvia the most abundant groups sampled. Assemblage structure varied across months, sites, and regions, with highest richness in September compared to May. Many taxa were unique to individual regions, with few documented in all regions. Finally, key drivers of assemblage structure included detritus tissue nitrogen content, depth, sea surface temperature, light intensity, as well as L. hyperborea canopy density and canopy biomass. Despite their dynamic composition and transient existence, accumulations of L. hyperborea detritus represent valuable repositories of biodiversity and represent an additional kelp forest component which influences secondary productivity, and potentially kelp forest food-web dynamics.

The structure and diversity of macroinvertebrate assemblages associated with the understudied pseudo-kelp Saccorhiza polyschides in the Western English Channel (UK).

We examined spatiotemporal variability in the structure of faunal assemblages associated with the warm-temperate pseudo-kelp Saccorhiza polyschides towards its range centre (Western English Channel, southwest UK), to better understand its role as a habitat-former in the northeast Atlantic. A total of 180 sporophytes and their associated fauna were sampled across three months, three sites, and two depths. Assemblage abundance and biomass varied markedly between three morpho-functional sporophyte components (i.e., holdfast, stipe, blade). We recorded rich and abundant macroinvertebrate assemblages, comprising nine phyla, 28 coarse taxonomic groups, and 57 species of molluscs, which consistently dominated assemblages. We observed pronounced seasonality in faunal assemblage structure, marked variability between sites and depths, and strong positive relationships between biogenic habitat availability and faunal abundance/biomass. S. polyschides sporophytes are short-lived and offer temporary, less-stable habitat compared with dominant perennial Laminaria species, so shifts in the relative abundances of habitat-formers will likely alter local biodiversity patterns.

Loss of the stress sensor GADD45A promotes stem cell activity and ferroptosis resistance in LGR4/HOXA9-dependent AML.

ABSTRACT: The overall prognosis of acute myeloid leukemia (AML) remains dismal, largely because of the inability of current therapies to kill leukemia stem cells (LSCs) with intrinsic resistance. Loss of the stress sensor growth arrest and DNA damage-inducible 45 alpha (GADD45A) is implicated in poor clinical outcomes, but its role in LSCs and AML pathogenesis is unknown. Here, we define GADD45A as a key downstream target of G protein-coupled receptor (LGR)4 pathway and discover a regulatory role for GADD45A loss in promoting leukemia-initiating activity and oxidative resistance in LGR4/HOXA9-dependent AML, a poor prognosis subset of leukemia. Knockout of GADD45A enhances AML progression in murine and patient-derived xenograft (PDX) mouse models. Deletion of GADD45A induces substantial mutations, increases LSC self-renewal and stemness in vivo, and reduces levels of reactive oxygen species (ROS), accompanied by a decreased response to ROS-associated genotoxic agents (eg, ferroptosis inducer RSL3) and acquisition of an increasingly aggressive phenotype on serial transplantation in mice. Our single-cell cellular indexing of transcriptomes and epitopes by sequencing analysis on patient-derived LSCs in PDX mice and subsequent functional studies in murine LSCs and primary AML patient cells show that loss of GADD45A is associated with resistance to ferroptosis (an iron-dependent oxidative cell death caused by ROS accumulation) through aberrant activation of antioxidant pathways related to iron and ROS detoxification, such as FTH1 and PRDX1, upregulation of which correlates with unfavorable outcomes in patients with AML. These results reveal a therapy resistance mechanism contributing to poor prognosis and support a role for GADD45A loss as a critical step for leukemia-initiating activity and as a target to overcome resistance in aggressive leukemia.

Ethical and Professional Decision-Making Capabilities of Artificial Intelligence Chatbots: Evaluating ChatGPT's Professional Competencies in Medicine.

Purpose: We examined the performance of artificial intelligence chatbots on the PREview Practice Exam, an online situational judgment test for professionalism and ethics. Methods: We used validated methodologies to calculate scores and descriptive statistics, χ2 tests, and Fisher's exact tests to compare scores by model and competency. Results: GPT-3.5 and GPT-4 scored 6/9 (76th percentile) and 7/9 (92nd percentile), respectively, higher than medical school applicant averages of 5/9 (56th percentile). Both models answered 95 + % of questions correctly. Conclusions: Chatbots outperformed the average applicant on PREview, suggesting their potential for healthcare training and decision-making and highlighting risks of online assessment delivery.

Mutant IDH inhibitors induce lineage differentiation in IDH-mutant oligodendroglioma.

A subset of patients with IDH-mutant glioma respond to inhibitors of mutant IDH (IDHi), yet the molecular underpinnings of such responses are not understood. Here, we profiled by single-cell or single-nucleus RNA-sequencing three IDH-mutant oligodendrogliomas from patients who derived clinical benefit from IDHi. Importantly, the tissues were sampled on-drug, four weeks from treatment initiation. We further integrate our findings with analysis of single-cell and bulk transcriptomes from independent cohorts and experimental models. We find that IDHi treatment induces a robust differentiation toward the astrocytic lineage, accompanied by a depletion of stem-like cells and a reduction of cell proliferation. Furthermore, mutations in NOTCH1 are associated with decreased astrocytic differentiation and may limit the response to IDHi. Our study highlights the differentiating potential of IDHi on the cellular hierarchies that drive oligodendrogliomas and suggests a genetic modifier that may improve patient stratification.

A flow cytometric assay to detect viability and persistence of Salmonella enterica subsp. enterica serotypes in nuclease-free water at 4 and 25°C.

Salmonella spp. is one of the most isolated microorganisms reported to be responsible for human foodborne diseases and death. Water constitutes a major reservoir where the Salmonella spp. can persist and go undetected when present in low numbers. In this study, we assessed the viability of 12 serotypes of Salmonella enterica subsp. enterica for 160 days in nuclease-free water at 4 and 25°C using flow cytometry and Tryptic Soy Agar (TSA) plate counts. The results show that all 12 serotypes remain viable after 160 days in distilled water using flow cytometry, whereas traditional plate counts failed to detect ten serotypes incubated at 25°C. Moreover, the findings demonstrate that 4°C constitutes a more favorable environment where Salmonella can remain viable for prolonged periods without nutrients. Under such conditions, however, Salmonella exhibits a higher susceptibility to all tested antibiotics and benzalkonium chloride (BZK). The pre-enrichment with Universal Pre-enrichment Broth (UP) and 1/10 × Tryptic Soy broth (1/10 × TSB) resuscitated all tested serotypes on TSA plates, nevertheless cell size decreased after 160 days. Furthermore, phenotype microarray (PM) analysis of S. Inverness and S. Enteritidis combined with principal component analysis (PCA) revealed an inter-individual variability in serotypes with their phenotype characteristics, and the impact of long-term storage at 4 and 25°C for 160 days in nuclease-free water. This study provides an insight to Salmonella spp. long-term survivability at different temperatures and highlights the need for powerful tools to detect this microorganism to reduce the risk of disease transmission of foodborne pathogens via nuclease-free water.

Auditor Choice and the Informativeness of 10-K Reports.

This study provides new evidence on the influential role of external auditors in enhancing the informativeness of form 10-K annual reports to shareholders. Specifically, we find that the client's choice of a Big 4 auditor (PwC, EY, KPMG, and Deloitte) versus a non-Big 4 auditor contributes to cross-sectional variations in 10-K disclosure volume. We also document that the benefit of enhanced disclosures provided by Big 4 auditors is more pronounced for audit clients with poorer accrual quality and those with higher information asymmetry. Furthermore, we introduce the portion of 10-K length unexplained by operating complexity and observable client characteristics as a new proxy for audit firm effort. Specifically, we find that abnormally long disclosures are associated with higher audit fees and longer audit report lag, which implies that an incremental level of audit effort can be inferred from the discretionary component of 10-K disclosures. As audit effort is costly, a greater volume of 10-K disclosures can be expected to be associated with an improvement in the quality of financial reporting. Overall, our findings show that auditors play more than a simple attestation role in the financial reporting process, and that the quality of financial reporting in a company's 10-K annual report is a joint product of the effort and decisions of both a company's managers and its auditors.

Thirty-four new species of Rhagovelia (Heteroptera: Veliidae) from the East Papua Composite Terrane, far eastern New Guinea.

Thirty-four new species of Rhagovelia are described from the East Papua Composite Terrane of far eastern New Guinea. The new taxa described from this area are as follows: R. yela, R. woa, and R. mbo from Rossel Island; R. tagula, R. kolukolu, and R. riu from Tagula Island; R. bwagabwaga from Misima Island; R. suloga from Woodlark Island; R. torrenticola and R. elongata from Goodenough Island; R. awaetowa from Fergusson Island; R. dibuwa from Normanby Island; R. basima from Fergusson and Normanby islands; R. kalawai from Sideia and Basilaki islands; R. guiagoila from Basilaki, Sideia and Sariba islands; R. tufi, R. bowutu, R. obscura, R. upalai, R. antap, R. goilala, R. udabe, R. watuti, R. peninsularis, R. auga, R. aviavi, R. tekadu, R. sapoi, R. mimani, R. dinga, R. ivimkana, R. loriae, R. grisea, and R. cheesmanae from the Owen Stanley Range of eastern New Guinea. Redescriptions are also provided for five previously described species occurring in this portion of New Guinea: R. peggiae Kirkaldy, R. hirsuta Lansbury, R. priori Lansbury, R. caesius Lansbury and R. aureospicata Lansbury. A regional key is provided for these 39 species of Rhagovelia occurring in the Papuan Peninsula and adjacent island groups, accompanied by figures of the male parameres and other diagnostic morphological structures, and distribution maps for all species.

GoT-Splice protocol for multi-omics profiling of gene expression, cell-surface proteins, mutational status, and RNA splicing in human cells.

Studying RNA splicing factor mutations is challenging due to difficulties in distinguishing wild-type and mutant cells within complex human tissues and inaccuracies associated with reconstructing splicing signals from short-read sequencing data. Here, we present Genotyping of Transcriptomes (GoT)-Splice, a protocol that overcomes these limitations by combining GoT with enhanced long-read single-cell transcriptome and cell-surface proteomics profiling. We describe steps for long-read library preparation and analysis, followed by cDNA re-amplification, enrichment of mutation of interest, sample indexing, and GoT library preparation. For complete details on the use and execution of this protocol, please refer to Cortés-López et al.1.

Brachial plexus birth injuries: a multi-speciality approach. Commentary and opinions.

The management of brachial plexus birth injuries (BPBI) remains controversial and ever evolving. In this article, studies are examined to provide further insight into the ongoing controversies and debates surrounding BPBI. The articles are diverse and examine the topics of aetiology, demographics, reliability versus accuracy of measurements and surgical management. The management of BPBI may differ depending on resources. Outcome measures may also vary depending on geography. Future research should focus on developing consensus-validated measures and reproducible surgical techniques. These can then guide further population-based research and provide guidelines to minimize the incidence of BPBI.

The Impact of PCAOB-Type Regulations on Auditors Under Different Legal Systems.

This article analyzes the impact of Public Company Accounting Oversight Board (PCAOB)-type regulatory oversight and legal liability on audit quality and social surplus. We show the conditions under which regulatory oversight can improve audit quality and social surplus, as compared with the impact of legal systems. Moreover, we demonstrate that regulatory oversight is not likely to substitute effectively for a legal system. This is the first study that analyzes the possible effects of an audit regulator on auditors under different legal systems, and our results enhance understanding of the complex relationship between regulatory oversight, a legal system, and social surplus.

Deciphering the Exact Sequence of Endogenous Soluble B Cell Maturation Antigen and Unbiased Quantitation in Multiple Myeloma Patient Samples by LC-MS.

BACKGROUND: B-cell maturation antigen is a pivotal therapeutic target for multiple myeloma (MM). Membrane-bound BCMA can be cleaved by γ-secretase and shed as soluble BCMA (sBCMA). sBCMA can act as a neutralizing sink to compete with drug, as well as serve as a diagnostic/prognostic biomarker for MM. Antibody-capture based methods, such as enzyme-linked immunosorbent assay (ELISA) and immunoaffinity-liquid chromatography-multiple reaction monitoring (IA-LC-MRM), have been reported and well adopted to measure sBCMA in clinical samples. However, both methods are biased by capturing antibodies. METHODS: We have used various LC-MS workflows to characterize and quantify endogenous sBCMA in MM patient samples, including bottom-up peptide mapping, intact analysis, IA-based, and reagent-free (RF)-LC-MRM quantitation. RESULTS: We have confirmed that sBCMA contains a variable N-terminus and a C-terminus that extends to the transmembrane domain, ending at amino acid 61. Leveraging an in-house synthesized G-1-61 sBCMA recombinant standard, we developed a RF-LC-MRM method for unbiased sBCMA quantitation in MM patient samples. By comparing the results from RF-LC-MRM with ELISA and IA-LC-MRM, we demonstrated that RF-LC-MRM measures a more complete pool of endogenous sBCMA compared to the antibody-based methods. CONCLUSIONS: This work fills the knowledge gap of the exact sequence of endogenous sBCMA for the first time, which differs from the current commercially available standard. Additionally, this work highlights the necessity of identifying the actual sequence of an endogenous soluble target such as sBCMA, both for bioanalytical purposes and to underpin pharmacodynamic measurements.

Can Medication be a Factor that Can Negatively Affect the Effect of Transcranial Magnetic Stimulation in Depression?

BACKGROUND: This study aims to evaluate the efficacy of transcranial magnetic stimulation (TMS) in patients with depression and whether concurrent psychotropic medication use negatively affects the treatment outcome of TMS. Patients' characteristics, predictors of treatment response, the relationship between demographics, and the selection of TMS as a treatment modality were also analyzed. STUDY QUESTION: Can psychotropic medication be a factor that can negatively affect the efficacy of TMS in patients with depression? STUDY DESIGN: This pilot-controlled study included 40 subjects from Romanian clinical practice who were treated with pharmacological treatment and TMS for major depressive disorder. The severity of depression and anxiety symptoms was measured using validated scales at baseline (day 1) and follow-up (day 30). DATA SOURCES: All patients' characteristics and information were collected manually from the clinic's medical records, deidentified, and then introduced into an electronic database for analysis. LIMITATIONS: Conducting the study in a clinical routine practice, it was not possible to include an active and/or sham control group. In addition, because TMS is not used as a monotherapy in this type of practice, we could not evaluate its safety and efficacy without concomitant pharmacological treatment. The study sample is small; therefore, the results cannot be generalized. RESULTS: Sixty percentage of patients (n = 24) included in this study obtained a clinical response, and 30% of patients (n = 12) obtained remission of depression. The group with pharmacological treatment obtained clinical responses in 80% of patients (n = 16) and remission of depression in 45% of patients (n = 9). The group with pharmacological treatment and TMS obtained clinical responses in 40% of patients (n = 8) and remission of depression in 15% (n = 3) of cases. CONCLUSIONS: The study results show a lack of efficacy for TMS as an adjunctive therapy to pharmacological treatment for patients with depression. In addition, a negative impact of psychotropic medication on TMS efficacy is observed in our study sample.