Single-fly genome assemblies fill major phylogenomic gaps across the Drosophilidae Tree of Life.

Long-read sequencing is driving rapid progress in genome assembly across all major groups of life, including species of the family Drosophilidae, a longtime model system for genetics, genomics, and evolution. We previously developed a cost-effective hybrid Oxford Nanopore (ONT) long-read and Illumina short-read sequencing approach and used it to assemble 101 drosophilid genomes from laboratory cultures, greatly increasing the number of genome assemblies for this taxonomic group. The next major challenge is to address the laboratory culture bias in taxon sampling by sequencing genomes of species that cannot easily be reared in the lab. Here, we build upon our previous methods to perform amplification-free ONT sequencing of single wild flies obtained either directly from the field or from ethanol-preserved specimens in museum collections, greatly improving the representation of lesser studied drosophilid taxa in whole-genome data. Using Illumina Novaseq X Plus and ONT P2 sequencers with R10.4.1 chemistry, we set a new benchmark for inexpensive hybrid genome assembly at US $150 per genome while assembling genomes from as little as 35 ng of genomic DNA from a single fly. We present 183 new genome assemblies for 179 species as a resource for drosophilid systematics, phylogenetics, and comparative genomics. Of these genomes, 62 are from pooled lab strains and 121 from single adult flies. Despite the sample limitations of working with small insects, most single-fly diploid assemblies are comparable in contiguity (>1 Mb contig N50), completeness (>98% complete dipteran BUSCOs), and accuracy (>QV40 genome-wide with ONT R10.4.1) to assemblies from inbred lines. We present a well-resolved multi-locus phylogeny for 360 drosophilid and 4 outgroup species encompassing all publicly available (as of August 2023) genomes for this group. Finally, we present a Progressive Cactus whole-genome, reference-free alignment built from a subset of 298 suitably high-quality drosophilid genomes. The new assemblies and alignment, along with updated laboratory protocols and computational pipelines, are released as an open resource and as a tool for studying evolution at the scale of an entire insect family.

Spt5 C-terminal repeat domain phosphorylation and length negatively regulate heterochromatin through distinct mechanisms.

Heterochromatin is a condensed chromatin structure that represses transcription of repetitive DNA elements and developmental genes, and is required for genome stability. Paradoxically, transcription of heterochromatic sequences is required for establishment of heterochromatin in diverse eukaryotic species. As such, components of the transcriptional machinery can play important roles in establishing heterochromatin. How these factors coordinate with heterochromatin proteins at nascent heterochromatic transcripts remains poorly understood. In the model eukaryote Schizosaccharomyces pombe (S. pombe), heterochromatin nucleation can be coupled to processing of nascent transcripts by the RNA interference (RNAi) pathway, or to other post-transcriptional mechanisms that are RNAi-independent. Here we show that the RNA polymerase II processivity factor Spt5 negatively regulates heterochromatin in S. pombe through its C-terminal domain (CTD). The Spt5 CTD is analogous to the CTD of the RNA polymerase II large subunit, and is comprised of multiple repeats of an amino acid motif that is phosphorylated by Cdk9. We provide evidence that genetic ablation of Spt5 CTD phosphorylation results in aberrant RNAi-dependent nucleation of heterochromatin at an ectopic location, as well as inappropriate spread of heterochromatin proximal to centromeres. In contrast, truncation of Spt5 CTD repeat number enhanced RNAi-independent heterochromatin formation and bypassed the requirement for RNAi. We relate these phenotypes to the known Spt5 CTD-binding factor Prf1/Rtf1. This separation of function argues that Spt5 CTD phosphorylation and CTD length restrict heterochromatin through unique mechanisms. More broadly, our findings argue that length and phosphorylation of the Spt5 CTD repeat array have distinct regulatory effects on transcription.

Gßγ subunits colocalize with RNA polymerase II and regulate transcription in cardiac fibroblasts.

Gßγ subunits mediate many different signaling processes in various compartments of the cell, including the nucleus. To gain insight into the functions of nuclear Gßγ signaling, we investigated the functional role of Gßγ signaling in the regulation of GPCR-mediated gene expression in primary rat neonatal cardiac fibroblasts. We identified a novel, negative, regulatory role for the Gß1γ dimer in the fibrotic response. Depletion of Gß1 led to derepression of the fibrotic response at the mRNA and protein levels under basal conditions and an enhanced fibrotic response after sustained stimulation of the angiotensin II type I receptor. Our genome-wide chromatin immunoprecipitation experiments revealed that Gß1 colocalized and interacted with RNA polymerase II on fibrotic genes in an angiotensin II-dependent manner. Additionally, blocking transcription with inhibitors of Cdk9 prevented association of Gßγ with transcription complexes. Together, our findings suggest that Gß1γ is a novel transcriptional regulator of the fibrotic response that may act to restrict fibrosis to conditions of sustained fibrotic signaling. Our work expands the role for Gßγ signaling in cardiac fibrosis and may have broad implications for the role of nuclear Gßγ signaling in other cell types.

Oxidative stress mediates associations between preoperative psychosocial phenotype and pain-related outcomes at 6 months following total knee arthroplasty: a longitudinal cohort study.

OBJECTIVE: Greater preoperative depression, anxiety, and pain catastrophizing are associated with more severe long-term pain following total knee arthroplasty (TKA). In a secondary analysis of previously reported data, we tested the hypothesis that these associations are mediated by oxidative stress (OS). DESIGN: A mixed between/within-subjects longitudinal cohort design. SETTING: A single academic medical center. SUBJECTS: Osteoarthritis patients (n = 91; 62.6% female) undergoing unilateral TKA. METHODS: We assessed depression, anxiety, and catastrophizing, as well as markers of central sensitization (widespread pain, temporal summation of pain) preoperatively. Blood samples were then obtained immediately prior to intraoperative tourniquet placement for quantification of in vivo biomarkers of systemic OS, F2-isoprostanes and isofurans. Post-TKA pain intensity (numeric rating scale worst pain [NRS], McGill Pain Questionnaire-2 [MPQ-2]) and function (PROMIS Pain Interference) were assessed at 6 months following TKA. RESULTS: Greater preoperative depression, catastrophizing, and widespread pain were associated with higher intraoperative combined OS (F2-isoprostanes+isofurans/2), which was in turn associated with higher post-TKA pain intensity and worse function (P < .05). All preoperative phenotype predictors except anxiety were correlated positively with post-TKA pain and/or function (P < .05). Bootstrapped mediation analyses revealed significant (P < .05) indirect (mediated) effects of depression (NRS Worst Pain, MPQ-2, PROMIS Pain Interference), anxiety (MPQ-2, PROMIS Pain Interference), and catastrophizing (PROMIS Pain Interference) on adverse long-term post-TKA outcomes via elevated OS. Central sensitization-related predictors demonstrated only direct effects (P < .05) on post-TKA outcomes that were independent of OS mechanisms. CONCLUSIONS: Results suggest that the adverse impact of depression, anxiety, and pain catastrophizing on post-TKA pain and functional outcomes are mediated in part by elevated OS.

Tourniquet does not affect intraoperative kinematics during total knee arthroplasty: Results of a prospective study using a robotic assistance system.

PURPOSE: Tourniquet use during total knee arthroplasty (TKA) remains controversial. There are limited data demonstrating the effect of tourniquet use on flexion and extension gaps. The use of a tourniquet can theoretically affect the kinematics of the knee joint, specifically the extension and flexion gaps and the laxity, by mechanically compressing the soft tissues including the muscles above the knee joint. Therefore, this study was designed to prospectively evaluate changes in flexion and extension gaps with and without the use of a tourniquet. METHODS: The following prospective study included 50 consecutive patients who underwent TKA using a surgical robot. The inclusion criteria were advanced osteoarthritis (OA) and varus-alignment or valgus-alignment <3° (hip-knee-ankle angle, standing long-leg X-ray), and the exclusion criteria were BMI >35 kg/m2 and mechanical axis in >3° valgus. A CR-TKA was performed, and the medial and lateral gaps (in mm) throughout the full range of motion in 10° increments were recorded. The procedure was conducted both with and without an applied tourniquet (350 mmHg). RESULTS: No significant differences were observed in the medial joint space. By contrast, the lateral gap showed significant differences in 10-20° of flexion (with a tourniquet 1.9 mm vs. without a tourniquet 2.1 mm, p = 0.018), 20-30° (1.6 vs. 1.8 mm, p = 0.02), 100-110° (0.9 vs. 1.1 mm, p = 0.021), and 110-120° (0.8 vs. 1 mm, p = 0.038). Thus, at the above degrees of flexion on the lateral side, there was a decrease in the mean of 0.2 mm with the use of a tourniquet. CONCLUSION: Although the use of a tourniquet showed a detectable change in the lateral gap in four 10° segments of flexion, clinical relevance with an average difference of 0.2 mm is not achieved. Thus, the use of a tourniquet in TKA can still be advocated based on the presented data. LEVEL OF EVIDENCE: Level I.

An alternative mechanism for skeletal muscle dysfunction in long-term post-viral lung disease.

Chronic lung disease is often accompanied by disabling extrapulmonary symptoms, notably skeletal muscle dysfunction and atrophy. Moreover, the severity of respiratory symptoms correlates with decreased muscle mass and in turn lowered physical activity and survival rates. Previous models of muscle atrophy in chronic lung disease often modeled chronic obstructive pulmonary disease (COPD) and relied on cigarette smoke exposure and LPS stimulation, but these conditions independently affect skeletal muscle even without accompanying lung disease. Moreover, there is an emerging and pressing need to understand the extrapulmonary manifestations of long-term post-viral lung disease (PVLD) as found in COVID-19. Here, we examine the development of skeletal muscle dysfunction in the setting of chronic pulmonary disease caused by infection due to the natural pathogen Sendai virus using a mouse model of PVLD. We identify a significant decrease in myofiber size when PVLD is maximal at 49 days after infection. We find no change in the relative types of myofibers, but the greatest decrease in fiber size is localized to fast-twitch-type IIB myofibers based on myosin heavy chain immunostaining. Remarkably, all biomarkers of myocyte protein synthesis and degradation (total RNA, ribosomal abundance, and ubiquitin-proteasome expression) were stable throughout the acute infectious illness and chronic post-viral disease process. Together, the results demonstrate a distinct pattern of skeletal muscle dysfunction in a mouse model of long-term PVLD. The findings thereby provide new insights into prolonged limitations in exercise capacity in patients with chronic lung disease after viral infections and perhaps other types of lung injury.NEW & NOTEWORTHY Our study used a mouse model of post-viral lung disease to study the impact of chronic lung disease on skeletal muscle. The model reveals a decrease in myofiber size that is selective for specific types of myofibers and an alternative mechanism for muscle atrophy that might be independent of the usual markers of protein synthesis and degradation. The findings provide a basis for new therapeutic strategies to correct skeletal muscle dysfunction in chronic respiratory disease.

Measuring hypertrophy in neonatal rat primary cardiomyocytes and human iPSC-derived cardiomyocytes.

In the heart, left ventricular hypertrophy is initially an adaptive mechanism that increases wall thickness to preserve normal cardiac output and function in the face of coronary artery disease or hypertension. Cardiac hypertrophy develops in response to pressure and volume overload but can also be seen in inherited cardiomyopathies. As the wall thickens, it becomes stiffer impairing the distribution of oxygenated blood to the rest of the body. With complex cellular signalling and transcriptional networks involved in the establishment of the hypertrophic state, several model systems have been developed to better understand the molecular drivers of disease. Immortalized cardiomyocyte cell lines, primary rodent and larger animal models have all helped understand the pathological mechanisms underlying cardiac hypertrophy. Induced pluripotent stem cell-derived cardiomyocytes are also used and have the additional benefit of providing access to human samples with direct disease relevance as when generated from patients suffering from hypertrophic cardiomyopathies. Here, we briefly review in vitro and in vivo model systems that have been used to model hypertrophy and provide detailed methods to isolate primary neonatal rat cardiomyocytes as well as to generate cardiomyocytes from human iPSCs. We also describe how to model hypertrophy in a "dish" using gene expression analysis and immunofluorescence combined with automated high-content imaging.

The Zebrafish Information Network: major gene page and home page updates.

The Zebrafish Information Network (ZFIN) (https://zfin.org/) is the database for the model organism, zebrafish (Danio rerio). ZFIN expertly curates, organizes, and provides a wide array of zebrafish genetic and genomic data, including genes, alleles, transgenic lines, gene expression, gene function, mutant phenotypes, orthology, human disease models, gene and mutant nomenclature, and reagents. New features at ZFIN include major updates to the home page and the gene page, the two most used pages at ZFIN. Data including disease models, phenotypes, expression, mutants and gene function continue to be contributed to The Alliance of Genome Resources for integration with similar data from other model organisms.

Posterior fossa ischemic infarction: single-center retrospective review of non-surgical and surgical cases.

Cerebellar ischemic stroke (CIS) is a morbid neurological event, with potentially fatal consequences. There is currently no objective standard of care regarding when surgical procedures are required for this entity. We retrospectively reviewed 763 patients with CIS, 247 patients of which had a stroke larger than 1 cm in greatest dimension on cranial imaging. In this subgroup, 11% of patients received ventriculostomy, 12% suboccipital craniectomy, and 9% mechanical endovascular thrombectomy. Various clinical and radiographic variables were examined for relationship to surgical procedures, 30-day mortality rate, and modified Rankin scores. The smallest volume of stroke requiring a surgical procedure was 15.5 mL3 (BrainLab Software). Patients receiving surgical procedures had a higher incidence of multi-territory infarctions, hydrocephalus, cistern compression, 4th ventricular compression, as well as younger age, lower admission GCS, higher admission NIHSS, and higher 30-day mortality/disability. Patients deemed to require surgical procedures for CIS have a higher expected morbidity and mortality than those not requiring surgery. Various clinical and radiographic variables, including stroke volume, can be used to guide selection of patients requiring surgery.

Identification of Demographic and Clinical Prognostic Factors in Traumatic Intraventricular Hemorrhage.

BACKGROUND: The presence of traumatic intraventricular hemorrhage (tIVH) following traumatic brain injury (TBI) is associated with worse neurological outcome. The mechanisms by which patients with tIVH have worse outcome are not fully understood and research is ongoing, but foundational studies that explore prognostic factors within tIVH populations are also lacking. This study aimed to further identify and characterize demographic and clinical variables within a subset of patients with TBI and tIVH that may be implicated in tIVH outcome. METHODS: In this observational study, we reviewed a large prospective TBI database to determine variables present on admission that predicted neurological outcome 6 months after injury. A review of 7,129 patients revealed 211 patients with tIVH on admission and 6-month outcome data. Hypothesized risk factors were tested in univariate analyses with significant variables (p < 0.05) included in logistic and linear regression models. Following the addition of either the Rotterdam computed tomography or Glasgow Coma Scale (GCS) score, we employed a backward selection process to determine significant variables in each multivariate model. RESULTS: Our study found that that hypotension (odds ratio [OR] = 0.35, 95% confidence interval [CI] = 0.13-0.94, p = 0.04) and the hemoglobin level (OR = 1.33, 95% CI = 1.09-1.63, p = 0.006) were significant predictors in the Rotterdam model, whereas only the hemoglobin level (OR = 1.29, 95% CI = 1.06-1.56, p = 0.01) was a significant predictor in the GCS model. CONCLUSIONS: This study represents one of the largest investigations into prognostic factors for patients with tIVH and demonstrates that admission hemoglobin level and hypotension are associated with outcomes in this patient population. These findings add value to established prognostic scales, could inform future predictive modeling studies, and may provide potential direction in early medical management of patients with tIVH.

Keep your cool: Overwintering physiology in response to urbanization in the acorn ant, Temnothorax curvispinosus.

Winter presents a challenge for survival, yet temperate ectotherms have remarkable physiological adaptations to cope with low-temperature conditions. Under recent climate change, rather than strictly relaxing pressure on overwintering survival, warmer winters can instead disrupt these low-temperature trait-environment associations, with negative consequences for populations. While there is increasing evidence of physiological adaptation to contemporary warming during the growing season, the effects of winter warming on physiological traits are less clear. To address this knowledge gap, we performed a common garden experiment using relatively warm-adapted versus cold-adapted populations of the acorn ant, Temnothorax curvispinosus, sampled across an urban heat island gradient, to explore the effects of winter conditions on plasticity and evolution of physiological traits. We found no evidence of evolutionary divergence in chill coma recovery nor in metabolic rate at either of two test temperatures (4 and 10 °C). Although we found the expected plastic response of increased metabolic rate under the 10 °C acute test temperature as compared with the 4 °C test temperature, this plastic response, (i.e., the acute thermal sensitivity of metabolic rate), was not different across populations. Surprisingly, we found that winter-acclimated urban ant populations exhibited higher heat tolerance compared with rural ant populations, and that the magnitude of divergence was comparable to that observed among growing-season acclimated ants. Finally, we found no evidence of differences between populations with respect to changes in colony size from the beginning to the end of the overwintering experiment. Together, these findings indicate that despite the evolution of higher heat tolerance that is often accompanied by losses in low-temperature tolerance, urban acorn ants have retained several components of low-temperature physiological performance when assessed under ecologically relevant overwintering conditions. Our study suggests the importance of measuring physiological traits under seasonally-relevant conditions to understand the causes and consequences of evolutionary responses to contemporary warming.

Lipidated peptides derived from intracellular loops 2 and 3 of the urotensin II receptor act as biased allosteric ligands.

Over the last decade, the urotensinergic system, composed of one G protein-coupled receptor and two endogenous ligands, has garnered significant attention as a promising new target for the treatment of various cardiovascular diseases. Indeed, this system is associated with various biomarkers of cardiovascular dysfunctions and is involved in changes in cardiac contractility, fibrosis, and hypertrophy contributing, like the angiotensinergic system, to the pathogenesis and progression of heart failure. Significant investment has been made toward the development of clinically relevant UT ligands for therapeutic intervention, but with little or no success to date. This system therefore remains to be therapeutically exploited. Pepducins and other lipidated peptides have been used as both mechanistic probes and potential therapeutics; therefore, pepducins derived from the human urotensin II receptor might represent unique tools to generate signaling bias and study hUT signaling networks. Two hUT-derived pepducins, derived from the second and the third intracellular loop of the receptor (hUT-Pep2 and [Trp1, Leu2]hUT-Pep3, respectively), were synthesized and pharmacologically characterized. Our results demonstrated that hUT-Pep2 and [Trp1, Leu2]hUT-Pep3 acted as biased ago-allosteric modulators, triggered ERK1/2 phosphorylation and, to a lesser extent, IP1 production, and stimulated cell proliferation yet were devoid of contractile activity. Interestingly, both hUT-derived pepducins were able to modulate human urotensin II (hUII)- and urotensin II-related peptide (URP)-mediated contraction albeit to different extents. These new derivatives represent unique tools to reveal the intricacies of hUT signaling and also a novel avenue for the design of allosteric ligands selectively targeting hUT signaling potentially.

Cognitive profile of mild traumatic brain injury patients requiring acute hospitalization - A UC davis cognitive screener (UCD-Cog) study.

OBJECTIVE: Identification of patients with mTBI at risk for developing persistent-post concussive syndromes should begin during the ED/inpatient evaluation due to frequent lack of post-discharge follow-up. The best method for evaluating cognitive deficits in these acute settings and how to utilize this information to optimize follow-up care is a matter of ongoing research. In this descriptive study, we present the cognitive profile of 214 hospitalized patients with mTBI using a novel cognitive and behavioral screener, the UCD-Cog. METHOD: A retrospective review of patients with mTBI requiring hospitalization who were enrolled in the UC Davis TBI Registry over the course of 1 year. RESULTS: Reasoning, executive function, and delayed recall were the most frequently impaired cognitive domains. GCS 13-14 was associated with higher numbers of impaired cognitive domains and frequencies of impairments in domains traditionally associated with post-concussive symptoms. Patients with abnormal UCD-Cog results, regardless of GCS, were recommended higher levels of post-discharge care and supervision. CONCLUSION: Inpatient cognitive profiles using the UCD-Cog were consistent with evaluations during the subacute/chronic phase of mTBI and supports the clinical utility of acute cognitive screeners for mTBI management. Future studies will determine how the acute cognitive assessments correlate with long-term mTBI outcomes.

Exploration of the Relationship Between Concussions and Depression Symptoms, Anxiety Symptoms, and Hazardous Drinking Among a Sample of College Students.

Objective: In clinical and athlete populations, research has found that experiencing a concussion (or traumatic brain injury) is correlated with experiencing other psychiatric conditions, including depression and alcohol problems. However, less is known about concussion comorbidity in other population segments. The purpose of this study is to examine the relationships between concussions and depression symptoms, anxiety symptoms, and hazardous drinking among a large sample of college students (N = 1776) enrolled in a mandatory health course. Methods: This study used an online health survey to examine concussion frequency (informal and formal diagnoses), sports-related concussions, depression symptoms, anxiety symptoms, and hazardous drinking in the sample. Bivariate and multivariate analyses were conducted to examine comorbid relationships between concussion frequency and the dependent variables of interest (anxiety symptoms, depression symptoms, and hazardous drinking). Results: We found that 691 (39.1%) participants indicated having at least one concussion. Analyses indicated that concussion frequency scores of both formal or informal diagnoses were significantly associated with scores of depression symptoms, anxiety symptoms, and hazardous drinking. When examining concussion frequency scores of only formal diagnoses, only hazardous drinking evidenced a statistically significant relationship. In addition, participants who had a sports concussion had significantly higher concussion frequency scores and hazardous drinking scores than those who have had a concussion that was not sport-related. Conclusions: The comorbid concussion relationships found in this study are consistent with those observed in clinical and athlete populations. It is important for college health professionals to be aware that concussion comorbidity is not limited to the athlete population and can impact the entire student body.

High-Content Single-Cell Förster Resonance Energy Transfer Imaging of Cultured Striatal Neurons Reveals Novel Cross-Talk in the Regulation of Nuclear Signaling by Protein Kinase A and Extracellular Signal-Regulated Kinase 1/2.

Genetically encoded biosensors can be used to track signaling events in living cells by measuring changes in fluorescence emitted by one or more fluorescent proteins. Here, we describe the use of genetically encoded biosensors based on Förster resonance energy transfer (FRET), combined with high-content microscopy, to image dynamic signaling events simultaneously in thousands of neurons in response to drug treatments. We first applied this approach to examine intercellular variation in signaling responses among cultured striatal neurons stimulated with multiple drugs. Using high-content FRET imaging and immunofluorescence, we identified neuronal subpopulations with unique responses to pharmacological manipulation and used nuclear morphology to identify medium spiny neurons within these heterogeneous striatal cultures. Focusing on protein kinase A (PKA) and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling in the cytoplasm and nucleus, we noted pronounced intercellular differences among putative medium spiny neurons, in both the magnitude and kinetics of signaling responses to drug application. Importantly, a conventional "bulk" analysis that pooled all cells in culture yielded a different rank order of drug potency than that revealed by single-cell analysis. Using a single-cell analytical approach, we dissected the relative contributions of PKA and ERK1/2 signaling in striatal neurons and unexpectedly identified a novel role for ERK1/2 in promoting nuclear activation of PKA in striatal neurons. This finding adds a new dimension of signaling crosstalk between PKA and ERK1/2 with relevance to dopamine D1 receptor signaling in striatal neurons. In conclusion, high-content single-cell imaging can complement and extend traditional population-level analyses and provides a novel vantage point from which to study cellular signaling. SIGNIFICANCE STATEMENT: High-content imaging revealed substantial intercellular variation in the magnitude and pattern of intracellular signaling events driven by receptor stimulation. Since individual neurons within the same population can respond differently to a given agonist, interpreting measures of intracellular signaling derived from the averaged response of entire neuronal populations may not always reflect what happened at the single-cell level. This study uses this approach to identify a new form of cross-talk between PKA and ERK1/2 signaling in the nucleus of striatal neurons.

Actin-binding protein profilin1 promotes aggressiveness of clear-cell renal cell carcinoma cells.

Clear-cell renal cell carcinoma (ccRCC), the most common subtype of renal cancer, has a poor clinical outcome. A hallmark of ccRCC is genetic loss-of-function of VHL (von Hippel-Lindau) that leads to a highly vascularized tumor microenvironment. Although many ccRCC patients initially respond to antiangiogenic therapies, virtually all develop progressive, drug-refractory disease. Given the role of dysregulated expressions of cytoskeletal and cytoskeleton-regulatory proteins in tumor progression, we performed analyses of The Cancer Genome Atlas (TCGA) transcriptome data for different classes of actin-binding proteins to demonstrate that increased mRNA expression of profilin1 (Pfn1), Arp3, cofilin1, Ena/VASP, and CapZ, is an indicator of poor prognosis in ccRCC. Focusing further on Pfn1, we performed immunohistochemistry-based classification of Pfn1 staining in tissue microarrays, which indicated Pfn1 positivity in both tumor and stromal cells; however, the vast majority of ccRCC tumors tend to be Pfn1-positive selectively in stromal cells only. This finding is further supported by evidence for dramatic transcriptional up-regulation of Pfn1 in tumor-associated vascular endothelial cells in the clinical specimens of ccRCC. In vitro studies support the importance of Pfn1 in proliferation and migration of RCC cells and in soluble Pfn1's involvement in vascular endothelial cell tumor cell cross-talk. Furthermore, proof-of-concept studies demonstrate that treatment with a novel computationally designed Pfn1-actin interaction inhibitor identified herein reduces proliferation and migration of RCC cells in vitro and RCC tumor growth in vivo Based on these findings, we propose a potentiating role for Pfn1 in promoting tumor cell aggressiveness in the setting of ccRCC.

Early Apixaban Use Following Stroke in Patients With Atrial Fibrillation: Results of the AREST Trial.

BACKGROUND AND PURPOSE: It is unknown when to start anticoagulation after acute ischemic stroke (AIS) from atrial fibrillation (AF). Early anticoagulation may prevent recurrent infarctions but may provoke hemorrhagic transformation as AF strokes are typically larger and hemorrhagic transformation-prone. Later anticoagulation may prevent hemorrhagic transformation but increases risk of secondary stroke in this time frame. Our aim was to compare early anticoagulation with apixaban in AF patients with stroke or transient ischemic attack (TIA) versus warfarin administration at later intervals. METHODS: AREST (Apixaban for Early Prevention of Recurrent Embolic Stroke and Hemorrhagic Transformation) was an open-label, randomized controlled trial comparing the safety of early use of apixaban at day 0 to 3 for TIA, day 3 to 5 for small-sized AIS (<1.5 cm), and day 7 to 9 for medium-sized AIS (≥1.5 cm, excluding full cortical territory), to warfarin, in a 1:1 ratio at 1 week post-TIA, or 2 weeks post-AIS. RESULTS: Although AREST ended prematurely after a national guideline focused update recommended direct oral anticoagulants over warfarin for AF, it revealed that apixaban had statistically similar yet generally numerically lower rates of recurrent strokes/TIA (14.6% versus 19.2%, P=0.78), death (4.9% versus 8.5%, P=0.68), fatal strokes (2.4% versus 8.5%, P=0.37), symptomatic hemorrhages (0% versus 2.1%), and the primary composite outcome of fatal stroke, recurrent ischemic stroke, or TIA (17.1% versus 25.5%, P=0.44). One symptomatic intracerebral hemorrhage occurred on warfarin, none on apixaban. Five asymptomatic hemorrhagic transformation occurred in each arm. CONCLUSIONS: Early initiation of anticoagulation after TIA, small-, or medium-sized AIS from AF does not appear to compromise patient safety. Potential efficacy of early initiation of anticoagulation remains to be determined from larger pivotal trials. Registration: URL: https://www.clinicaltrials.gov/; Unique identifier: NCT02283294.

Natural Selection on Adults Has Trait-Dependent Consequences for Juvenile Evolution in Dragonflies.

AbstractAlthough natural selection often fluctuates across ontogeny, it remains unclear what conditions enable selection in one life-cycle stage to shape evolution in others. Organisms that undergo metamorphosis are useful for addressing this topic because their highly specialized life-cycle stages cannot always evolve independently despite their dramatic life-history transition. Using a comparative study of dragonflies, we examined three conditions that are hypothesized to allow selection in one stage to affect evolution in others. First, we tested whether lineages with less dramatic metamorphosis (e.g., hemimetabolous insects) lack the capacity for stage-specific evolution. Rejecting this hypothesis, we found that larval body shape evolves independently from selection on adult shape. Next, we evaluated whether stage-specific evolution is limited for homologous and/or coadapted structures. Indeed, we found that selection for larger wings is associated with the evolution of coadapted larval sheaths that store developing wing tissue. Finally, we assessed whether stage-specific evolution is restricted for traits linked to a single biochemical pathway. Supporting this hypothesis, we found that species with more wing melanization in the adult stage have evolved weaker melanin immune defenses in the larval stage. Thus, our results collectively show that natural selection in one stage imposes trait-dependent constraints on evolution in others.

Myogenic Cell Expression of Intercellular Adhesion Molecule-1 Contributes to Muscle Regeneration after Injury.

This study investigated intercellular adhesion molecule-1 (ICAM-1), a membrane protein that mediates cell-to-cell adhesion and communication, as a mechanism through which the inflammatory response facilitates muscle regeneration after injury. Toxin-induced muscle injury to tibialis anterior muscles of wild-type mice caused ICAM-1 to be expressed by a population of satellite cells/myoblasts and myofibers. Myogenic cell expression of ICAM-1 contributed to the restoration of muscle structure after injury, as regenerating myofibers were more abundant and myofiber size was larger for wild-type compared with Icam1-/- mice during 28 days of recovery. Contrastingly, restoration of muscle function after injury was similar between the genotypes. ICAM-1 facilitated the restoration of muscle structure after injury through mechanisms involving the regulation of myofiber branching, protein synthesis, and the organization of nuclei within myofibers after myogenic cell fusion. These findings provide support for a paradigm in which ICAM-1 expressed by myogenic cells after muscle injury augments their adhesive and fusogenic properties, which, in turn, facilitates regenerative and hypertrophic processes that restore structure to injured muscle.

Physiological adaptation to cities as a proxy to forecast global-scale responses to climate change.

Cities are emerging as a new venue to overcome the challenges of obtaining data on compensatory responses to climatic warming through phenotypic plasticity and evolutionary change. In this Review, we highlight how cities can be used to explore physiological trait responses to experimental warming, and also how cities can be used as human-made space-for-time substitutions. We assessed the current literature and found evidence for significant plasticity and evolution in thermal tolerance trait responses to urban heat islands. For those studies that reported both plastic and evolved components of thermal tolerance, we found evidence that both mechanisms contributed to phenotypic shifts in thermal tolerance, rather than plastic responses precluding or limiting evolved responses. Interestingly though, for a broader range of studies, we found that the magnitude of evolved shifts in thermal tolerance was not significantly different from the magnitude of shift in those studies that only reported phenotypic results, which could be a product of evolution, plasticity, or both. Regardless, the magnitude of shifts in urban thermal tolerance phenotypes was comparable to more traditional space-for-time substitutions across latitudinal and altitudinal clines in environmental temperature. We conclude by considering how urban-derived estimates of plasticity and evolution of thermal tolerance traits can be used to improve forecasting methods, including macrophysiological models and species distribution modelling approaches. Finally, we consider areas for further exploration including sub-lethal performance traits and thermal performance curves, assessing the adaptive nature of trait shifts, and taking full advantage of the environmental thermal variation that cities generate.