Rapid microtubule self-assembly kinetics.

Microtubule assembly is vital for many fundamental cellular processes. Current models for microtubule assembly kinetics assume that the subunit dissociation rate from a microtubule tip is independent of free subunit concentration. Total-Internal-Reflection-Fluorescence (TIRF) microscopy experiments and data from a laser tweezers assay that measures in vitro microtubule assembly with nanometer resolution, provides evidence that the subunit dissociation rate from a microtubule tip increases as the free subunit concentration increases. These data are consistent with a two-dimensional model for microtubule assembly, and are explained by a shift in microtubule tip structure from a relatively blunt shape at low free concentrations to relatively tapered at high free concentrations. We find that because both the association and the dissociation rates increase at higher free subunit concentrations, the kinetics of microtubule assembly are an order-of-magnitude higher than currently estimated in the literature.

Understanding the pathophysiology of idiopathic intracranial hypertension (IIH): a review of recent developments.

Idiopathic intracranial hypertension (IIH) is a condition of significant morbidity and rising prevalence. It typically affects young people living with obesity, mostly women of reproductive age, and can present with headaches, visual abnormalities, tinnitus and cognitive dysfunction. Raised intracranial pressure without a secondary identified cause remains a key diagnostic feature of this condition, however, the underlying pathophysiological mechanisms that drive this increase are poorly understood. Previous theories have focused on cerebrospinal fluid (CSF) hypersecretion or impaired reabsorption, however, the recent characterisation of the glymphatic system in many other neurological conditions necessitates a re-evaluation of these hypotheses. Further, the impact of metabolic dysfunction and hormonal dysregulation in this population group must also be considered. Given the emerging evidence, it is likely that IIH is triggered by the interaction of multiple aetiological factors that ultimately results in the disruption of CSF dynamics. This review aims to provide a comprehensive update on the current theories regarding the pathogenesis of IIH.

GATA4 Regulates Developing Endocardium Through Interaction With ETS1.

BACKGROUND: The pioneer transcription factor (TF) GATA4 (GATA Binding Protein 4) is expressed in multiple cardiovascular lineages and is essential for heart development. GATA4 lineage-specific occupancy in the developing heart underlies its lineage specific activities. Here, we characterized GATA4 chromatin occupancy in cardiomyocyte and endocardial lineages, dissected mechanisms that control lineage specific occupancy, and analyzed GATA4 regulation of endocardial gene expression. METHODS: We mapped GATA4 chromatin occupancy in cardiomyocyte and endocardial cells of embryonic day 12.5 (E12.5) mouse heart using lineage specific, Cre-activated biotinylation of GATA4. Regulation of GATA4 pioneering activity was studied in cell lines stably overexpressing GATA4. GATA4 regulation of endocardial gene expression was analyzed using single cell RNA sequencing and luciferase reporter assays. RESULTS: Cardiomyocyte-selective and endothelial-selective GATA4 occupied genomic regions had features of lineage specific enhancers. Footprints within cardiomyocyte- and endothelial-selective GATA4 regions were enriched for NKX2-5 (NK2 homeobox 5) and ETS1 (ETS Proto-Oncogene 1) motifs, respectively, and both of these TFs interacted with GATA4 in co-immunoprecipitation assays. In stable NIH3T3 cell lines expressing GATA4 with or without NKX2-5 or ETS1, the partner TFs re-directed GATA4 pioneer binding and augmented its ability to open previously inaccessible regions, with ETS1 displaying greater potency as a pioneer partner than NKX2-5. Single-cell RNA sequencing of embryonic hearts with endothelial cell-specific Gata4 inactivation identified Gata4-regulated endocardial genes, which were adjacent to GATA4-bound, endothelial regions enriched for both GATA4 and ETS1 motifs. In reporter assays, GATA4 and ETS1 cooperatively stimulated endothelial cell enhancer activity. CONCLUSIONS: Lineage selective non-pioneer TFs NKX2-5 and ETS1 guide the activity of pioneer TF GATA4 to bind and open chromatin and create active enhancers and mechanistically link ETS1 interaction to GATA4 regulation of endocardial development.

Sarcomeres regulate murine cardiomyocyte maturation through MRTF-SRF signaling.

The paucity of knowledge about cardiomyocyte maturation is a major bottleneck in cardiac regenerative medicine. In development, cardiomyocyte maturation is characterized by orchestrated structural, transcriptional, and functional specializations that occur mainly at the perinatal stage. Sarcomeres are the key cytoskeletal structures that regulate the ultrastructural maturation of other organelles, but whether sarcomeres modulate the signal transduction pathways that are essential for cardiomyocyte maturation remains unclear. To address this question, here we generated mice with cardiomyocyte-specific, mosaic, and hypomorphic mutations of α-actinin-2 (Actn2) to study the cell-autonomous roles of sarcomeres in postnatal cardiomyocyte maturation. Actn2 mutation resulted in defective structural maturation of transverse-tubules and mitochondria. In addition, Actn2 mutation triggered transcriptional dysregulation, including abnormal expression of key sarcomeric and mitochondrial genes, and profound impairment of the normal progression of maturational gene expression. Mechanistically, the transcriptional changes in Actn2 mutant cardiomyocytes strongly correlated with those in cardiomyocytes deleted of serum response factor (SRF), a critical transcription factor that regulates cardiomyocyte maturation. Actn2 mutation increased the monomeric form of cardiac α-actin, which interacted with the SRF cofactor MRTFA and perturbed its nuclear localization. Overexpression of a dominant-negative MRTFA mutant was sufficient to recapitulate the morphological and transcriptional defects in Actn2 and Srf mutant cardiomyocytes. Together, these data indicate that Actn2-based sarcomere organization regulates structural and transcriptional maturation of cardiomyocytes through MRTF-SRF signaling.

External Load Fluctuations Across an Amateur Athletic Union Basketball Season.

ABSTRACT: Kutson, CW, Russell, JL, Strack, D, Coutts, AJ, and McLean, BD. External load fluctuations across an Amateur Athletic Union basketball season. J Strength Cond Res 38(3): 592-598, 2024-Amateur Athletic Union (AAU) competitions are an important component of the developmental pathway for youth basketball athletes. Despite its relative importance, there is currently a paucity of research investigating the physical demands in AAU basketball. Therefore, the purpose of this study was to examine the physical demands encountered over the course of an AAU basketball season. External training load was quantified using inertial sensors (Catapult T6) from one male AAU basketball team (age: 17.5 ± 0.5 years, height: 197.3 ± 10.0 cm, and mass: 89.4 ± 11.6 kg) over the course of the 2021 AAU season and categorized post hoc into high-, medium-, and low-minute groups based on mean playing minutes. After player categorization, 2 linear mixed models were constructed, one for PlayerLoad (PL) and one for duration, to examine the differences across player category, month of the season, and activity types (practices or games). The results show that the highest training loads were encountered by high-minute players, who had total PLs of 9,766 ± 1,516 AU, 13,207 ± 2,561 AU, and 7,071 ± 2,122 AU during April, May, and June, respectively. Highly variable training loads were also evident over the course of a season, with peak PL values as high as 4,921 AU per week. Practitioners should be aware that AAU basketball players experience variable loads throughout the season, which peak around congested competition/tournament periods. In addition, players with high game minutes accumulate the most load over the course of a season. This information may be used to better inform planning and periodizing strategies during developmental phases.

The Relationship Between Mental Fatigue and Shooting Performance Over the Course of a National Collegiate Athletic Association Division I Basketball Season.

ABSTRACT: Daub, BD, McLean, BD, Heishman, AD, Peak, KM, and Coutts, AJ. The relationship between mental fatigue and shooting performance over the course of a National Collegiate Athletic Association Division I basketball season. J Strength Cond Res 38(2): 334-341, 2024-The aim of this investigation was to examine the presence of mental fatigue and concurrent changes in shooting performance across various experimental weeks throughout a National Collegiate Athletic Association (NCAA) basketball season. Fifteen elite male NCAA Division I collegiate basketball players (age 20.2 ± 1.2 years, height 199.3 ± 7.1 cm, and body mass 93.1 ± 8.6 kg) volunteered for this study. Mental fatigue and basketball shooting performance was evaluated at 4 timepoints with varying seasonal demands: high game volume (GAME), high academic load (ACADEMIC), no games and no academic load (PRACTICE), and standard number of games and academic requirements (TYPICAL). Subjective mental fatigue increased significantly ( p ≤ 0.05) from Pre to Post brief psychomotor vigilance test (PVT-B) measurements at the end of the ACADEMIC week ( p = 0.002, d = 1.51) and from beginning to end of the ACADEMIC week ( p < 0.001, d = 2.21). Ratings of mental effort were significantly increased during the ACADEMIC week ( p < 0.001, d = 1.67). Recovery stress questionnaire (REST-Q) showed significant differences between week GAME and ACADEMIC with an increase in Social Stress ( p = 0.001, d = 0.84), Fatigue ( p = 0.021, d = 1.12), Disturbed Breaks ( p = 0.024, d = 0.57), and Emotional Exhaustion ( p = 0.035, d = 0.75). Lower shooting performance was observed during the ACADEMIC week from Pre to Post ( p = 0.009, d = 0.35) and higher scores Pre to Post in the TYPICAL week ( p = 0.008, d = 0.25). Basketball shooting performance was significantly reduced after increased levels of mental fatigue stemming from added academic stress. In addition, an increase in sport-specific training or games had no effect on subsequent basketball shooting performance. Special consideration should be given by coaches around examination periods because the existence of academic stressors can influence basketball shooting performance.

New Probe of Inflationary Gravitational Waves: Cross-Correlations of Lensed Primary CMB B-Modes with Large-Scale Structure.

We propose a new probe of inflationary gravitational waves (IGWs): the cross-correlation of the lensing of inflationary B-mode polarization with a large-scale structure (LSS) tracer, which can also be a cosmic microwave background (CMB) lensing map. This is equivalent to measuring a three-point function of two CMB B-modes and an LSS tracer. We forecast expected 1σ constraints on the tensor-to-scalar ratio r, albeit with a simplistic foreground treatment, and find constraints of σ_{r}≃7×10^{-3} from the correlation of CMB-S4-Deep B-mode lensing and LSST galaxies, σ_{r}≃5×10^{-3} from the correlation of CMB-S4-Deep B-mode lensing and CMB-S4-Deep CMB lensing, and σ_{r}≃10^{-2} from the correlation of LiteBIRD B-mode lensing and CMB-S4-Wide lensing. Because this probe is inherently non-Gaussian, simple Gaussian foregrounds will not produce any biases to the measurement of r. While a detailed investigation of non-Gaussian foreground contamination for different cross-correlations will be essential, this observable has the potential to be a useful probe of IGWs, which, due to different sensitivity to many potential sources of systematic errors, can be complementary to standard methods for constraining r.

Canonical Hubble-Tension-Resolving Early Dark Energy Cosmologies Are Inconsistent with the Lyman-α Forest.

Current cosmological data exhibit discordance between indirect and some direct inferences of the present-day expansion rate H_{0}. Early dark energy (EDE), which briefly increases the cosmic expansion rate prior to recombination, is a leading scenario for resolving this "Hubble tension" while preserving a good fit to cosmic microwave background (CMB) data. However, this comes at the cost of changes in parameters that affect structure formation in the late-time universe, including the spectral index of scalar perturbations n_{s}. Here, we present the first constraints on axionlike EDE using data from the Lyman-α forest, i.e., absorption lines imprinted in background quasar spectra by neutral hydrogen gas along the line of sight. We consider two independent measurements of the one-dimensional Lyα forest flux power spectrum from the Sloan Digital Sky Survey (SDSS eBOSS) and from the MIKE/HIRES and X-Shooter spectrographs. We combine these with a baseline dataset comprised of Planck CMB data and baryon acoustic oscillation (BAO) measurements. Combining the eBOSS Lyα data with the CMB and BAO dataset reduces the 95% confidence level (C.L.) upper bound on the maximum fractional contribution of EDE to the cosmic energy budget f_{EDE} from 0.07 to 0.03 and constrains H_{0}=67.9_{-0.4}^{+0.4} km/s/Mpc (68% C.L.), with maximum a posteriori value H_{0}=67.9 km/s/Mpc. Similar results are obtained for the MIKE/HIRES and X-Shooter Lyα data. Our Lyα-based EDE constraints yield H_{0} values that are in >4σ tension with the SH0ES distance-ladder measurement and are driven by the preference of the Lyα forest data for n_{s} values lower than those required by EDE cosmologies that fit Planck CMB data. Taken at face value, the Lyα forest severely constrains canonical EDE models that could resolve the Hubble tension.

Lack of Useful Predictors of Dignity-Related Distress Among the Critically Ill as Assessed With the Patient Dignity Inventory.

BACKGROUND: Many intensive care unit patients are awake (ie, alert and engaging in conversation), actively experiencing many facets of their critical care. The Patient Dignity Inventory can be used to elicit sources of distress in these patients. We examined the administrative question as to which awake intensive care unit patients should be evaluated and potentially treated (eg, through palliative care consultation) for distress. Should the decision to screen for distress be based on patient demographics or treatment conditions? METHODS: This was a retrospective cohort study of 155 adult patients from 5 intensive care units of one hospital from 2019 to 2020. Each patient had ≥48 hours without delirium, dementia, or sedation. The Patient Dignity Inventory has 25 items to which patients responded on a 1 (not a problem) to 5 (an overwhelming problem) scale. Multiple complete, stepwise forward, and stepwise backward logistic regression models were created among patient and treatment variables for predicting thresholds of the mean among the 25 items. RESULTS: There were 50% (78/155; 95% confidence interval [CI], 42-58) of patients with significant dignity-related distress (mean score ≥1.60). There were 34% (52/155; CI, 26-42) of patients with severe dignity-related distress (mean score ≥1.92; previously associated with often feeling like wanting to die). Models including combinations of vasopressor medication (protective of distress), tracheostomy (greater risk of distress), and female gender (greater risk of distress) had some predictive value. However, all combinations of potential predictors had misclassification rates significantly >20%. CONCLUSIONS: Identification of subsets of patients with little potential benefit to screening for dignity-related distress would have a reduced workload of palliative care team members (eg, nurses or social workers). Our results show that this is impractical. Given that approximately one-third of critical care patients who are alert and without delirium demonstrate severe dignity-related distress, all such patients with prolonged intensive care unit length of stay should probably be evaluated for distress.

Racial and Ethnic Patient Care Disparities in Anesthesiology: History, Current State, and a Way Forward.

Disparities in patient care and outcomes are well-documented in medicine but have received comparatively less attention in anesthesiology. Those disparities linked to racial and ethnic identity are pervasive, with compelling evidence in operative anesthesiology, obstetric anesthesiology, pain medicine, and critical care. This narrative review presents an overview of disparities in perioperative patient care that is grounded in historical context followed by potential solutions for mitigating disparities and inequities.

Sensitivity of Countermovement Jump Variables in Professional Rugby Union Players Within a Playing Season.

ABSTRACT: Howarth, DJ, McLean, BD, Cohen, DD, and Coutts, AJ. Sensitivity of countermovement jump variables in professional rugby union players within a playing season. J Strength Cond Res 37(7): 1463-1469, 2023-The aim of this study was to explore the measurement sensitivity of a wide range of countermovement jump (CMJ) variables to a full European professional rugby union season. A secondary purpose was to compare 3 different data treatment methods for the calculation of CMJ variables. Twenty-nine professional rugby union players (mean ± SD; age 24 ± 4 years, height 183.7 ± 8.0 cm, body mass 101.6 ± 10.7 kg) completed a minimum of 12 CMJ testing sessions on Thursdays-a day preceded by a rest day and a minimum of 96 hours after a match-throughout a season. Measurement sensitivity, quantified by signal-to-noise ratio (SNR), was determined for 74 CMJ variables and was calculated by dividing the signal, (week-to-week variation expressed as a coefficient of variation [CV%]) by the noise (interday test/retest reliability expressed as CV%). We also identified variables which had no overlap between the 95% confidence intervals (CIs) for the signal and the noise. The 3 data treatment methods for comparison were (a) mean output across 3 jump trials (Mean3), (b) single output from the trial with the highest jump (BestJH), and (c) the trial with the highest flight time to contraction time ratio (BestFTCT). Most variables had an SNR >1.0 (Mean3 = 60/74; BestFTCT = 59/74; BestJH = 48/74). Fewer variables displayed a nonoverlap of 95% CIs (Mean3 = 23/60; BestFTCT = 22/59; BestJH = 16/48). Most CMJ variables during a professional rugby season demonstrated a signal that exceeded measured noise (SNR > 1.0) and that using the Mean3 or BestFTCT data treatment methods yields a greater number of variables considered sensitive within a season (i.e., SNR > 1.0) than when using BestJH. We also recommend the calculation of the 95% CIs for both signal and noise, with nonoverlap indicative of a greater probability that the responsiveness of the variable at team level (i.e., SNR) also applies at the individual level. As sensitivity analysis is cohort and environment specific, practitioners should conduct a sensitivity analysis using internal signal and noise data to inform their own monitoring protocols.

Finding the Signal in the Noise-Interday Reliability and Seasonal Sensitivity of 84 Countermovement Jump Variables in Professional Basketball Players.

ABSTRACT: Mercer, RAJ, Russell, JL, McGuigan, LC, Coutts, AJ, Strack, DS, and McLean, BD. Finding the signal in the noise-interday reliability and seasonal sensitivity of 84 countermovement jump variables in professional basketball players. J Strength Cond Res 37(2): 394-402, 2023-This study examined the measurement characteristics of countermovement jump (CMJ) variables in basketball athletes using different variable selection criteria. Test-retest reliability (noise) and seasonal variability (signal) CMJ data were collected from 13 professional basketball athletes playing for the same club throughout 1 competitive season. Interday reliability (coefficient of variation [CV] and intraclass correlation coefficients) were calculated over 3 preseason tests conducted on 3 consecutive days. To evaluate sensitivity, signal-to-noise ratio (SNR) was calculated by dividing seasonal variability (CV) from 8 in-season CMJ tests (collected from November to February) by preseason reliability (CV). Players performed 3 CMJs each testing day, and 3 data analysis techniques were applied: a single variable from the trial with either the best jump height (BestJH; calculated by flight time) or the best flight time to contraction time (BestFT:CT) and mean output across 3 jumps (Mean3). Mean3 was the most reliable data analysis technique, with 79 and 82 of 84 variables displaying lower interday CVs compared with BestJH and BestFT:CT, respectively. Overall, many CMJ measures display seasonal changes that are greater than the inherent noise, with 77 variables producing SNR of >1.00 for Mean3 compared with 65 and 58 variables for BestJH and BestFT:CT, respectively. To improve reliability and sensitivity, it is recommended that practitioners use the average of multiple CMJ trials and regularly reassess measurement characteristics specific to their cohort and environment.

Cornelia de Lange syndrome and the Cohesin complex: Abstracts from the 9th Biennial Scientific and Educational Virtual Symposium 2020.

Cornelia de Lange syndrome (CdLS) is a spectrum disorder due to variants in genes of the cohesin protein complex. The following abstracts are from the Cornelia de Lange Syndrome Scientific and Educational Symposium held virtually in October 2020. Aspects of behavior, including autistic features, impulsivity, adaptive skills, executive function, and anxiety are described. Applied behavioral analysis is a promising approach for autism, and an N-acetylcysteine trial is proposed. Children below 6 years with CdLS have an increased number of and further travel to medical providers, with insurance type comprising a significant barrier. Speech, language, and feeding abilities fall significantly below expectations for age in CdLS. Augmentative alternative communication can yield potential barriers as well as interesting benefits. Developmentally, studies in animal models further elucidate the mechanisms and roles of cohesin: link with mediator transcriptional complex; facilitation of enhancer-promoter communication; regulation of gene expression; allocation of cells to germ layers; and repair of spontaneous DNA damage in placental cells. Genome and RNA sequencing can help identify the molecular cause in the 20% of individuals with suspected CdLS and negative testing. The phenotypes in individuals with variants in the SMC1A gene are distinct, and that with intractable seizures has been further evaluated. AMA CME credits provided by GBMC, Baltimore, MD. All studies approved by an ethics committee.

Genetic selection of Eimeria parasites in the chicken for improvement of poultry health: implications for drug resistance and live vaccine development.

Apicomplexan parasites of the genus Eimeria are widespread in poultry flocks and can cause the intestinal disease coccidiosis. Early studies, concerned with intraspecific variation in oocyst morphology, indicated that phenotypic changes may be induced by selection experiments conducted in vivo. Genetic selection driven by targeted selection for specific phenotypes has contributed to our understanding of the phenomenon of drug resistance and the development of live attenuated vaccines. Our present knowledge regarding genetics of Eimeria is largely based upon the utilization of such selected strains as genetic markers. Practical advantages of working with Eimeria spp. in the chicken are discussed. The selection of drug-resistant strains by serial propagation has provided useful information regarding the mechanisms of drug resistance and likely longevity of anticoccidial drugs when introduced in the field. Selection experiments to develop precocious strains of Eimeria and growth in chicken embryos have contributed to the development of safe and effective live attenuated vaccines for the control of coccidiosis. Establishment of protocols for genetic complementation by transient or stable transfection of Eimeria is now supporting direct manipulation of parasite genotypes, creating opportunities to expand the range and value of live parasite vaccines. Procedures for developing drug-resistant and precocious lines of Eimeria and/or genetic markers described here are likely to prove useful for researchers investigating the propensity for resistance development to novel compounds and the development of new attenuated vaccines. Such investigations can be helpful in providing a better understanding of biochemical and molecular aspects of the biology of these parasites.

Establishing the Noise: Interday Ecological Reliability of Countermovement Jump Variables in Professional Rugby Union Players.

ABSTRACT: Howarth, DJ, Cohen, DD, McLean, BD, and Coutts, AJ. Establishing the noise: interday ecological reliability of countermovement jump variables in professional rugby union players. J Strength Cond Res 36(11): 3159-3166, 2022-The purpose of this study was to examine the interday "ecological" reliability of a wide range of ground reaction force-derived countermovement jump (CMJ) variables. Thirty-six male, professional rugby union players performed 3 CMJs on 4 separate days over an 8-day period during the first week of preseason. We calculated reliability for 86 CMJ variables across 5 interday combinations using 2 criteria: mean output across 3 jump trials (Mean 3 ) and single output from the highest jump (Best JH ). Interday coefficient of variation (CV) of the 86 variables in each CMJ phase, for Mean 3 and Best JH , respectively, ranged between concentric = 2-11% and 2-13%; eccentric = 1-45% and 1-107%; and landing = 4-32% and 6-45%. Mean 3 interday CV was lower in all 86 variables across every interday combination, compared with Best JH . CVs were lower in our cohort than previous studies, particularly for eccentric phase variables. There was no meaningful difference between interday conditions, suggesting any 2-day combination conducted within the first 8 days of preseason, represents a measure of "noise." We did not apply arbitrary reliability "cut-offs" used in previous work (e.g., CV <10%); therefore, our analysis provides reference reliability for a wide range of CMJ variables. However, we recommend that practitioners assess reliability in their athletes, as it is likely to be environment, protocol, and cohort specific.

The Polar Flagellar Transcriptional Regulatory Network in Vibrio campbellii Deviates from Canonical Vibrio Species.

Swimming motility is a critical virulence factor in pathogenesis for numerous Vibrio species. Vibrio campbellii DS40M4 is a wild-type isolate that has been recently established as a highly tractable model strain for bacterial genetics studies. We sought to exploit the tractability and relevance of this strain for characterization of flagellar gene regulation in V. campbellii. Using comparative genomics, we identified homologs of V. campbellii flagellar and chemotaxis genes conserved in other members of the Vibrionaceae and determined the transcriptional profile of these loci using differential RNA-seq. We systematically deleted all 63 predicted flagellar and chemotaxis genes in V. campbellii and examined their effects on motility and flagellum production. We specifically focused on the core regulators of the flagellar hierarchy established in other vibrios: RpoN (σ54), FlrA, FlrC, and FliA. Our results show that V. campbellii transcription of flagellar and chemotaxis genes is governed by a multitiered regulatory hierarchy similar to other motile Vibrio species. However, there are several critical differences in V. campbellii: (i) the σ54-dependent regulator FlrA is dispensable for motility; (ii) the flgA, fliEFGHIJ, flrA, and flrBC operons do not require σ54 for expression; and (iii) FlrA and FlrC coregulate class II genes. Our model proposes that the V. campbellii flagellar transcriptional hierarchy has three classes of genes, in contrast to the four-class hierarchy in Vibrio cholerae. Our genetic and phenotypic dissection of the V. campbellii flagellar regulatory network highlights the differences that have evolved in flagellar regulation across the Vibrionaceae. IMPORTANCE Vibrio campbellii is a Gram-negative bacterium that is free-living and ubiquitous in marine environments and is an important global pathogen of fish and shellfish. Disruption of the flagellar motor significantly decreases host mortality of V. campbellii, suggesting that motility is a key factor in pathogenesis. Using this model organism, we identified >60 genes that encode proteins with predicted structural, mechanical, or regulatory roles in function of the single polar flagellum in V. campbellii. We systematically tested strains containing single deletions of each gene to determine the impact on motility and flagellum production. Our studies have uncovered differences in the regulatory network and function of several genes in V. campbellii compared to established systems in Vibrio cholerae and Vibrio parahaemolyticus.

Identification of the Rigid Core for Aged Liquid Droplets of an RNA-Binding Protein Low Complexity Domain.

The biomolecular condensation of proteins with low complexity sequences plays a functional role in RNA metabolism and a pathogenic role in neurodegenerative diseases. The formation of dynamic liquid droplets brings biomolecules together to achieve complex cellular functions. The rigidification of liquid droplets into ß-strand-rich hydrogel structures composed of protein fibrils is thought to be purely pathological in nature. However, low complexity sequences often harbor multiple fibril-prone regions with delicately balanced functional and pathological interactions. Here, we investigate the maturation of liquid droplets formed by the low complexity domain of the TAR DNA-binding protein 43 (TDP-43). Solid state nuclear magnetic resonance measurements on the aged liquid droplets identify residues 365-400 as the structured core, which are squarely outside the region between residues 311-360 thought to be most important for pathological fibril formation and aggregation. The results of this study suggest that multiple segments of this low complexity domain are prone to form fibrils and that stabilization of ß-strand-rich structure in one segment precludes the other region from adopting a rigid fibril structure.

Drosophila emerins control LINC complex localization and transcription to regulate myonuclear position.

Mispositioned nuclei are a hallmark of skeletal muscle disease. Many of the genes that are linked to Emery-Dreifuss muscular dystrophy (EDMD) encode proteins that are critical for nuclear movement in various cells, suggesting that disruptions in nuclear movement and position may contribute to disease progression. However, how these genes are coordinated to move nuclei is not known. Here, we focussed on two different emerin proteins in Drosophila, Bocksbeutel and Otefin, and their effects on nuclear movement. Although nuclear position was dependent on both, elimination of either Bocksbeutel or Otefin produced distinct phenotypes that were based in differential effects on the KASH-domain protein Klarsicht. Specifically, loss of Bocksbeutel reduced Klarsicht localization to the nucleus and resulted in a disruption in nuclear separation. Loss of Otefin increased the transcription of Klarsicht and led to premature separation of nuclei and their positioning closer to the edge of the muscle. Consistent with opposing functions, nuclear position is normal in otefin; bocksbeutel double mutants. These data indicate emerin-dependent regulation of Klarsicht levels in the nuclear envelope is a critical determinant of nuclear position.

The quorum-sensing systems of Vibrio campbellii DS40M4 and BB120 are genetically and functionally distinct.

Vibrio campbellii BB120 (previously classified as Vibrio harveyi) is a fundamental model strain for studying quorum sensing in vibrios. A phylogenetic evaluation of sequenced Vibrio strains in Genbank revealed that BB120 is closely related to the environmental isolate V. campbellii DS40M4. We exploited DS40M4's competence for exogenous DNA uptake to rapidly generate greater than 30 isogenic strains with deletions of genes encoding BB120 quorum-sensing system homologues. Our results show that the quorum-sensing circuit of DS40M4 is distinct from BB120 in three ways: (i) DS40M4 does not produce an acyl homoserine lactone autoinducer but encodes an active orphan LuxN receptor, (ii) the quorum regulatory small RNAs (Qrrs) are not solely regulated by autoinducer signalling through the response regulator LuxO and (iii) the DS40M4 quorum-sensing regulon is much smaller than BB120 (~100 genes vs. ~400 genes, respectively). Using comparative genomics to expand our understanding of quorum-sensing circuit diversity, we observe that conservation of LuxM/LuxN proteins differs widely both between and within Vibrio species. These strains are also phenotypically distinct: DS40M4 exhibits stronger interbacterial cell killing, whereas BB120 forms more robust biofilms and is bioluminescent. These results underscore the need to examine wild isolates for a broader view of bacterial diversity in the marine ecosystem.