Selective Cdk9 inhibition resolves neutrophilic inflammation and enhances cardiac regeneration in larval zebrafish.

Sustained neutrophilic inflammation is detrimental for cardiac repair and associated with adverse outcomes following myocardial infarction (MI). An attractive therapeutic strategy to treat MI is to reduce or remove infiltrating neutrophils to promote downstream reparative mechanisms. CDK9 inhibitor compounds enhance the resolution of neutrophilic inflammation; however, their effects on cardiac repair/regeneration are unknown. We have devised a cardiac injury model to investigate inflammatory and regenerative responses in larval zebrafish using heartbeat-synchronised light-sheet fluorescence microscopy. We used this model to test two clinically approved CDK9 inhibitors, AT7519 and flavopiridol, examining their effects on neutrophils, macrophages and cardiomyocyte regeneration. We found that AT7519 and flavopiridol resolve neutrophil infiltration by inducing reverse migration from the cardiac lesion. Although continuous exposure to AT7519 or flavopiridol caused adverse phenotypes, transient treatment accelerated neutrophil resolution while avoiding these effects. Transient treatment with AT7519, but not flavopiridol, augmented wound-associated macrophage polarisation, which enhanced macrophage-dependent cardiomyocyte number expansion and the rate of myocardial wound closure. Using cdk9-/- knockout mutants, we showed that AT7519 is a selective CDK9 inhibitor, revealing the potential of such treatments to promote cardiac repair/regeneration.

Inhibition of soluble epoxide hydrolase by natural isothiocyanates.

GOAL: The long-term goal of our research is to develop safe and effective soluble epoxide hydrolase (sEH) inhibitors. The objective of this study is to evaluate the potency and selectivity of six natural isothiocyanates (ITCs) as sEH inhibitors. METHODS: Molecular docking was used to model likely interactions between the ligands and receptors. The sEH inhibitory activity was tested using a validated fluorescence-based assay and PHOME as a substrate. To evaluate their selectivity as sEH inhibitors, the inhibitory potential of the ITCs was determined on microsomal epoxide hydrolase (mEH) and cytochrome P450 (CYP) enzymes in human liver microsomes. Probe substrates such as styrene oxide (mEH substrate) and established substrates for CYP2A6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 were used in this study. The metabolites of these substrates were analyzed using validated LC-MS/MS and HPLC-UV assays. RESULTS: Molecular Docking revealed significant differences in binding site preference among the ITCs in silico and pointed to important interactions between the ligands and the catalytic residues of the sEH enzyme. In vitro, the ITCs showed varying degrees of sEH inhibition, but sulforaphane (SFN) and phenyl isothiocyanate (PITC) were the most potent inhibitors with IC50 values of 3.65 and 7.5 µM, respectively. mEH was not significantly inhibited by any of the ITCs. Erucin and iberin were the only ITCs that did not inhibit the activity of any of the tested CYP enzymes. CONCLUSION: Our results demonstrate that natural ITCs have the potential to offer safe, selective, and potent sEH inhibition.

Fast algorithm for 3D volume reconstruction from light field microscopy datasets.

Light field microscopy can capture 3D volume datasets in a snapshot, making it a valuable tool for high-speed 3D imaging of dynamic biological events. However, subsequent computational reconstruction of the raw data into a human-interpretable 3D+time image is very time-consuming, limiting the technique's utility as a routine imaging tool. Here we derive improved equations for 3D volume reconstruction from light field microscopy datasets, leading to dramatic speedups. We characterize our open-source Python implementation of these algorithms and demonstrate real-world reconstruction speedups of more than an order of magnitude compared with established approaches. The scale of this performance improvement opens up new possibilities for studying large timelapse datasets in light field microscopy.

The bacterial curli system possesses a potent and selective inhibitor of amyloid formation.

Curli are extracellular functional amyloids that are assembled by enteric bacteria during biofilm formation and host colonization. An efficient secretion system and chaperone network ensures that the major curli fiber subunit, CsgA, does not form intracellular amyloid aggregates. We discovered that the periplasmic protein CsgC was a highly effective inhibitor of CsgA amyloid formation. In the absence of CsgC, CsgA formed toxic intracellular aggregates. In vitro, CsgC inhibited CsgA amyloid formation at substoichiometric concentrations and maintained CsgA in a non-ß-sheet-rich conformation. Interestingly, CsgC inhibited amyloid assembly of human α-synuclein, but not Aß42, in vitro. We identified a common D-Q-Φ-X0,1-G-K-N-ζ-E motif in CsgC client proteins that is not found in Aß42. CsgC is therefore both an efficient and selective amyloid inhibitor. Dedicated functional amyloid inhibitors may be a key feature that distinguishes functional amyloids from disease-associated amyloids.

Characterization of the Native Disulfide Isomers of the Novel χ-Conotoxin PnID: Implications for Further Increasing Conotoxin Diversity.

χ-Conotoxins are known for their ability to selectively inhibit norepinephrine transporters, an ability that makes them potential leads for treating various neurological disorders, including neuropathic pain. PnID, a peptide isolated from the venom of Conus pennaceus, shares high sequence homology with previously characterized χ-conotoxins. Whereas previously reported χ-conotoxins seem to only have a single native disulfide bonding pattern, PnID has three native isomers due to the formation of different disulfide bond patterns during its maturation in the venom duct. In this study, the disulfide connectivity and three-dimensional structure of these disulfide isomers were explored using regioselective synthesis, chromatographic coelution, and solution-state nuclear magnetic resonance spectroscopy. Of the native isomers, only the isomer with a ribbon disulfide configuration showed pharmacological activity similar to other χ-conotoxins. This isomer inhibited the rat norepinephrine transporter (IC50 = 10 ± 2 µM) and has the most structural similarity to previously characterized χ-conotoxins. In contrast, the globular isoform of PnID showed more than ten times less activity against this transporter and the beaded isoform did not display any measurable biological activity. This study is the first report of the pharmacological and structural characterization of an χ-conotoxin from a species other than Conus marmoreus and is the first report of the existence of natively-formed conotoxin isomers.

Using Confocal Microscopy and Pigment Analyses to Detect Adverse Insecticide Effects in non-target Freshwater Diatom species - a proof-of-concept Using Nitzschia palea.

The persistence of insecticides in aquatic environments is a cause of concern and to date hardly any studies have focused on the effects that DDT and deltamethrin have on non-target freshwater diatom communities. The application of diatoms in ecotoxicological studies is well acknowledged and therefore this study used laboratory bioassays to determine the effects that DDT and deltamethrin have on a monoculture of a diatom indicator species, Nitzschia palea. The insecticides affected the morphology of chloroplasts at all exposure concentrations. These effects were a maximum reduction in chlorophyll concentrations (4.8% and 2.3%), cell viability (51% and 42%), and increases in cell deformities (3.6% and 1.6%) following exposure to DDT and deltamethrin respectively. Based on the results we propose that methods, such as confocal microscopy, chlorophyll-α analysis and cell deformities are useful tools in assessing the effects of insecticides on diatoms.

Survival analysis on rare events using group-regularized multi-response Cox regression.

MOTIVATION: The prediction performance of Cox proportional hazard model suffers when there are only few uncensored events in the training data. RESULTS: We propose a Sparse-Group regularized Cox regression method to improve the prediction performance of large-scale and high-dimensional survival data with few observed events. Our approach is applicable when there is one or more other survival responses that 1. has a large number of observed events; 2. share a common set of associated predictors with the rare event response. This scenario is common in the UK Biobank dataset where records for a large number of common and less prevalent diseases of the same set of individuals are available. By analyzing these responses together, we hope to achieve higher prediction performance than when they are analyzed individually. To make this approach practical for large-scale data, we developed an accelerated proximal gradient optimization algorithm as well as a screening procedure inspired by Qian et al. AVAILABILITYANDIMPLEMENTATION: https://github.com/rivas-lab/multisnpnet-Cox.

Snapshot volumetric imaging with engineered point-spread functions.

The biological world involves intracellular and intercellular interactions that occur at high speed, at multiple scales and in three dimensions. Acquiring 3D images, however, typically requires a compromise in either spatial or temporal resolution compared to 2D imaging. Conventional 2D fluorescence imaging provides high spatial resolution but requires plane-by-plane imaging of volumes. Conversely, snapshot methods such as light-field microscopy allow video-rate imaging, but at the cost of spatial resolution. Here we introduce 3D engineered point-spread function microscopy (3D-EPM), enabling snapshot imaging of real-world 3D extended biological structures while retaining the native resolution of the microscope in space and time. Our new computational recovery strategy is the key to volumetrically reconstructing arbitrary 3D structures from the information encapsulated in 2D raw EPM images. We validate our technique on both point-like and extended samples, and demonstrate its power by imaging the intracellular motion of chloroplasts undergoing cyclosis in a sample of Egeria densa. Our technique represents a generalised computational methodology for 3D image recovery which is readily adapted to a diverse range of existing microscopy platforms and engineered point-spread functions. We therefore expect it to find broad applicability in the study of rapid biological dynamics in 3D.

Diatom type material in permanent slides does not need to be permanently unavailable for electron microscopy examination.

The inability to thoroughly examine diatom type material existing only in resin-mounted permanent slides is a common frustration for diatomologists. Here, we present an efficient, inexpensive, and straightforward technique to extract siliceous exoskeletons of diatoms from permanent slides prepared with commonly used mounting media. Van Heurck's slide constituting an isotype duplicate of Proschkinia bulnheimii var. belgica, an entity with an uncertain taxonomic status, was deconstructed to allow thorough examination using scanning electron microscopy. Numerous specimens of the taxon, including intact frustules and specimens with complete areola occlusions, were recovered and documented. The extracted diatom material was sufficient to prepare two scanning electron microscopy specimens and reconstruct the permanent slide. The proposed approach may help overcome many of the taxonomic bottlenecks associated with the low resolution of information provided by older diatom descriptions and permanent slide observations.

On Sea Turtle-associated Craspedostauros (Bacillariophyta), with Description of Three Novel Species.

The current study focuses on four species from the primarily marine diatom genus Craspedostauros that were observed growing attached to numerous sea turtles and sea turtle-associated barnacles from Croatia and South Africa. Three of the examined taxa, C. danayanus sp. nov., C. legouvelloanus sp. nov., and C. macewanii sp. nov., are described based on morphological and, whenever possible, molecular characteristics. The new taxa exhibit characters not previously observed in other members of the genus, such as the presence of more than two rows of cribrate areolae on the girdle bands, shallow perforated septa, and a complete reduction of the stauros. The fourth species, C. alatus, itself recently described from museum sea turtle specimens, is reported for the first time from loggerhead sea turtles rescued in Europe. A 3-gene phylogenetic analysis including DNA sequence data for three sea turtle-associated Craspedostauros species and other marine and epizoic diatom taxa indicated that Craspedostauros is monophyletic and sister to Achnanthes. This study, being based on a large number of samples and animal specimens analyzed and using different preservation and processing methods, provides new insights into the ecology and biogeography of the genus and sheds light on the level of intimacy and permanency in the host-epibiont interaction within the epizoic Craspedostauros species.

"Books Frighted Them Terribly": The Perils of Propagating Fear and the Ethics of Writing about Disease in Defoe's A Journal of the Plague Year.

This article challenges the view that Daniel Defoe's A Journal of the Plague Year (1722) exploited or augmented the plague's horrors. It demonstrates that Defoe denounced writers who sensationalized plague and explores his use of A Journal to debunk terrifying accounts of the disease published during the Plague of Marseille (1720-22). Section one explores how Defoe's opposition to inciting fear was shaped by medical beliefs that fear increased susceptibility to disease and his observations about fear's socioeconomic repercussions. Section two examines his conviction that "Books frighted [people] terribly" and A Journal's attempts to discredit macabre images and tales circulated in contemporary plague writing. The conclusion addresses the relevance of Defoe's observations in the context of modern anxiety about pandemic disease, the resonance of his conclusions with recent scholarship on the media's augmentation of panic prior to pandemics, and the ethical questions A Journal poses for those writing about infectious diseases.

Twin-Airy Point-Spread Function for Extended-Volume Particle Localization.

The localization of point sources in optical microscopy enables nm-precision imaging of single-molecules and biological dynamics. We report a new method of localization microscopy using twin Airy beams that yields precise 3D localization with the key advantages of extended depth range, higher optical throughput, and potential for imaging higher emitter densities than are possible using other techniques. A precision of better than 30 nm was achieved over a depth range in excess of 7 µm using a 60×, 1.4 NA objective. An illustrative application to extended-depth-range blood-flow imaging in a live zebrafish is also demonstrated.

Extreme Fermi Surface Smearing in a Maximally Disordered Concentrated Solid Solution.

We show that the Fermi surface can survive the presence of extreme compositional disorder in the equiatomic alloy Ni_{0.25}Fe_{0.25}Co_{0.25}Cr_{0.25}. Our high-resolution Compton scattering experiments reveal a Fermi surface which is smeared across a significant fraction of the Brillouin zone (up to 40% of 2π/a). The extent of this smearing and its variation on and between different sheets of the Fermi surface have been determined, and estimates of the electron mean free path and residual resistivity have been made by connecting this smearing with the coherence length of the quasiparticle states.

'[Her] hostess is anxious to have her back when she is cured': The impact of the evacuation of children on wartime local services, England, 1939-1945.

World War II had a profound, but uneven, impact on the delivery of services designed to support the bodies and minds of English children. This article, which is based on a study of a rural local authority located in North-West England, explores the influence of World War II on children's welfare services. Drawing on detailed case files relating to individual children and reports published by local and national policy makers, the article advances three related arguments which together nuance existing understandings of the conflict and its longer-term consequences. First, the article argues that many of the problems associated with evacuees were already familiar to medical and social work professionals. This awareness has important consequences for how we conceptualise the wartime proposals that attracted policy makers' attention. Second, the article shows that the arrival of evacuees into reception areas initially resulted in an expansion of children's services. A fuller understanding of Britain's welfare state, however, must acknowledge that local authorities continued to wield significant influence over the delivery of specialist services once the conflict ended. As a result, the priorities of local officials could lead to the needs of looked after children being overlooked despite wartime improvements to children's services. Finally, the article argues that amidst the totality of World War II, the British state remained unwilling to intrude on the rights of parents to influence the care of their children. Closer examination of the implementation of evacuation and the experiences of individuals reveals that important tensions existed between the state appointed experts and the civilians they were tasked with supporting.

Comparison of the Lateral Retention Forces on Sessile, Pendant, and Inverted Sessile Drops.

We compare the lateral retention forces on sessile drops (which are drops that are placed on top of a solid surface), pendant drops (which are drops that are placed on the underside of the surface), and inverted sessile drops (which are drops that are first placed on top and then on the underside of the surface by flipping the surface). We have found experimentally that the retention force on a truly pendant drop is always smaller than that on a sessile drop. However, the retention force on an inverted sessile drop is comparable to, and usually larger than, that on a sessile drop. Thus, the retention force on a drop depends not only on whether it is placed on top or on bottom of a surface, but also on the history of drop deposition, since such history affects the width, the shape, and the contact angles of the drop.

The Effect of ARDS on Survival: Do Patients Die From ARDS or With ARDS?

OBJECTIVE:: To investigate the contribution of acute respiratory distress syndrome (ARDS) in of itself to mortality among ventilated patients. DESIGN AND SETTING:: A longitudinal retrospective study of ventilated intensive care unit (ICU) patients. PATIENTS:: The analysis included patients ventilated for more than 48 hours. Patients were classified as having ARDS on admission (early-onset ARDS), late-onset ARDS (ARDS not present during the first 24 hours of admission), or no ARDS. Primary outcomes were mortality at 28 days, and secondary outcomes were 2-year mortality rate from ICU admission. RESULTS:: A total of 1411 ventilated patients were enrolled: 41% had ARDS on admission, 28.5% developed ARDS during their ICU stay, and 30.5% did not meet the ARDS criteria prior to ICU discharge or death. The non-ARDS group was used as the control. We also divided the cohort based on the severity of ARDS. After adjusting for covariates, mortality risk at 28 days was not significantly different among the different groups. Both early- and late-onset ARDS as well as the severity of ARDS were found to be significant risk factors for 2 years from ICU survival. CONCLUSION:: Among patients who were ventilated on ICU admission, neither the presence, the severity, or the timing of ARDS contribute independently to the short-term mortality risk. However, acute respiratory distress syndrome does contribute significantly to 2-year mortality risk. This suggests that patients may not die acutely from ARDS itself but rather from the primary disease, and during the acute phase of ARDS, clinicians should focus on improving treatment strategies for the diseases that led to ARDS.

High-dimensional regression adjustments in randomized experiments.

We study the problem of treatment effect estimation in randomized experiments with high-dimensional covariate information and show that essentially any risk-consistent regression adjustment can be used to obtain efficient estimates of the average treatment effect. Our results considerably extend the range of settings where high-dimensional regression adjustments are guaranteed to provide valid inference about the population average treatment effect. We then propose cross-estimation, a simple method for obtaining finite-sample-unbiased treatment effect estimates that leverages high-dimensional regression adjustments. Our method can be used when the regression model is estimated using the lasso, the elastic net, subset selection, etc. Finally, we extend our analysis to allow for adaptive specification search via cross-validation and flexible nonparametric regression adjustments with machine-learning methods such as random forests or neural networks.

S100B and Inflammatory Cytokine Levels in Blood as Potential Markers of Blood-Brain Barrier Damage and Psychiatric Impairment in Comorbid Hepatitis C Viral Infection and Alcohol Use Disorder.

BACKGROUND: Hepatitis C virus (HCV) infection and alcohol use disorder (AUD) both adversely affect the immune system resulting in alterations in immune cell signaling and inflammatory processes. The aim of this study was to investigate how comorbid AUD contributes to abnormalities in inflammatory mediators and psychiatric impairments in adults with HCV. METHODS: Alcohol use, mood, and inflammatory factors were evaluated at 3 time points (baseline, week 4, and week 12) in Veterans with HCV, with (n = 42) and without (n = 13) comorbid AUD. Peripheral indices of immune activation, blood-brain barrier (BBB) damage (S100 calcium-binding protein B [S100B]), liver function, and viral load were measured using immunoassays and polymerase chain reaction assays. RESULTS: Comorbid AUD was associated with increased symptoms of depression and anxiety, elevated levels of liver enzymes, and altered expression of inflammatory factors. Alcohol consumption was positively correlated with the severity of psychiatric symptoms. Univariate analysis identified significant group differences in interleukin (IL)-8 (p = 0.006), IL-10 (p = 0.03), and S100B (p = 0.048), with increased levels in participants with AUD, which persisted over time despite reductions in alcohol use and no significant change in HCV viral load. Statistically significant effects of study group or time were not found for the other immune factors assessed. Exploratory receiver operating characteristic curve analysis evaluated the ability of IL-8, IL-10, and S100B to differentiate between levels of alcohol consumption and generated biomarker cutoff values used to identify low risk and unhealthy alcohol use groups. CONCLUSIONS: These results demonstrate that HCV and comorbid AUD are associated with greater psychiatric impairments, potentially resulting from increased inflammation, dysregulated cytokine expression, and compromised BBB function. Alcohol-induced BBB damage may increase the risk of neuropathological consequences within the context of chronic HCV infection.

Statistical learning and selective inference.

We describe the problem of "selective inference." This addresses the following challenge: Having mined a set of data to find potential associations, how do we properly assess the strength of these associations? The fact that we have "cherry-picked"--searched for the strongest associations--means that we must set a higher bar for declaring significant the associations that we see. This challenge becomes more important in the era of big data and complex statistical modeling. The cherry tree (dataset) can be very large and the tools for cherry picking (statistical learning methods) are now very sophisticated. We describe some recent new developments in selective inference and illustrate their use in forward stepwise regression, the lasso, and principal components analysis.

Post-Selection Inference for ℓ1-Penalized Likelihood Models.

We present a new method for post-selection inference for ℓ1 (lasso)-penalized likelihood models, including generalized regression models. Our approach generalizes the post-selection framework presented in Lee et al. (2013). The method provides p-values and confidence intervals that are asymptotically valid, conditional on the inherent selection done by the lasso. We present applications of this work to (regularized) logistic regression, Cox's proportional hazards model and the graphical lasso. We do not provide rigorous proofs here of the claimed results, but rather conceptual and theoretical sketches.