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Background: The increase in reports of resistance to macrocyclic lactones in the canine heartworm, Dirofilaria immitis is alarming. While DNA based tests have been well-validated, they can be expensive. In a previous study, we showed that two biochemical tests adapted to a 96- well plate format and read in a spectrophotometer could detect differences among lab validated D. immitis isolates. The two tests- Resazurin reduction and Hoechst 33342 efflux—detect metabolism and P-glycoprotein activity respectively in microfilariae isolated from infected dog blood. Methods: Our objective was to optimize the two assays further by testing various assay parameters in D. immitis isolates not tested previously. We tested microfilarial seeding density, incubation time and the effect of in vitro treatment with ivermectin and doxycycline in five other D. immitis isolates—JYD-34, Big Head, Berkeley, Georgia III and LOL. All assays were performed in 3 technical replicates and 2–4 biological replicates. To understand the molecular basis of the assays, we also performed qPCR for selected drug metabolism and elimination associated genes of the ABC transporter and cytochrome P450 gene families. Results: Metabolism and ABC transporter activity as detected by these assays varied between strains. Anthelmintic status (resistant or susceptible) did not correlate with metabolism or P-gp efflux. Basal transcriptional variations were found between strains in ABC transporter and cytochrome P450 genes. Conclusions: These assays provide a greater understanding of the biochemical variation among isolates of D. immitis, which can be exploited in the future to develop in vitro diagnostic tests capable of differentiating susceptible and resistant isolates. [ABSTRACT FROM AUTHOR]

This study investigates the predictability of downslope windstorms located in Santa Barbara County, California, locally referred to as Sundowner winds, from both observed relationships and a high-resolution, operational numerical weather prediction model. We focus on April 2022, during which the Sundowner Winds Experiment (SWEX) was conducted. We further refine our study area to the Montecito region owing to some of the highest wind measurements occurring at or near surface station MTIC1, situated on the coast-facing slope overlooking the area. Fires are not uncommon in this area, and the difficulty of egress makes the population particularly vulnerable. Area forecasters often use the sea-level pressure difference (Δ SLP) between Santa Barbara Airport (KSBA) and locations to the north such as Bakersfield (KBFL) to predict Sundowner windstorm occurrence. Our analysis indicates that Δ SLP by itself is prone to high false alarm rates and offers little information regarding downslope wind onset, duration, or magnitude. Additionally, our analysis shows that the high-resolution rapid refresh (HRRR) model has limited predictive skill overall for forecasting winds in the Montecito area. The HRRR, however, skillfully predicts KSBA-KBFL Δ SLP, as does GraphCast, a machine learning weather prediction model. Using a logistic regression model we were able to predict the occurrence of winds exceeding 9 m s − 1 with a high probability of detection while minimizing false alarm rates compared to other methods analyzed. This provides a refined and easily computed algorithm for operational applications. [ABSTRACT FROM AUTHOR]

A novel group of biocidal compounds are the Crystal 3D (Cry) and Cytolytic (Cyt) proteins produced by Bacillus thuringiensis (Bt). Some Bt Cry proteins have a selective nematocidal activity, with Cry5B being the most studied. Cry5B kills nematode parasites by binding selectively to membrane glycosphingolipids, then forming pores in the cell membranes of the intestine leading to damage. Cry5B selectively targets multiple species of nematodes from different clades and has no effect against mammalian hosts. Levamisole is a cholinergic anthelmintic that acts by selectively opening L-subtype nicotinic acetylcholine receptor ion-channels (L-AChRs) that have been found on muscles of nematodes. A synergistic nematocidal interaction between levamisole and Cry5B at the whole-worm level has been described previously, but the location, mechanism and time-course of this synergism is not known. In this study we follow the timeline of the effects of levamisole and Cry5B on the Ca2+ levels in enterocyte cells in the intestine of Ascaris suum using fluorescence imaging. The peak Ca2+ responses to levamisole were observed after approximately 10 minutes while the peak responses to activated Cry5B were observed after approximately 80 minutes. When levamisole and Cry5B were applied simultaneously, we observed that the responses to Cry5B were bigger and occurred sooner than when it was applied by itself. It is proposed that the synergism is due to the cytoplasmic Ca2+ overload that is induced by the combination of levamisole opening Ca2+ permeable L-subtype nAChRs and the Ca2+ permeable Cry5B toxin pores produced in the enterocyte plasma membranes. The effect of levamisole potentiates and speeds the actions of Cry5B that gives rise to bigger Ca2+ overloads that accelerates cell-death of the enterocytes. Author summary: The neglected tropical diseases are a diverse set of infectious diseases which are common in low-income populations of Asia, Africa and the Americas. They include soil-transmitted helminth (STH) infections produced by the parasitic nematode Ascaris lumbricoides (= Ascaris suum). There are no effective vaccines, so mass drug administration (MDA) to control and prevent infection is the only practical option. With the limited number of anthelmintic drugs available for treatment, there is an increasing concern about the development of resistance. The use of combinations of anthelmintics, particularly if they are additive, is important for the delay of the onset of resistance. Here we describe synergistic interactions of levamisole and Cry5B mediated by Ca++ in A. suum intestine enterocytes. We have been able to make these new observations because of our access to A. suum parasites and development of our techniques for studying Ca++ signaling in the intestine of this nematode parasite, a site of the interaction of these two anthelmintic drugs. Our study provides new insights for development of effective STH combination therapies of anthelmintic drugs for slowing development of resistance with continued mass drug administration and understanding the nematode parasite intestine as a major target for combinations of anthelmintic drugs. [ABSTRACT FROM AUTHOR]

The transmembrane protein claudin‐1 is critical for formation of the epidermal barrier structure called tight junctions (TJ) and has been shown to be important in multiple disease states. These include neonatal ichthyosis and sclerosing cholangitis syndrome, atopic dermatitis and various viral infections. To develop a model to investigate the role of claudin‐1 in different disease settings, we used CRISPR/Cas9 to generate human immortalized keratinocyte (KC) lines lacking claudin‐1 (CLDN1 KO). We then determined whether loss of claudin‐1 expression affects epidermal barrier formation/function and KC differentiation/stratification. The absence of claudin‐1 resulted in significantly reduced barrier function in both monolayer and organotypic cultures. CLDN1 KO cells demonstrated decreases in gene transcripts encoding the barrier protein filaggrin and the differentiation marker cytokeratin‐10. Marked morphological differences were also observed in CLDN1 KO organotypic cultures including diminished stratification and reduced formation of the stratum granulosum. We also detected increased proliferative KC in the basale layer of CLDN1 KO organotypic cultures. These results further support the role of claudin‐1 in epidermal barrier and suggest an additional role of this protein in appropriate stratification of the epidermis. [ABSTRACT FROM AUTHOR]

Coastal Santa Barbara is among the most exposed communities to wildfire hazards in Southern California. Downslope, dry, and gusty windstorms are frequently observed on the south-facing slopes of the Santa Ynez Mountains that separate the Pacific Ocean from the Santa Ynez valley. These winds, known as "Sundowners," peak after sunset and are strong throughout the night and early morning. The Sundowner Winds Experiment (SWEX) was a field campaign funded by the National Science Foundation that took place in Santa Barbara, California, between 1 April and 15 May 2022. It was a collaborative effort of 10 institutions to advance understanding and predictability of Sundowners, while providing rich datasets for developing new theories of downslope windstorms in coastal environments with similar geographic and climatic characteristics. Sundowner spatiotemporal characteristics are controlled by complex interactions among atmospheric processes occurring upstream (Santa Ynez valley), and downstream due to the influence of a cool and stable marine boundary layer. SWEX was designed to enhance spatial measurements to resolve local circulations and vertical structure from the surface to the midtroposphere and from the Santa Barbara Channel to the Santa Ynez valley. This article discusses how SWEX brought cutting-edge science and the strengths of multiple ground-based and mobile instrument platforms to bear on this important problem. Among them are flux towers, mobile and stationary lidars, wind profilers, ceilometers, radiosondes, and an aircraft equipped with three lidars and a dropsonde system. The unique features observed during SWEX using this network of sophisticated instruments are discussed here. [ABSTRACT FROM AUTHOR]

Objective: Heterogeneous brief non-pharmacological interventions and guidelines exist to treat the burgeoning presentations to both emergency department and inpatient settings, for those in a crisis of mental ill-health. We systematically reviewed the literature to create a taxonomy of these brief non-pharmacological interventions, and review their evaluation methods and effectiveness. Method: We conducted a systematic review across Cochrane, CINAHL, DARE, Embase, MEDLINE, PsycINFO databases. Studies meeting quality criteria, using Joanna Briggs Institute tools, were eligible. Interventions were categorised, and outcomes synthesised. Results: Thirty-nine studies were included: 8 randomised controlled trials, 17 quasi-experimental, 11 qualitative studies, and 3 file audits. Taxonomy produced six coherent intervention types: Skills-focussed, Environment-focussed, Special Observation, Psychoeducation, Multicomponent Group and Multicomponent Individual. Despite this, a broad and inconsistent range of outcome measures reflected different outcome priorities and prevented systematic comparison of different types of intervention or meta-analysis. Few brief non-pharmacological interventions had consistent evidential support: sensory modulation rooms consistently improved distress in inpatient settings. Short admissions may reduce suicide attempts and readmission, if accompanied by psychotherapy. Suicide-specific interventions in emergency departments may improve depressive symptoms, but not suicide attempt rates. There was evidence that brief non-pharmacological interventions did not reduce incidence of self-harm on inpatient wards. We found no evidence for frequently used interventions such as no-suicide contracting, special observation or inpatient self-harm interventions. Conclusion: Categorising brief non-pharmacological interventions is feasible, but an evidence base for many is severely limited if not missing. Even when there is evidence, the inconsistency in outcomes often precludes clinicians from making inferences, although some interventions show promise. [ABSTRACT FROM AUTHOR]

Background: Suicidal thoughts and behaviours (STB) are a common reason for presentation to emergency departments and general hospitals. A meta‐analysis of the strength of clinical risk factors for subsequent suicide might aid understanding of suicidal behaviour and help suicide prevention. Methods: We conducted a meta‐analysis of cohort and controlled studies on clinical risk factors and later suicide among people presenting to emergency departments and general hospitals with STB. Data were extracted from papers meeting inclusion criteria, published in Medline, PsycINFO, and Embase between 1 January 1960 and 10 October 2022 using papers located with the search terms ((suicide*).m_titl AND (emergency* OR accident and emergency OR casualty OR general hospital OR toxicology service).mp) or were indexed in PubMed and had titles located with the search terms (suicide* OR self‐harm OR self‐harm OR self‐injury OR self‐injury OR self‐poisoning OR self‐poisoning OR overdose OR para‐suicide OR parasuicide [title/abstract]) AND (Emergency department OR emergency room OR Casualty OR general hospital OR toxicology OR accident and emergency [all fields]). Data about the association between clinical risk factors and suicide extracted from three or more studies were included in a random‐effects meta‐analysis of the odds of later death by suicide. The study was registered in PROSPERO and conducted according to MOOSE and PRISMA guidelines. Results: Seventy‐five studies reported on 741,624 people, of which 19,649 died by suicide (2.65%). Male sex (odds ratio (OR) = 1.99) and age (OR = 2.01) were the most consistently reported risk factors. The strongest associations with subsequent death by suicide related to violent self‐harm methods at the hospital presentation, including: unspecified violent method (OR = 4.97), any violent method (OR = 4.57) and the specific violent methods of drowning (OR = 4.32), hanging (OR = 4.26), and use of firearms (OR = 10.08). Patients categorised as higher risk using suicide prediction scales or any other method that combined risk factors had moderately increased odds of suicide (OR = 2.58). Younger age, Black and Hispanic ethnicity, overdose, a diagnosis of adjustment disorder, and the absence of any psychiatric diagnosis were protective against suicide. Conclusions: Most risk factors for suicide among people who have presented with STB are not strongly associated with later suicide. The strongest risk factors relate to self‐harm methods. In the absence of clear indicators of future suicide, all people presenting with suicidality warrant a thorough assessment of their needs, and further research is needed before we can meaningfully categorise people with STB according to suicide risk. [ABSTRACT FROM AUTHOR]

Purpose: Innate immune activation plays a critical role in the onset and progression of many diseases. While positron emission tomography (PET) imaging provides a non-invasive means to visualize and quantify such immune responses, most available tracers are not specific for innate immune cells. To address this need, we developed [18F]OP-801 by radiolabeling a novel hydroxyl dendrimer that is selectively taken up by reactive macrophages/microglia and evaluated its ability to detect innate immune activation in mice following lipopolysaccharide (LPS) challenge. Procedures: OP-801 was radiolabeled in two steps: [18F]fluorination of a tosyl precursor to yield [18F]3-fluoropropylazide, followed by a copper-catalyzed click reaction. After purification and stability testing, [18F]OP-801 (150-250 μCi) was intravenously injected into female C57BL/6 mice 24 h after intraperitoneal administration of LPS (10 mg/kg, n=14) or saline (n=6). Upon completing dynamic PET/CT imaging, mice were perfused, and radioactivity was measured in tissues of interest via gamma counting or autoradiography. Results: [18F]OP-801 was produced with >95% radiochemical purity, 12–52 μCi/μg specific activity, and 4.3±1.5% decay-corrected yield. Ex vivo metabolite analysis of plasma samples (n=4) demonstrated high stability in mice (97±3% intact tracer >120 min post-injection). PET/CT images of mice following LPS challenge revealed higher signal in organs known to be inflamed in this context, including the liver, lung, and spleen. Gamma counting confirmed PET findings, showing significantly elevated signal in the same tissues compared to saline-injected mice: the liver (p=0.009), lung (p=0.030), and spleen (p=0.004). Brain PET/CT images (summed 50–60 min) revealed linearly increasing [18F]OP-801 uptake in the whole brain that significantly correlated with murine sepsis score (r=0.85, p<0.0001). Specifically, tracer uptake was significantly higher in the brain stem, cortex, olfactory bulb, white matter, and ventricles of LPS-treated mice compared to saline-treated mice (p<0.05). Conclusion: [18F]OP-801 is a promising new PET tracer for sensitive and specific detection of activated macrophages and microglia that warrants further investigation. [ABSTRACT FROM AUTHOR]

Background: Toxocara canis is a cosmopolitan parasite of dogs that is transmitted transplacentally to puppies resulting in widespread shedding of eggs in the environment. However, it is not clear if there are dominant parasite genotypes that are more common, pathogenic, or likely to be zoonotic. Methods/principle findings: Sequences of mitochondrial cox1 gene from adult worms were used to compare parasites from the United States with submitted sequences from parasites isolated from dogs in different countries. Our analysis revealed at least 55 haplotypes. While we expected the North American worms to form a distinct cluster, we found haplotypes of T. canis reported elsewhere existing in this population. Interestingly, combining the sequence data from our study with the available GenBank data, analysis of cox1 sequences results in five distinct clades that are not geographically defined. Conclusions: The five clades of T. canis revealed in this study potentially have unique life histories, traits, or host preferences. Additional investigation is needed to see if these distinct clades represent cryptic species with clinically useful attributes or genotypes with taxonomic value. Evaluation of common mitochondrial genes may reveal distinct populations of zoonotic T. canis. Author summary: Toxocara canis is a cosmopolitan ascarid nematode of dogs. In humans, following ingestion of eggs, larvae of this parasite cause visceral larva migrans and ocular toxocariasis. Toxocara canis continues to persist in the United States, with a high level of seropositivity observed in people. However, relatively little data is available to describe 1) genetic isolates of the parasite that have been transmitted from dogs to humans or 2) geographically isolated clusters of T. canis that may form taxonomic groups with unique properties. In this study, we collected adult T. canis from dogs in four different states and applied phylogenetic and haplotypic analyses to compare parasite cox1 sequences with those reported globally. Our hypothesis was that we would discover new haplotypes that were unique to the United States. However, our data demonstrated very little geographic clustering of isolates. Instead, maximum likelihood trees and median joining networks supported the conclusion that nearly all haplotypes reported thus far worldwide fell within 5 distinct clades. Analysis of additional genes and parasite traits could reveal distinct clades of T. canis with specific clinical significance such as those predisposed to causing disease in humans. [ABSTRACT FROM AUTHOR]

Little is known about whether type 1 (IFNγ), 2 (IL-4/IL-13), or 3 (IL-17A/IL-22) cytokines affect the susceptibility of keratinocytes (KC) to viruses. These immune pathways predominate in various skin diseases: lupus, atopic dermatitis (AD), and psoriasis, respectively. Janus kinase inhibitors (JAKi) are approved to treat both AD and psoriasis, and are in clinical development for lupus. We evaluated whether these cytokines alter viral susceptibility of KC and determined if this effect is modulated by treatment with JAKi. Viral susceptibility to vaccinia virus (VV) or herpes simplex virus-1 (HSV-1) ± JAKi was assessed in immortalized and primary human KC pretreated with cytokines. Exposure to type 2 (IL-4 + IL-13) or the type 3 (IL-22) cytokines significantly increased KC viral susceptibility. Specifically, there was a peak increase of 12.2 ± 3.1-fold (IL-4 + IL-13) or 7.7 ± 2.8-fold (IL-22) in VV infection as measured by plaque number. Conversely, IFNγ significantly reduced susceptibility to VV (63.1 ± 64.4-fold). The IL-4 + IL-13-induced viral susceptibility was reduced (44 ± 16%) by JAK1 inhibition, while the IL-22-enhanced viral susceptibility was diminished (76 ± 19%) by TYK2 inhibition. IFNγ-mediated resistance to viral infection was reversed by JAK2 inhibition (366 ± 294% increase in infection). Cytokines expressed in AD skin (IL-4, IL-13, IL-22) increase KC viral susceptibility while IFNγ is protective. JAKi that target JAK1 or TYK2 reversed cytokine-enhanced viral susceptibility, while JAK2 inhibition reduced the protective effects of IFNγ. [ABSTRACT FROM AUTHOR]

The nematode parasite intestine absorbs nutrients, is involved in innate immunity, can metabolize xenobiotics and as we show here, is also a site of action of the anthelmintic, diethylcarbamazine. Diethylcarbamazine (DEC) is used to treat lymphatic filariasis and activates TRP-2, GON-2 & CED-11 TRP channels in Brugia malayi muscle cells producing spastic paralysis. DEC also has stimulatory effects on ascarid nematode parasites. Using PCR techniques, we detected, in Ascaris suum intestine, message for: Asu-trp-2, Asu-gon-2, Asu-ced-11, Asu-ocr-1, Asu-osm-9 and Asu-trpa-1. Comparison of amino-acid sequences of the TRP channels of B. malayi, and A. suum revealed noteworthy similarity, suggesting that the intestine of Ascaris will also be sensitive to DEC. We used Fluo-3AM as a Ca2+ indicator and observed characteristic unsteady time-dependent increases in the Ca2+ signal in the intestine in response to DEC. Application of La3+ and the TRP channel inhibitors, 2-APB or SKF 96365, inhibited DEC mediated increases in intracellular Ca2+. These observations are important because they emphasize that the nematode intestine, in addition to muscle, is a site of action of DEC as well as other anthelmintics. DEC may also enhance the Ca2+ toxicity effects of other anthelmintics acting on the intestine or, increase the effects of other anthelmintics that are metabolized and excreted by the nematode intestine. [ABSTRACT FROM AUTHOR]

We analyzed meteorological conditions that occurred during the December 2021 Boulder, Colorado, downslope windstorm. This event is of particular interest due to the ignition and spread of the Marshall Fire, which quickly became the most destructive wildfire in Colorado history. Observations indicated a rapid onset of fast winds with gusts as high as 51 m/s that generally remained confined to the east-facing slopes and foothills of the Rockies, similar to previous Boulder windstorms. After about 12 h, the windstorm shifted into a second, less intense phase. Midtropospheric winds above northwestern Colorado weakened prior to the onset of strong surface winds and the event strength started waning as stronger winds moved back into the area. Forecasts from NOAA high-resolution operational models initialized more than a few hours prior to windstorm onset did not simulate the start time, development rate and/or maximum strength of the windstorm correctly, and day-ahead runs even failed to develop strong downslope windstorms at all. Idealized modeling confirmed that predictability was limited by errors on the synoptic scale affecting the midtropospheric wind conditions representing the Boulder windstorm's inflow environment. Gust forecasts for this event were critically evaluated. [ABSTRACT FROM AUTHOR]

Individuals with underlying chronic skin conditions, notably atopic dermatitis (AD), are disproportionately affected by infections from members of the herpesviridae, papovaviridae, and poxviridae families. Many patients with AD experience recurrent, widespread cutaneous viral infections that can lead to viremia, serious organ complications, and even death. Little is known about how the type 2 inflammatory environment observed in the skin of AD patients impacts the susceptibility of epidermal cells (keratinocytes) to viral pathogens. Herein, we studied the susceptibility of keratinocytes to the prototypical poxvirus, vaccinia virus (VV)—the causative agent of eczema vaccinatum—under conditions that simulate the epidermal environment observed in AD. Treatment of keratinocytes with type 2 cytokines (IL-4 and -13) to simulate the inflammatory environment or a tight junction disrupting peptide to mirror the barrier disruption observed in AD patients, resulted in a differentiation-dependent increase in susceptibility to VV. Furthermore, pan JAK inhibition was able to diminish the VV susceptibility occurring in keratinocytes exposed to type 2 cytokines. We propose that in AD, the increased viral susceptibility of keratinocytes leads to enhanced virus production in the skin, which contributes to the rampant dissemination and pathology seen within patients. [ABSTRACT FROM AUTHOR]

The extracellular virion (EV) form of Orthopoxviruses is required for cell-to-cell spread and pathogenesis, and is the target of neutralizing antibodies in the protective immune response. EV have a double envelope that contains several unique proteins that are involved in its intracellular envelopment and/or subsequent infectivity. One of these, F13, is involved in both EV formation and infectivity. Here, we report that replacement of vaccinia virus F13L with the molluscum contagiosum virus homolog, MC021L results in the production of EV particles with significantly increased levels of EV glycoproteins, which correlate with a small plaque phenotype. Using a novel fluorescence-activated virion sorting assay to isolate EV populations based on glycoprotein content we determine that EV containing either higher or lower levels of glycoproteins are less infectious, suggesting that there is an optimal concentration of glycoproteins in the outer envelope that is required for maximal infectivity of EV. This optimal glycoprotein concentration was required for lethality and induction of pathology in a cutaneous model of animal infection, but was not required for induction of a protective immune response. Therefore, our results demonstrate that there is a sensitive balance between glycoprotein incorporation, infectivity, and pathogenesis, and that manipulation of EV glycoprotein levels can produce vaccine vectors in which pathologic side effects are attenuated without a marked diminution in induction of protective immunity. Author summary: Viral glycoproteins are critical determinants of host cell tropism, immunity, and pathogenesis. Vaccinia virus was used for the most successful immunization program in history, and poxviruses continue to be used as vaccine vectors. Here, we report that vaccinia virus extracellular virion (EV) protein F13 plays an important, previously unappreciated, role in controlling glycoprotein incorporation, and that there is a direct relationship between glycoprotein concentrations and subsequent infectivity. Crucially, manipulation of the EV glycoprotein concentrations altered pathogenesis and lethality in an in vivo infection model, but did not markedly alter the induced immune response. These results have important implications that inform the design of safer and more efficacious poxvirus-based vaccine vectors by altering glycoprotein content. [ABSTRACT FROM AUTHOR]