Mixtures of phthalates disrupt expression of genes related to lipid metabolism and peroxisome proliferator-activated receptor signaling in mouse granulosa cells.

Phthalates are a class of known endocrine disrupting chemicals that are found in common everyday products. Several studies associate phthalate exposure with detrimental effects on ovarian functions, including growth and development of the follicle and production of steroid hormones. We hypothesized that dysregulation of the ovary by phthalates may be mediated by phthalate toxicity towards granulosa cells, a major cell type in ovarian follicles responsible for key steps of hormone production and nourishing the developing oocyte. To test the hypothesis that phthalates target granulosa cells, we harvested granulosa cells from adult CD-1 mouse ovaries and cultured them for 96 hours in vehicle control, a phthalate mixture, or a phthalate metabolite mixture (0.1-100 µg/mL). After culture, we measured metabolism of the phthalate mixture into monoester metabolites by the granulosa cells, finding that granulosa cells do not significantly contribute to ovarian metabolism of phthalates. Immunohistochemistry of phthalate metabolizing enzymes in whole ovaries confirmed that these enzymes are not strongly expressed in granulosa cells of antral follicles and that ovarian metabolism of phthalates likely occurs primarily in the stroma. RNA sequencing of treated granulosa cells identified 407 differentially expressed genes, with overrepresentation of genes from lipid metabolic processes, cholesterol metabolism, and peroxisome proliferator-activated receptor (PPAR) signaling pathways. Expression of significantly differentially expressed genes related to these pathways were confirmed using qPCR. Our results agree with previous findings that phthalates and phthalate metabolites have different effects on the ovary and interfere with PPAR signaling in granulosa cells.

Validation of a bupropion, dextromethorphan, and omeprazole cocktail for simultaneous phenotyping of cytochrome P450 2B11, 2D15, and 3A12 activities in dogs.

OBJECTIVE: Develop a cytochrome P450 (CYP) phenotyping cocktail for dogs using specific substrates for hepatic P450 enzymes CYP2B11, CYP2D15, and CYP3A12 and determine whether alternative sampling methods (saliva and urine) or single time point samples could be used instead of multiple blood sampling. ANIMALS: 12 healthy client-owned dogs (8 females and 4 males) from February 2019 to May 2019. METHODS: In a randomized crossover study, dogs received oral administration of the probe drug bupropion (75 mg), dextromethorphan (30 mg), or omeprazole (40 mg) alone or as a 3-drug combination (Program in Individualized Medicine [PrIMe] cocktail) to evaluate simultaneous phenotyping of CYP2B11, CYP2D15, and CYP3A12. Pharmacokinetic profiles for the probe drugs and metabolites were determined using plasma, saliva, and urine. Dogs received probe drugs alone or combined. Pharmacokinetic profiles up to 6 hours postdose for the probe drugs and metabolites were determined using plasma, saliva, and urine. RESULTS: The PrIMe cocktail was well tolerated. There was no statistically significant interaction between the probe drugs when administered together. Single time point plasma metabolic ratios at 4 hours postdose for all probe drugs strongly correlated with the corresponding area under the plasma concentration-versus-time curve (AUC) ratios. Saliva AUC metabolic ratios for CYP3A12 and CYP2D15 and 6-hour urine for CYP2B11 and CYP2D15 were correlated with plasma AUC ratios. CONCLUSIONS: The PrIMe cocktail can be used for simultaneous CYP phenotyping using plasma 4-hour single time point sample metabolic ratios. Saliva and urine sampling are suitable for specific CYPs. CLINICAL RELEVANCE: The PrIMe cocktail has potential as a useful tool in dogs to detect clinically important CYP-mediated drug-drug interactions, identify novel pharmacogenes, determine the drug-metabolizing phenotype of individual dogs, aid in individualized dose selection, and evaluate the effects of various physiological states on drug metabolism.

De Novo TANGLED1 Recruitment to Aberrant Cell Plate Fusion Sites in Maize.

Division plane positioning is critical for proper growth and development in many organisms. In plants, the division plane is established before mitosis, by accumulation of a cytoskeletal structure called the preprophase band (PPB). The PPB is thought to be essential for recruitment of division site localized proteins, which remain at the division site after the PPB disassembles. Here, we show that a division site localized protein, TANGLED1 (TAN1), is recruited independently of the PPB to the cell cortex at sites, by the plant cytokinetic machinery, the phragmoplast. TAN1 recruitment to de novo sites on the cortex is partially dependent on intact actin filaments and the myosin XI motor protein OPAQUE1 (O1). These data imply a yet unknown role for TAN1 and possibly other division site localized proteins during the last stages of cell division when the phragmoplast touches the cell cortex to complete cytokinesis.

Pharmacogenomics of poor drug metabolism in greyhounds: Canine P450 oxidoreductase genetic variation, breed heterogeneity, and functional characterization.

Greyhounds metabolize cytochrome P450 (CYP) 2B11 substrates more slowly than other dog breeds. However, CYP2B11 gene variants associated with decreased CYP2B11 expression do not fully explain reduced CYP2B11 activity in this breed. P450 oxidoreductase (POR) is an essential redox partner for all CYPs. POR protein variants can enhance or repress CYP enzyme function in a CYP isoform and substrate dependent manner. The study objectives were to identify POR protein variants in greyhounds and determine their effect on coexpressed CYP2B11 and CYP2D15 enzyme function. Gene sequencing identified two missense variants (Glu315Gln and Asp570Glu) forming four alleles, POR-H1 (reference), POR-H2 (570Glu), POR-H3 (315Gln, 570Glu) and POR-H4 (315Gln). Out of 68 dog breeds surveyed, POR-H2 was widely distributed across multiple breeds, while POR-H3 was largely restricted to greyhounds and Scottish deerhounds (35% allele frequencies), and POR-H4 was rare. Three-dimensional protein structure modelling indicated significant effects of Glu315Gln (but not Asp570Glu) on protein flexibility through loss of a salt bridge between Glu315 and Arg519. Recombinant POR-H1 (reference) and each POR variant (H2-H4) were expressed alone or with CYP2B11 or CYP2D15 in insect cells. No substantial effects on POR protein expression or enzyme activity (cytochrome c reduction) were observed for any POR variant (versus POR-H1) when expressed alone or with CYP2B11 or CYP2D15. Furthermore, there were no effects on CYP2B11 or CYP2D15 protein expression, or on CYP2D15 enzyme kinetics by any POR variant (versus POR-H1). However, Vmax values for 7-benzyloxyresorufin, propofol and bupropion oxidation by CYP2B11 were significantly reduced by coexpression with POR-H3 (by 34-37%) and POR-H4 (by 65-72%) compared with POR-H1. Km values were unaffected. Our results indicate that the Glu315Gln mutation (common to POR-H3 and POR-H4) reduces CYP2B11 enzyme function without affecting at least one other major canine hepatic P450 (CYP2D15). Additional in vivo studies are warranted to confirm these findings.

Identification of Intestinal Lamina Propria Plasma Cells by Surface Transmembrane Activator and CAML Interactor Expression.

Plasma cells secrete an abundance of Abs and are a crucial component of our immune system. The intestinal lamina propria harbors the largest population of plasma cells, most of which produce IgA. These Abs can bind to beneficial gut bacteria to reinforce intestinal homeostasis and provide protection against enteric pathogens. Plasma cells downregulate many cell-surface proteins commonly used to identify B cells. In mice, expression of the surface marker CD138 has been widely used to identify plasma cells in lymph nodes, bone marrow, and spleen. Intestinal plasma cells require liberation via extensive tissue processing involving treatment with collagenase. We report that detection of CD138 surface expression is reduced following collagenase treatment. Using a mouse in which yellow fluorescent protein expression is controlled by the plasma cell requisite transcription factor Blimp-1, we show that surface detection of transmembrane activator and CAML interactor captures a significant proportion of Ab-secreting plasma cells in the intestinal lamina propria and gut-draining mesenteric lymph nodes. Additionally, we describe a flow cytometry panel based on the detection of surface markers to identify murine B cell subsets in the intestinal lamina propria and, as a proof of concept, combine it with a cutting-edge fate-tracking system to characterize the fate of germinal center B cells activated in early life. By identifying plasma cells and other key intestinal B subsets in a manner compatible with several downstream applications, including sorting and culturing and in vitro manipulations, this efficient and powerful approach can enhance studies of mucosal immunity.

PHARMACOKINETICS OF SINGLE INTRAMUSCULAR ADMINISTRATION OF CEFTIOFUR CRYSTALLINE FREE ACID IN AMERICAN ALLIGATORS (ALLIGATOR MISSISSIPPIENSIS).

A pharmacokinetic study of ceftiofur crystalline free acid sterile oil suspension (CCFA) was performed in six apparently healthy juvenile American alligators (Alligator mississippiensis). A single intramuscular dose of 30 mg/kg was administered in the triceps muscle. Blood samples were collected prior to treatment and at 4, 12, 24, 48, 72, 120, 144, 192, 288, and 366 h post administration. Plasma samples were analyzed for ceftiofur equivalent concentrations using liquid chromatography-mass spectrometry. Pharmacokinetic parameters were determined by noncompartmental analysis. Mean peak plasma concentration was 23.2 µg/ml (range, 16.0-27.9), median time to maximum concentration was 72 h (range, 72-120), mean area under the curve from 0 to 366 h postdose was 4.24 h · mg/ml (range, 3.54-4.97), and mean terminal half-life was 143 h (range, 90.8-220). Plasma concentrations were maintained above the minimum inhibitory concentration for this study of 2.0 µg/ml, which was established from similar CCFA pharmacokinetic studies in other reptilian species, through the end of the data collection of 366 h. Because of prolonged plasma concentrations, a dosing interval could not be established in this study. Future studies should include extended collection time points and multidose studies to determine dosing regimens.

Thwarted Belonging and Perceived Burdensomeness During Middle and Older Adulthood: The Role of Generativity.

Using a sample of middle-aged and older adults, this research explores associations between generativity and two key risk factors for suicide: thwarted belonging (T.B.) and perceived burdensomeness (P.B.). These variables are typically studied as predictors of suicide; the current study is unique in examining their psychosocial correlates. Erikson described, generativity as a psychosocial construct that characterizes adult well-being in mid-life, conceptualized as the sense one has successfully guided and contributed to the younger generation through mentoring. Using the Midlife in the United States Survey (MIDUS), the current analyses indicate that generativity is associated with lower levels of P.B. and T.B., even after accounting for measures of hopelessness, depressive symptoms, financial stability, perceived neighborhood quality, chronic health conditions, and respondent's demographic characteristics including gender and age. Results are discussed in terms of applications for suicide-risk prevention, and with regard to the promotion of positive psychosocial development across the lifespan.

Military Nursing Evidence-Based Practice Summit: A Descriptive Analysis of EBP Barriers and Solutions Within the Military Healthcare System.

BACKGROUND: Evidence-based practice (EBP) is an innovative systematic problem-solving methodology that incorporates the best research evidence into clinical practice to improve patient outcomes, job satisfaction, and reduced healthcare costs. Although there are significant advances to implement EBP into military healthcare and operational settings, many barriers and challenges still exist. Civilian healthcare organizations have examined barriers and solutions to integrate EBP into clinical practice, but limited data exists to identify barriers and solutions to integrate EBP into military healthcare settings. Advancing the implementation of EBPs within military healthcare settings has the power to transform the administrative processes of healthcare management and most importantly, the delivery of healthcare for service members and beneficiaries. The purpose of this article is to present findings from a qualitative descriptive research study which analyzed data obtained during an EBP military summit. METHODS: A qualitative descriptive research study was used to examine EBP barriers and solutions to implement EBP in military healthcare settings. Participants attended a virtual 1-day military EBP summit (n = 182). As part of the summit, participants were invited to voluntarily participate in focus groups. Focus groups were conducted to gain an understanding of EBP barriers and solutions from military and civilian nurses and medics with interest and experience conducting EBP within military healthcare settings (n = 42). Focus group discussions were transcribed and analyzed by the study team. RESULTS: The study analysis identified six themes: leadership, command culture, EBP barriers (specific to MTF/operational environments), communication, infrastructure support, and outcome measures. Sub-themes identified additional dimensions military-specific barriers and solutions within the six identified themes. CONCLUSIONS: The results of this research study identify actionable tasks and recommendations to advance EBP within the military healthcare system. EBP is currently underutilized in the military healthcare system, and supportive implementation of EBP can be accomplished through enhanced leadership engagement, changing command culture, addressing EBP barriers, infrastructure, communication planning, and integration of existing national clinical and financial outcome measures. Given the critical need to further transition of military healthcare to evidence-based data driven decisions, the knowledge gained from this study can optimize readiness and advance healthcare delivered to service members and beneficiaries within the military healthcare system.

Improvement in severe asthma patients receiving biologics and factors associated with persistent insufficient control: a real-life national study.

BACKGROUND: Biological therapies have revolutionized the treatment of severe asthma with type 2 inflammation. Although such treatments are very effective in reducing exacerbation and the dose of oral steroids, little is known about the persistence of symptoms in severe asthma patients treated with biologics. PURPOSE: We aim to describe asthma control and healthcare consumption of severe asthma patients treated with biologics. DESIGN: The Second Souffle study is a real-life prospective observational study endorsed by the Clinical Research Initiative in Severe Asthma: a Lever for Innovation & Science Network. METHODS: Adults with a confirmed diagnosis of severe asthma for at least 12 months' duration were enrolled in the study. A self-administered questionnaire including the Asthma Control Questionnaire (ACQ), Asthma Quality of Life Questionnaire (AQLQ) and a compliance evaluation test was given to the patients. Healthcare consumption within 12 months prior to enrolment was documented. In patients receiving biologics, doctors indicated whether the patients were biologic responders or non-responders. RESULTS: The characteristics of 431 patients with severe asthma were analysed. Among them, 409 patients (94.9%) presented asthma with type 2 inflammation (T2 high) profile, and 297 (72.6%) patients with a T2 high phenotype were treated with a biologic. Physicians estimated that 88.2% of patients receiving biologics were responders. However, asthma control was only achieved in 25.3% of those patients (ACQ > 0.75). A high proportion of patients (77.8%) identified as responders to biologics were not controlled according to the ACQ score. About 50% of patients continue to use oral corticosteroids either daily (25.2%) or more than three times a year for at least three consecutive days (25.6%). Gastro-oesophageal Reflux Disease (GERD) and Obstructive Sleep Apnoea syndrome (OSA) were identified as independent factors associated with uncontrolled asthma. CONCLUSION: Although a high proportion of severe asthma patients respond to biologics, only 25.3% have controlled asthma. GERD and OSA are independent factors of uncontrolled asthma.

Subcellular positioning during cell division and cell plate formation in maize.

Introduction: During proliferative plant cell division, the new cell wall, called the cell plate, is first built in the middle of the cell and then expands outward to complete cytokinesis. This dynamic process requires coordinated movement and arrangement of the cytoskeleton and organelles. Methods: Here we use live-cell markers to track the dynamic reorganization of microtubules, nuclei, endoplasmic reticulum, and endomembrane compartments during division and the formation of the cell plate in maize leaf epidermal cells. Results: The microtubule plus-end localized protein END BINDING1 (EB1) highlighted increasing microtubule dynamicity during mitosis to support rapid changes in microtubule structures. The localization of the cell-plate specific syntaxin KNOLLE, several RAB-GTPases, as well as two plasma membrane localized proteins was assessed after treatment with the cytokinesis-specific callose-deposition inhibitor Endosidin7 (ES7) and the microtubule-disrupting herbicide chlorpropham (CIPC). While ES7 caused cell plate defects in Arabidopsis thaliana, it did not alter callose accumulation, or disrupt cell plate formation in maize. In contrast, CIPC treatment of maize epidermal cells occasionally produced irregular cell plates that split or fragmented, but did not otherwise disrupt the accumulation of cell-plate localized proteins. Discussion: Together, these markers provide a robust suite of tools to examine subcellular trafficking and organellar organization during mitosis and cell plate formation in maize.

Human interactions with bats and bat coronaviruses in rural Côte d'Ivoire.

Bats are presumed reservoirs of diverse α- and ß- coronaviruses (CoVs) and understanding the diversity of bat-CoVs and the role bats play in CoV transmission is highly relevant in the context of the current COVID pandemic. We sampled bats in Côte d'Ivoire (2016-2018) living at ecotones between anthropogenic and wild habitats in the Marahoué National Park, a recently encroached protected area, to detect and characterize the CoVs circulating in bats and humans. A total of 314 bats were captured, mostly during the rainy season (78%), and CoV RNA was detected in three of the bats (0.96%). A CoV RNA sequence similar to Chaerephon bat coronavirus/Kenya/KY22/2006 (BtKY22) was found in a Chaerephon cf. pumilus and a Mops sp. fecal swab, while a CoV RNA sequence similar to the two almost identical Kenya bat coronaviruses BtKY55 and BtKY56 (BtKY55/56) was detected in an Epomops buettikoferi oral swab. Phylogenetic analyses indicated differences in the degree of evolutionary host-virus co-speciation for BtKY22 and BtKY55/56. To assess potential for human exposure to these viruses, we conducted human syndromic and community-based surveillance in clinics and high-risk communities. We collected data on participant characteristics, livelihoods, animal contact, and high-risk behaviors that may be associated with exposure to zoonotic diseases. We then collected biological samples for viral testing from 401 people. PCR testing of these biological samples revealed no evidence of CoV infection among the enrolled individuals. We identified higher levels of exposure to bats in people working in crop production and in hunting, trapping and fishing. Finally, we used the 'Spillover' risk-ranking tool to assess the potential for viral spillover and concluded that, while there is no evidence to suggest imminent risk of spillover for these CoVs, their host range and other traits suggest caution and vigilance are warranted in people with high exposure risk.

Early visual perceptual processing is altered in obsessive-compulsive disorder.

OBJECTIVE: Existing studies have shown changes in attention and emotion processing of disorder-relevant visual stimuli in those with obsessive compulsive disorder (OCD). However, early visual processing in OCD has not been assessed, as previous studies did not examine the entire time course of visual processing but instead assessed potential differences in pre-determined visual evoked potentials (VEPs). This study investigates the entire visual processing stream in OCD compared to healthy age-matched controls (HC) using emotionally-neutral visual stimuli and a data-driven rather than hypothesis-driven approach. METHODS: 35 HC and 26 participants with OCD underwent EEG recording while completing a modified Eriksen flanker task. Permutation-controlled t-tests were used to identify group differences in the data's full time course of visual evoked potentials. Baseline-corrected amplitudes at time points where the groups were significantly different were analyzed using ANCOVAs with BDI, BAI, and SNAP-inattentiveness scores included as covariates. RESULTS: This analysis identified enhanced P1 amplitudes to two visual stimuli (the initial flanker and the stimulus), corresponding to time windows of 65-93 ms and 157-187 ms post-flanker presentation in the OCD group compared to controls. Group (OCD vs. HC) was the strongest predictor of VEP amplitude during both time windows, with no significant influences of any covariates. CONCLUSIONS: This study showed an enhanced P1 component in people with OCD to neutral visual stimuli, potentially reflecting either inefficient or excessive early visual processing in this population. Additional inquiry is necessary to determine whether altered visual processing is associated with the sensory hypervigilance observed in those with OCD. SIGNIFICANCE: This work identifies early visual processing alterations in OCD using neutral stimuli and a data-driven approach.

Focused Wound Care Handoff Improves Burn Center Physician-Nursing Communication and Wound Care Education.

Burn patients require changing wound care routines dependent on wound characteristics and operative interventions. Order discrepancies on electronic medical systems and poor communication between providers leads to incorrect wound care treatment which can be harmful to the complex burn patient. By dedicating a daily wound care discussion for each patient involving integral components of the team: physician, charge nurse, and wound care technicians, enhanced communication amongst team members and improved patient care was noted. A single-center burn unit conducted pre- and postintervention survey of nursing staff measuring various components of wound care. The time spent on the wound care discussions were measured daily. Additional time required to conduct the rounds were minimal with nurse reported increased clarification in patient care without additional work burden. Thus, focused wound care meetings assist with communication between providers, clarification of wound care needs, and avoidance of errors without increasing strain on the team.

Hoarding symptoms are associated with higher rates of disability than other medical and psychiatric disorders across multiple domains of functioning.

BACKGROUND: Hoarding symptoms are associated with functional impairment, though investigation of disability among individuals with hoarding disorder has largely focused on clutter-related impairment to home management activities and difficulties using space because of clutter. This analysis assesses disability among individuals with hoarding symptoms in multiple domains of everyday functioning, including cognition, mobility, self-care, interpersonal and community-level interactions, and home management. The magnitude of the association between hoarding and disability was compared to that of medical and psychiatric disorders with documented high disability burden, including major depressive disorder (MDD), diabetes, and chronic pain. METHODS: Data were cross-sectionally collected from 16,312 adult participants enrolled in an internet-based research registry, the Brain Health Registry. Pearson's chi-square tests and multivariable logistic regression models were used to quantify the relationship between hoarding and functional ability relative to MDD, diabetes, and chronic pain. RESULTS: More than one in ten participants endorsed clinical (5.7%) or subclinical (5.7%) hoarding symptoms (CHS and SCHS, respectively). After adjusting for participant demographic characteristics and psychiatric and medical comorbidity, CHS and SCHS were associated with increased odds of impairment in all domains of functioning. Moderate to extreme impairment was endorsed more frequently by those with CHS or SCHS compared to those with self-reported MDD, diabetes, and/or chronic pain in nearly all domains (e.g., difficulty with day-to-day work or school: CHS: 18.7% vs. MDD: 11.8%, p < 0.0001) except mobility and self-care. While those with current depressive symptoms endorsed higher rates of impairment than those with hoarding symptoms, disability was most prevalent among those endorsing both hoarding and comorbid depressive symptoms. CONCLUSIONS: Hoarding symptoms are associated with profound disability in all domains of functioning. The burden of hoarding is comparable to that of other medical and psychiatric illnesses with known high rates of functional impairment. Future studies should examine the directionality and underlying causality of the observed associations, and possibly identify target interventions to minimize impairment associated with hoarding symptomatology.

Carry That Weight! The Challenge of Managing Weight Changes During Inpatient Admission for Patients With Burn Injuries ≥20% TBSA.

The hypermetabolic state of patients with ≥20% total body surface area (TBSA) causes loss of muscle mass and compromised immune function with delayed wound healing. Weight loss is most severe in patients with ≥20% TBSA with initial weight gain due to fluid resuscitation. The American Burn Association (ABA) proposed quality measures for burn injury admissions, including weight loss from admission to discharge. We assessed how our outcomes adhere to these measures and if they correlate with previously described results. We retrospectively reviewed adult admissions with ≥20% TBSA burn injuries from 2016 to 2021. Four groups were established based on %TBSA: 20% to 29% (Group 1), 30% to 39% (Group 2), 40% to 59% (Group 3), and ≥60% (Group 4). We assessed weight changes from admission to discharge and performed multivariate analyses to account for age, sex, total surgeries, and length of stay. Data from 123 patients revealed 40 with 20% to 29% TBSA, 29 with 30% to 39% TBSA, 33 with 40% to 59% TBSA, 21 with ≥60% TBSA. A significant difference in weight loss was observed when comparing Groups 1 and 2 and Groups 3 and 4 (Group 1: -3.63%, Group 2: -2%, Group 3: -9.28%, Group 4: -13.85%; P-value ≤ .05). Groups 3 and 4 had significantly longer lengths of stay compared to Groups 1 and 2 (Group 1: 32.16, Group 2: 37.5, Group 3: 71.13, Group 4: 87.18; P-value ≤ .01). Most patients that experienced weight loss during their admission had <15% weight loss. We found no significant difference in outcomes for patients receiving oxandrolone vs not. The mean weight change was -11% for patients with an overall weight loss and +5% for patients with an overall weight gain. The significant difference between the two groups was admission body mass index (BMI; loss: 30.4 kg/m2, gain: 26.0 kg/m2; P-value ≤ .05). Patients with ≥20% TBSA suffer weight changes, likely due to metabolic disturbances. Increased length of stay and higher %TBSA may be associated with greater weight loss. Patients experiencing weight gain had lower admission BMI suggesting that patients with higher BMI are more prone to weight loss. Our findings support that patients with %TBSA ≥40 are unique, requiring specialized nutritional protocols and metabolic analysis.

A KARRIKIN INSENSITIVE2 paralog in lettuce mediates highly sensitive germination responses to karrikinolide.

Karrikins (KARs) are chemicals in smoke that can enhance germination of many plants. Lettuce (Lactuca sativa) cv. Grand Rapids germinates in response to nanomolar karrikinolide (KAR1). Lettuce is much less responsive to KAR2 or a mixture of synthetic strigolactone analogs, rac-GR24. We investigated the molecular basis of selective and sensitive KAR1 perception in lettuce. The lettuce genome contains two copies of KARRIKIN INSENSITIVE2 (KAI2), which in Arabidopsis (Arabidopsis thaliana) encodes a receptor that is required for KAR responses. LsKAI2b is more highly expressed than LsKAI2a in dry achenes and during early stages of imbibition. Through cross-species complementation assays in Arabidopsis, we found that an LsKAI2b transgene confers robust responses to KAR1, but LsKAI2a does not. Therefore, LsKAI2b likely mediates KAR1 responses in lettuce. We compared homology models of KAI2 proteins from lettuce and a fire-follower, whispering bells (Emmenanthe penduliflora). This identified pocket residues 96, 124, 139, and 161 as candidates that influence the ligand specificity of KAI2. Further support for the importance of these residues was found through a broader comparison of pocket residues among 281 KAI2 proteins from 184 asterid species. Almost all KAI2 proteins had either Tyr or Phe identity at position 124. Genes encoding Y124-type KAI2 are more broadly distributed in asterids than in F124-type KAI2. Substitutions at residues 96, 124, 139, and 161 in Arabidopsis KAI2 produced a broad array of responses to KAR1, KAR2, and rac-GR24. This suggests that the diverse ligand preferences observed among KAI2 proteins in plants could have evolved through relatively few mutations.

Knowledge, attitudes, and practices associated with zoonotic disease transmission risk in North Sulawesi, Indonesia.

BACKGROUND: Hunters, vendors, and consumers are key actors in the wildlife trade value chain in North Sulawesi, Indonesia, and potentially face an elevated risk of exposure to zoonotic diseases. Understanding the knowledge, attitudes, and practices (KAP) associated with the risk of zoonotic disease transmission in these communities is therefore critical for developing recommendations to prevent or mitigate zoonotic outbreaks in the future. METHODS: Qualitative and quantitative methods were combined to understand KAP associated zoonotic diseases transmission risk in communities involved in the wildlife trade in North Sulawesi. Qualitative data were collected through semi-structured ethnographic interviews and focus group discussions (FGDs) while quantitative data were collected using questionnaires. We conducted 46 ethnographic interviews and 2 FGDs in 2016, and 477 questionnaire administrations in 2017-2018 in communities from five districts in North Sulawesi. We also collected biological specimens, including nasal swab, oropharyngeal swab, and blood, from 254 participants. The study sites were targeted based on known wildlife consumption and trade activities. The participants for qualitative data collection were purposively selected while participants for quantitative data collection were randomly selected. Biological samples were tested for five viral families including Coronaviridae, Filoviridae, Flaviviridae, Orthomyxoviridae and Paramyxoviridae. RESULTS: Knowledge regarding disease transmission from animals to humans was similar across the participants in qualitative focus groups, including knowledge of rabies and bird flu as zoonotic diseases. However, only a small fraction of the participants from the quantitative group (1%) considered that contact with wild animals could cause sickness. Our biological specimen testing identified a single individual (1/254, 0.004%) who was sampled in 2018 with serological evidence of sarbecovirus exposure. Overall, participants were aware of some level of risk in working with open wounds while slaughtering or butchering an animal (71%) but most did not know what the specific risks were. However, significant differences in the attitudes or beliefs around zoonotic disease risk and health seeking behaviors were observed across our study sites in North Sulawesi. CONCLUSIONS: Our study showed variable levels of knowledge, attitudes, and practices associated with the risk of zoonotic disease transmission among study participants. These findings can be used to develop locally responsive recommendations to mitigate zoonotic disease transmission.

Behavioral-biological surveillance of emerging infectious diseases among a dynamic cohort in Thailand.

BACKGROUND: Interactions between humans and animals are the key elements of zoonotic spillover leading to zoonotic disease emergence. Research to understand the high-risk behaviors associated with disease transmission at the human-animal interface is limited, and few consider regional and local contexts. OBJECTIVE: This study employed an integrated behavioral-biological surveillance approach for the early detection of novel and known zoonotic viruses in potentially high-risk populations, in an effort to identify risk factors for spillover and to determine potential foci for risk-mitigation measures. METHOD: Participants were enrolled at two community-based sites (n = 472) in eastern and western Thailand and two hospital (clinical) sites (n = 206) in northeastern and central Thailand. A behavioral questionnaire was administered to understand participants' demographics, living conditions, health history, and animal-contact behaviors and attitudes. Biological specimens were tested for coronaviruses, filoviruses, flaviviruses, influenza viruses, and paramyxoviruses using pan (consensus) RNA Virus assays. RESULTS: Overall 61/678 (9%) of participants tested positive for the viral families screened which included influenza viruses (75%), paramyxoviruses (15%), human coronaviruses (3%), flaviviruses (3%), and enteroviruses (3%). The most salient predictors of reporting unusual symptoms (i.e., any illness or sickness that is not known or recognized in the community or diagnosed by medical providers) in the past year were having other household members who had unusual symptoms and being scratched or bitten by animals in the same year. Many participants reported raising and handling poultry (10.3% and 24.2%), swine (2%, 14.6%), and cattle (4.9%, 7.8%) and several participants also reported eating raw or undercooked meat of these animals (2.2%, 5.5%, 10.3% respectively). Twenty four participants (3.5%) reported handling bats or having bats in the house roof. Gender, age, and livelihood activities were shown to be significantly associated with participants' interactions with animals. Participants' knowledge of risks influenced their health-seeking behavior. CONCLUSION: The results suggest that there is a high level of interaction between humans, livestock, and wild animals in communities at sites we investigated in Thailand. This study highlights important differences among demographic and occupational risk factors as they relate to animal contact and zoonotic disease risk, which can be used by policymakers and local public health programs to build more effective surveillance strategies and behavior-focused interventions.

KARRIKIN UP-REGULATED F-BOX 1 (KUF1) imposes negative feedback regulation of karrikin and KAI2 ligand metabolism in Arabidopsis thaliana.

SignificanceKarrikins are chemicals in smoke that stimulate regrowth of many plants after fire. However, karrikin responses are not limited to species from fire-prone environments and can affect growth after germination. Putatively, this is because karrikins mimic an unknown signal in plants, KAI2 ligand (KL). Karrikins likely require modification in plants to become bioactive. We identify a gene, KUF1, that appears to negatively regulate biosynthesis of KL and metabolism of a specific karrikin. KUF1 expression increases in response to karrikin or KL signaling, thus forming a negative feedback loop that limits further activation of the signaling pathway. This discovery will advance understanding of how karrikins are perceived and how smoke-activated germination evolved. It will also aid identification of the elusive KL.