The von Hippel-Lindau protein forms fibrillar amyloid assemblies that are mitigated by the anti-amyloid molecule Purpurin.

The von Hippel-Lindau protein (pVHL) is a tumor suppressor involved in oxygen regulation via dynamic nucleocytoplasmic shuttling. It plays a crucial role in cell survival by degrading hypoxia-inducible factors (HIFs). Mutations in the VHL gene cause angiogenic tumors, characterized as VHL syndrome. However, aggressive tumors involving wild-type pVHL have also been described but the underlying mechanism remains to be revealed. We have previously shown that pVHL possesses several short amyloid-forming motifs, making it aggregation-prone. In this study, using a series of biophysical assays, we demonstrated that a pVHL-derived fragment (pVHL104-140) that harbors the nuclear export motif and HIF binding site, forms amyloid-like fibrillar structures in vitro by following secondary-nucleation-based kinetics. The peptide also formed amyloids at acidic pH that mimics the tumor microenvironment. We, subsequently, validated the amyloid formation by pVHL in vitro. Using the Curli-dependent amyloid generator (C-DAG) expression system, we confirmed the amyloidogenesis of pVHL in bacterial cells. The pVHL amyloids are an attractive target for therapeutics of the VHL syndrome. Accordingly, we demonstrated in vitro that Purpurin is a potent inhibitor of pVHL fibrillation. The amyloidogenic behavior of wild-type pVHL and its inhibition provide novel insights into the molecular underpinning of the VHL syndrome and its possible treatment.

Pf-Phospho: a machine learning-based phosphorylation sites prediction tool for Plasmodium proteins.

Even though several in silico tools are available for prediction of the phosphorylation sites for mammalian, yeast or plant proteins, currently no software is available for predicting phosphosites for Plasmodium proteins. However, the availability of significant amount of phospho-proteomics data during the last decade and advances in machine learning (ML) algorithms have opened up the opportunities for deciphering phosphorylation patterns of plasmodial system and developing ML-based phosphosite prediction tools for Plasmodium. We have developed Pf-Phospho, an ML-based method for prediction of phosphosites by training Random Forest classifiers using a large data set of 12 096 phosphosites of Plasmodium falciparum and Plasmodium bergei. Of the 12 096 known phosphosites, 75% of sites have been used for training/validation of the classifier, while remaining 25% have been used as completely unseen test data for blind testing. It is encouraging to note that Pf-Phospho can predict the kinase-independent phosphosites with 84% sensitivity, 75% specificity and 78% precision. In addition, it can also predict kinase-specific phosphosites for five plasmodial kinases-PfPKG, Plasmodium falciparum, PfPKA, PfPK7 and PbCDPK4 with high accuracy. Pf-Phospho (http://www.nii.ac.in/pfphospho.html) outperforms other widely used phosphosite prediction tools, which have been trained using mammalian phosphoproteome data. It also has been integrated with other widely used resources such as PlasmoDB, MPMP, Pfam and recently available ML-based predicted structures by AlphaFold2. Currently, Pf-phospho is the only bioinformatics resource available for ML-based prediction of phospho-signaling networks of Plasmodium and is a user-friendly platform for integrative analysis of phospho-signaling along with metabolic and protein-protein interaction networks.

Hepatic Vitamin A Concentrations and Association with Infectious Causes of Child Death.

OBJECTIVES: To assess postmortem vitamin A (VA) concentrations in children under 5 years of age and evaluate the association between VA deficiency (VAD) and infectious causes of death (CoD). STUDY DESIGN: In this cross-sectional study from the Child Health and Mortality Prevention Surveillance (CHAMPS) Network, liver biopsies collected within 72 hours of death were analyzed from 405 stillbirths and children under 5 years in Kenya and South Africa. Total liver VA (TLVA) concentrations were quantified using ultra-performance liquid chromatography, and cutoffs of ≤0.1 µmol/g, >0.1 to <0.7 µmol/g, ≥0.7 to <1.0 µmol/g, and ≥1.0 µmol/g were used to define VAD, adequate VA status, high VA, and hypervitaminosis A, respectively. CoD were determined by expert panel review. RESULTS: Among 366 liver samples with viable extraction, pooled prevalences of VAD, adequacy, high VA, and hypervitaminosis were 34.2%, 51.1%, 6.0%, and 8.7%, respectively. VAD was more common among neonates compared with stillbirths, infants, or children, and among those with low birthweight (LBW), underweight, or stunting (P < .05). When adjusting for site, age, and sex, there was no significant association of VAD with increased infectious CoD (OR 1.9, 95% confidence interval [CI] 0.9, 3.8, P = .073). In stratified analyses, VA deficient boys, but not girls, had an increased risk of infectious CoD (OR 3.4, 95% CI 1.3, 10.3, P = .013). CONCLUSIONS: Definitive postmortem assessment of VA status identified both VAD and VA excess among children under 5 years of age in Kenya and South Africa. VAD in boys was associated with increased risk of infectious mortality. Our findings may inform a transition from universal VA supplementation (VAS) to targeted strategies in certain countries.

Formulation of Functional Liquid Diets Under IDDSI Levels 0 to 2 for Dysphagia Patients: Physico-Chemical, Sensory, Antioxidant and Electromyographic Oral Processing Analyses.

Dysphagia is a significant health concern especially amongst the old age population. It is an ailment brought on by the weakening of the swallowing muscles. To reduce the risk of choking in dysphagia patients, the food is usually diluted to suit their swallowing ability. But dilution results in reducing the nutritional density of the foods thus causing undernutrition and malnutrition in patients. In this study, functional liquid diets were formulated under International Dysphagia Diet Standardization Initiative (IDDSI) levels 0-2. The developed diets were analysed for their proximate composition, colour, antioxidant and sensory properties. Antioxidant activities were determined using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS+), 2,2-diphenyl-1-picrylhydrazyl (DPPH), ferric reducing antioxidant power (FRAP) and total phenolic content (TPC) methods. The highest ABTS+ value was observed in pumpkin puree (level-2) i.e. 98.59%. Black carrot juice (level-1) showed the highest DPPH free radical scavenging activity and FRAP value viz. 88.43% and 689.33 µM TE/g, respectively. Electromyography (EMG) is an upcoming technique of food texture evaluation which provides real-time information about food oral processing. In this study, an EMG was conducted to measure the myoelectrical activity of human suprahyoid and masseter muscles by placing electrodes on the skin's surface during the oral processing of liquid. The EMG parameters correlated significantly with viscosity, ease of swallowing and IDDSI levels of the formulated diets. Hence EMG can be used as a tool for design and development of textured-modified diets for dysphagia patients. The sensory scores of formulated diets in this study were high indicating that these liquid diets may be incorporated into the diet plans of dysphagia patients.

First ever reported case of right-sided rupture of eventration of the diaphragm: Thoraco-laparoscopic approach.

ABSTRACT: Diaphragmatic eventration (DE) is an abnormal condition where a portion or the entire hemidiaphragm elevates due to insufficient muscle or nerve function while maintaining its anatomical attachments. On the other hand, congenital diaphragmatic hernias occur due to the abnormal development of muscular entities of the diaphragm, resulting in the displacement of abdominal contents into the thorax. The difference between diaphragmatic hernia and eventration is important as there is no true defect in DE. Ruptured eventration of the diaphragm is a rare entity, with only three cases reported in adults in literature till date, all on the left side. We report the first case of ruptured eventration of the diaphragm on the right side, which was repaired by a combination of laparoscopy and thoracoscopy and with double-mesh placement.

SMMPPI: a machine learning-based approach for prediction of modulators of protein-protein interactions and its application for identification of novel inhibitors for RBD:hACE2 interactions in SARS-CoV-2.

Small molecule modulators of protein-protein interactions (PPIs) are being pursued as novel anticancer, antiviral and antimicrobial drug candidates. We have utilized a large data set of experimentally validated PPI modulators and developed machine learning classifiers for prediction of new small molecule modulators of PPI. Our analysis reveals that using random forest (RF) classifier, general PPI Modulators independent of PPI family can be predicted with ROC-AUC higher than 0.9, when training and test sets are generated by random split. The performance of the classifier on data sets very different from those used in training has also been estimated by using different state of the art protocols for removing various types of bias in division of data into training and test sets. The family-specific PPIM predictors developed in this work for 11 clinically important PPI families also have prediction accuracies of above 90% in majority of the cases. All these ML-based predictors have been implemented in a freely available software named SMMPPI for prediction of small molecule modulators for clinically relevant PPIs like RBD:hACE2, Bromodomain_Histone, BCL2-Like_BAX/BAK, LEDGF_IN, LFA_ICAM, MDM2-Like_P53, RAS_SOS1, XIAP_Smac, WDR5_MLL1, KEAP1_NRF2 and CD4_gp120. We have identified novel chemical scaffolds as inhibitors for RBD_hACE PPI involved in host cell entry of SARS-CoV-2. Docking studies for some of the compounds reveal that they can inhibit RBD_hACE2 interaction by high affinity binding to interaction hotspots on RBD. Some of these new scaffolds have also been found in SARS-CoV-2 viral growth inhibitors reported recently; however, it is not known if these molecules inhibit the entry phase.

Ligand-based designing of DPP-4 inhibitors via hybridization; synthesis, docking, and biological evaluation of pyridazine-acetohydrazides.

A series of novel pyridazine-acetohydrazide hybrids were designed, synthesized, and evaluated for their in vitro and in vivo antihyperglycemic activity. In this context, pyridazine-acetohydrazides (6a-6p) were synthesized by coupling substituted aldehyde with 2-(5-cyano-6-oxo-3,4-diphenylpyridazine-1-6H-yl) acetohydrazide, which was prepared via the reaction of pyridazine ester with hydrazine hydrate. The molecular docking study was carried out to examine the binding affinities and interaction of designed compounds against the DPP-4 enzyme. Compounds 6e, 6f, 6l, and 6n exhibited interaction with active residue. In silico ADMET properties, and toxicity studies corroborated that compounds were found to have good bioavailability and less toxic. The synthesized compounds were further estimated for in vitro DPP-4 activity. Compounds 6e and 6l were found as the most effective DPP-4 inhibitor in this series with IC50 values (6.48, 8.22 nM) when compared with sitagliptin (13.02 nM). According to the toxicity assay compound, 6l showed very less toxicity at a higher concentration so further selected for the in vivo antihyperglycemic activity.

In silico and in vivo assessment of developmental toxicity, oxidative stress response & Na+/K+-ATPase activity in zebrafish embryos exposed to cypermethrin.

Cypermethrin (CYP), a synthetic type II pyrethroid pesticide, is extensively used to control pests in industrial, domestic, and agricultural environments. However, its indiscriminate use leads to a potential threat to aquatic organisms. Although several reports focussed on developmental toxicity effects, a concise study combining cardiotoxicity along with Na+/K+-ATPase activity and molecular docking of developmental proteins with CYP was lacking. This present study was designed to address this gap to comprehend the impact of CYP exposure (0, 25, 100 and 200 µg/L) on embryonic zebrafish. As a result, CYP delayed the hatching rate, reduced heart rate, increased mortality rate and induced numerous morphological abnormalities. Subsequently, CYP induced oxidative stress in treated zebrafish embryos with the concomitant increase in antioxidant enzymes (SOD and CAT) and malondialdehyde production. In addition, an alteration in AChE, NO content and Na+/K+-ATPase activity was observed, suggesting a disruption in cardiac development and ion regulation. Furthermore, AO staining showed notable apoptotic cells which are supported by alteration in apoptosis-related gene expressions. Moreover, to explore the putative targets of CYP, computational docking with developmental proteins (WNT3A, WNT8A, GATA-4, Nkx 2-5 and ZHE1) showed strong interactions and binding. Taken together, our findings provide a better understanding of assessing the ecotoxicological risk information and the mode of action underlying the development of teleost fishes following CYP exposure. Meanwhile, the pioneering nature of this study is to emphasize the future use of Na+/K+-ATPase activity as a potential toxicity biomarker and in silico molecular docking studies to complement developmental toxicity findings.

Superparamagnetic Iron-Oxide Nanoparticles Synthesized via Green Chemistry for the Potential Treatment of Breast Cancer.

In the emerging field of nanomedicine, nanoparticles have been widely considered as drug carriers and are now used in various clinically approved products. Therefore, in this study, we synthesized superparamagnetic iron-oxide nanoparticles (SPIONs) via green chemistry, and the SPIONs were further coated with tamoxifen-conjugated bovine serum albumin (BSA-SPIONs-TMX). The BSA-SPIONs-TMX were within the nanometric hydrodynamic size (117 ± 4 nm), with a small poly dispersity index (0.28 ± 0.02) and zeta potential of -30.2 ± 0.09 mV. FTIR, DSC, X-RD, and elemental analysis confirmed that BSA-SPIONs-TMX were successfully prepared. The saturation magnetization (Ms) of BSA-SPIONs-TMX was found to be ~8.31 emu/g, indicating that BSA-SPIONs-TMX possess superparamagnetic properties for theragnostic applications. In addition, BSA-SPIONs-TMX were efficiently internalized into breast cancer cell lines (MCF-7 and T47D) and were effective in reducing cell proliferation of breast cancer cells, with IC50 values of 4.97 ± 0.42 µM and 6.29 ± 0.21 µM in MCF-7 and T47D cells, respectively. Furthermore, an acute toxicity study on rats confirmed that these BSA-SPIONs-TMX are safe for use in drug delivery systems. In conclusion, green synthesized superparamagnetic iron-oxide nanoparticles have the potential to be used as drug delivery carriers and may also have diagnostic applications.

Identification of numerous novel disease-causing variants in patients with inherited retinal diseases, combining careful clinical-functional phenotyping with systematic, broad NGS panel-based genotyping.

Purpose: The widespread consensus is that genotyping is essential for patients with inherited retinal disease (IRD). Given the numerous ongoing gene therapy clinical trials for IRDs, identifying the pathogenic mutation in these patients has potential important therapeutic implications. In this study, we demonstrate how we identified with a high degree of confidence numerous novel disease-causing mutations, deletions, and duplications in a large consecutive IRD case series by using a judicious combination of careful, in-depth clinical-functional phenotyping to guide and integrate our genotyping approach. Methods: We conducted a retrospective analysis of data between November 2016 and March 2018 from the Duke Center for Retinal Degenerations and Ophthalmic Genetic Diseases IRD patient database, which encompassed 378 IRD cases that had not yet been previously genotyped. With the exception of some patients who presented with classical clinical-functional phenotypes that allowed for targeted gene testing, all other subjects systematically underwent next-generation sequencing-based broad, IRD-focused panel testing. Most cases were also tested for parental allele phase. Results were reviewed vis-à-vis the clinical-functional phenotypes for reconciliation and potential addition of supplemental testing such as deletion/duplication microarrays or copy number variant (CNV) analysis. Supplemental testing was driven by an IRD specialist-laboratory consensus, and decisions were clinically or genetically driven or both. Results: By judiciously using this two-way approach and leveraging to its full potential the benefits of careful, in-depth clinical-functional phenotyping by an experienced IRD specialist, more than 80% of the cases in this series were successfully genotyped. We also identified with a high degree of confidence 52 novel disease-causing mutations, deletions, and duplications. Conclusions: The combination of meticulous, expert clinical-functional phenotyping studies with systematic next-generation sequencing panel-based genotyping and microarray deletion/duplication testing or CNV analysis as applicable in accordance with the above-mentioned consensus was extremely effective at the diagnostic end, reduced costs, and saved time. IRD specialist-laboratory two-way interactions and case discussions would augment the efficacy of this approach and improve the diagnostic yield in successfully solving and genotyping IRD cases.

Mobile Health Services for COVID-19: Counseling, Testing, and Vaccination for Medically Underserved Populations.

Mobile health units can improve access to preventive health services, especially for medically underserved populations. However, there is little published experience of mobile health units being used to expand access to COVID-19 vaccination. In concert with local public health departments and community members, we implemented a mobile COVID-19 health unit and deployed it to 12 predominantly low-income and racial/ethnic minority communities in Massachusetts. We describe the success and challenges of this innovative program in expanding access to COVID-19 vaccination. (Am J Public Health. 2022;112(11):1556-1559. https://doi.org/10.2105/AJPH.2022.307021).

Expanding COVID-19 vaccine access to underserved populations through implementation of mobile vaccination units.

COVID-19 has disproportionately impacted underserved populations, including racial/ethnic minorities. Prior studies have demonstrated that mobile health units are effective at expanding preventive services for hard-to-reach populations, but this has not been studied in the context of COVID-19 vaccination. Our objective was to determine if voluntary participants who access mobile COVID-19 vaccination units are more likely to be racial/ethnic minorities and adolescents compared with the general vaccinated population. We conducted a cross-sectional study of individuals who presented to three different mobile COVID-19 vaccination units in the Greater Boston area from May 20, 2021, to August 18, 2021. We acquired data regarding the general vaccinated population in the state and of target communities from the Massachusetts Department of Public Health. We used chi-square testing to compare the demographic characteristics of mobile vaccination unit participants and the general state and community populations that received COVID-19 vaccines during the same time period. We found that during this three-month period, mobile vaccination units held 130 sessions and administered 2622 COVID-19 vaccine doses to 1982 unique participants. The median (IQR) age of participants was 31 (16-46) years, 1016 (51%) were female, 1575 (80%) were non-White, and 1126 (57%) were Hispanic. Participants in the mobile vaccination units were more likely to be younger (p < 0.001), non-White race (p < 0.001), and Hispanic ethnicity (p < 0.001) compared with the general vaccinated population of the state and target communities. This study suggests that mobile vaccination units have the potential to improve access to COVID-19 vaccination for diverse populations.

Interest in Electronic Cigarettes for Smoking Cessation Among Adults With Opioid Use Disorder in Buprenorphine Treatment: A Mixed-Methods Investigation.

INTRODUCTION: Individuals in treatment for opioid use disorder (OUD) have high smoking rates and limited success with Food and Drug Administration (FDA)-approved cessation aids, suggesting need for novel approaches. Electronic cigarettes (e-cigarettes) might benefit this population, but e-cigarettes' acceptability for tobacco reduction or cessation among smokers in OUD treatment is not known. METHODS: A cross-sectional mixed-methods study of 222 adults in OUD treatment with buprenorphine in the Boston, Massachusetts metropolitan area was conducted in 2020. We used quantitative and qualitative data to investigate individuals' experience with and interest in e-cigarettes and other methods for smoking cessation and assessed factors associated with interest in e-cigarette use. RESULTS: One hundred sixty (72%) of the 222 participants were past 30-day cigarette smokers. They most frequently reported having ever used nicotine replacement therapy (NRT; 83%) and e-cigarettes (71%) for smoking cessation and most often indicated interest in using NRT (71%) and e-cigarettes (44%) for future smoking cessation. In multiple logistic regression analysis, interest in using e-cigarettes for future smoking cessation was independently associated with having ever used e-cigarettes for smoking cessation, current e-cigarette use, and perceiving e-cigarettes to be less harmful than cigarettes (ps < .05). In qualitative data, many current vapers/former smokers reported that e-cigarettes had been helpful for quitting cigarettes. For current smokers who currently or formerly vaped, frequently reported challenges in switching to e-cigarettes were concerns about replacing one addiction with another and e-cigarettes not adequately substituting for cigarettes. CONCLUSIONS: E-cigarettes had a moderate level of acceptability for smoking cessation among cigarette smokers in OUD treatment. More research is warranted to test the efficacy of this approach. IMPLICATIONS: Individuals in treatment for opioid use disorder (OUD) have high smoking rates and limited success with existing smoking cessation tools, suggesting a need for novel cessation treatment approaches. In this mixed-methods study of individuals receiving medication treatment for OUD with buprenorphine in Massachusetts in 2020, we found a moderate level of acceptability of e-cigarettes for smoking cessation.

Click inspired novel pyrazole-triazole-persulfonimide & pyrazole-triazole-aryl derivatives; Design, synthesis, DPP-4 inhibitor with potential anti-diabetic agents.

This work presented the first report on designing, synthesizing of novel pyrazole-triazole-persulfonimide (7a-i) and pyrazole-triazole-aryl derivatives (8a-j) via click reaction using CuI catalyst and evaluated for their anti-diabetic activity and DPP-4 inhibitory effect. Click reactions went smoothly with CuI catalyst in the presence of tridentate chelating ligands and produced copper-free target pyrazole-triazole-persulfonimide analogues in excellent yield at RT. The designed compounds were docked against DPP-4 enzyme and showed excellent interaction with active amino acids residue. Further, all novel pyrazole-triazole-persulfonimide and pyrazole-triazole derivatives were subjected to enzyme-based in vitro DPP-4 inhibitory activity. Based on the SAR study DPP-4 inhibitory capacity compounds 7f (9.52 nM) and 8h (4.54 nM) possessed the significant inhibition of DPP-4. Finally compounds 7f and 8h were evaluated for their in vivo anti-diabetic activity using STZ induced diabetic mice model, and 8h showed a significant diabetic control effect compared to the sitagliptin drug. These studies demonstrated that the novel pyrazole-triazole-persulfonimide and pyrazole-triazole-aryl derivatives might be used as the leading compounds to develop novel DPP-4 inhibitors as potential anti-diabetic agents.

Prevalence of Cannabis Use and Cannabis Route of Administration among Massachusetts Adults in Buprenorphine Treatment for Opioid Use Disorder.

BACKGROUND: Recent prevalence estimates of cannabis use among individuals receiving medication treatment for OUD (MOUD) are lacking, and no study has characterized cannabis route of administration (cROA) in this population. These knowledge gaps are relevant because cannabis' effects and health outcomes vary by cROA and the availability and perceptions of cROA (e.g., vaping devices) are changing. METHODS: The Vaping In Buprenorphine-treated patients Evaluation (VIBE) cross-sectional survey assessed the prevalence and correlates of cannabis use and cROA among adults receiving buprenorphine MOUD from 02/20 to 07/20 at five community health centers in Massachusetts, a state with legal recreational and medical cannabis use. RESULTS: Among the 92/222 (41%) respondents reporting past 30-day cannabis use, smoking was the most common cROA (75%), followed by vaping (38%), and eating (26%). Smoking was more often used as a single cROA vs. in combination others (p = 0.01), whereas vaping, eating, and dabbing were more often used in combination with another cROA (all p < 0.05). Of the 39% of participants reporting multiple cROA, smoking and vaping (61%), and smoking and eating (50%), were the most prevalent combinations. Nonwhite race (vs. white) and current cigarette smoking (vs. no nicotine use) were associated with past 30-day cannabis use in multiple logistic regression. CONCLUSIONS: Prevalence of past 30-day cannabis use among individuals receiving buprenorphine MOUD in Massachusetts in 2020 was nearly double the prevalence of cannabis use in Massachusetts' adult general population in 2019 (21%). Our data are consistent with state and national data showing smoking as the most common cROA.

Molecular changes associated with migratory departure from wintering areas in obligate songbird migrants.

Day length regulates the development of spring migratory and subsequent reproductive phenotypes in avian migrants. This study used molecular approaches, and compared mRNA and proteome-wide expression in captive redheaded buntings that were photostimulated under long-day (LD) conditions for 4 days (early stimulated, LD-eS) or for ∼3 weeks until each bird had shown 4 successive nights of Zugunruhe (stimulated, LD-S); controls were maintained under short days. After ∼3 weeks of LD, photostimulated indices of the migratory preparedness (fattening, weight gain and Zugunruhe) were paralleled with upregulated expression of acc, dgat2 and apoa1 genes in the liver, and of cd36, fabp3 and cpt1 genes in the flight muscle, suggesting enhanced fatty acid (FA) synthesis and transport in the LD-S state. Concurrently, elevated expression of genes involved in the calcium ion signalling and transport (camk1 and atp2a2; camk2a in LD-eS), cellular stress (hspa8 and sod1, not nos2) and metabolic pathways (apoa1 and sirt1), but not of genes associated with migratory behaviour (adcyap1 and vps13a), were found in the mediobasal hypothalamus (MBH). Further, MBH-specific quantitative proteomics revealed that out of 503 annotated proteins, 28 were differentially expressed (LD-eS versus LD-S: 21 up-regulated and 7 down-regulated) and they enriched five physiological pathways that are associated with FA transport and metabolism. These first comprehensive results on gene and protein expression suggest that changes in molecular correlates of FA transport and metabolism may aid the decision for migratory departure from wintering areas in obligate songbird migrants.

Magnetic Resonance Imaging Provides Useful Diagnostic Information Following Equivocal Ultrasound in Children With Suspected Appendicitis [Formula: see text].

PURPOSE: In Canada, ultrasonography is the primary imaging modality for children with suspected appendicitis, yet equivocal studies are common. Magnetic resonance imaging provides promise as an adjunct imaging strategy. The primary objective of this study was to determine the proportion of children with suspected appendicitis and equivocal ultrasound where magnetic resonance imaging determined a diagnosis. METHODS: A prospective consecutive cohort of children aged 5-17 years presenting to a tertiary pediatric Emergency Department with suspected appendicitis were enrolled. Participants underwent diagnostic and management strategies according to our local suspected appendicitis pathway, followed by magnetic resonance (Siemens Avanto 1.5 Tesla) imaging. Sub-specialty pediatric radiologists reported all images. RESULTS: Magnetic resonance imaging was performed in 101 children with suspected appendicitis. The mean age was 11.9 (SD 3.4) years and median Pediatric Appendicitis Score was 6 [IQR 4,8]. Ultrasonography was completed in 98/101 (97.0%). Of 53/98 (54.1%) with equivocal ultrasound, magnetic resonance imaging provided further diagnostic information in 41 (77.4%; 10 positive, 31 negative; 12 remained equivocal). Secondary findings of appendicitis on magnetic resonance imaging in children with equivocal ultrasound included abdominal free fluid (24, 45.3%), peri-appendiceal fluid (12, 22.6%), intraluminal appendiceal fluid (9, 17.0%), fat stranding (8, 15.1%), appendicolith (2, 3.8%), and peri-appendiceal abscess (1, 1.9%). The observed agreement between magnetic resonance imaging results and final diagnosis was 94.9% (kappa = 0.89).

Role of pyruvate kinase M2 in oxidized LDL-induced macrophage foam cell formation and inflammation.

Pyruvate kinase M2 (PKM2) links metabolic and inflammatory dysfunction in atherosclerotic coronary artery disease; however, its role in oxidized LDL (Ox-LDL)-induced macrophage foam cell formation and inflammation is unknown and therefore was studied. In recombinant mouse granulocyte-macrophage colony-stimulating factor-differentiated murine bone marrow-derived macrophages, early (1-6 h) Ox-LDL treatment induced PKM2 tyrosine 105 phosphorylation and promotes its nuclear localization. PKM2 regulates aerobic glycolysis and inflammation because PKM2 shRNA or Shikonin abrogated Ox-LDL-induced hypoxia-inducible factor-1α target genes lactate dehydrogenase, glucose transporter member 1, interleukin 1ß (IL-1ß) mRNA expression, lactate, and secretory IL-1ß production. PKM2 inhibition significantly increased Ox-LDL-induced ABCA1 and ABCG1 protein expression and NBD-cholesterol efflux to apoA1 and HDL. PKM2 shRNA significantly inhibited Ox-LDL-induced CD36, FASN protein expression, DiI-Ox-LDL binding and uptake, and cellular total cholesterol, free cholesterol, and cholesteryl ester content. Therefore, PKM2 regulates lipid uptake and efflux. DASA-58, a PKM2 activator, downregulated LXR-α, ABCA1, and ABCG1, and augmented FASN and CD36 protein expression. Peritoneal macrophages showed similar results. Ox-LDL induced PKM2- SREBP-1 interaction and FASN expression in a PKM2-dependent manner. Therefore, this study suggests a role for PKM2 in Ox-LDL-induced aerobic glycolysis, inflammation, and macrophage foam cell formation.

MEDEA-interacting protein LONG-CHAIN BASE KINASE 1 promotes pattern-triggered immunity in Arabidopsis thaliana.

KEY MESSAGE: Arabidopsis LONG-CHAIN BASE KINASE 1 (LCBK1) interacts with MEDEA, a component of PCR2 complex that negatively regulates immunity. LCBK1 phosphorylates phytosphingosine and thereby promotes stomatal immunity against bacterial pathogens. Arabidopsis polycomb-group repressor complex2 (PRC2) protein MEDEA (MEA) suppresses both pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). MEA represses the expression of RPS2 and thereby attenuates AvrRpt2 effector-mediated ETI. However, the mechanism of MEA-mediated PTI diminution was not known. By screening the Arabidopsis cDNA library using yeast-2-hybrid interaction, we identified LONG-CHAIN BASE KINASE1 (LCBK1) as an MEA-interacting protein. We found that lcbk1 mutants are susceptible to virulent bacterial pathogens, such as Pseudomonas syringae pv maculicola (Psm) and P. syringae pv tomato (Pst) but not the avirulent strain of Pst that carries AvrRpt2 effector. Pathogen inoculation induces LCBK1 expression, especially in guard cells. We found that LCBK1 has a positive regulatory role in stomatal closure after pathogen inoculation. WT plants close stomata within an hour of Pst inoculation or flg22 (a 22 amino acid peptide from bacterial flagellin protein that activates PTI) treatment, but not lcbk1 mutants. LCBK1 phosphorylates phytosphingosine (PHS). Exogenous application of phosphorylated PHS (PHS-P) induces stomatal closure and rescues loss-of-PTI phenotype of lcbk1 mutant plants. MEA overexpressing (MEA-Oex) plants are defective, whereas loss-of-function mea-6 mutants are hyperactive in PTI-induced stomatal closure. Exogenous application of PHS-P rescues loss-of-PTI in MEA-Oex plants. Results altogether demonstrate that LCBK1 is an interactor of MEA that positively regulates PTI-induced stomatal closure in Arabidopsis.

Ift172 conditional knock-out mice exhibit rapid retinal degeneration and protein trafficking defects.

Intraflagellar transport (IFT) is a bidirectional transport process that occurs along primary cilia and specialized sensory cilia, such as photoreceptor outersegments. Genes coding for various IFT components are associated with ciliopathies. Mutations in IFT172 lead to diseases ranging from isolated retinal degeneration to severe syndromic ciliopathies. In this study, we created a mouse model of IFT172-associated retinal degeneration to investigate the ocular disease mechanism. We found that depletion of IFT172 in rod photoreceptors leads to a rapid degeneration of the retina, with severely reduced electroretinography (ERG) responses by 1 month and complete outer-nuclear layer (ONL) degeneration by 2 months. We investigated molecular mechanisms of degeneration and show that IFT172 protein reduction leads to mislocalization of specific photoreceptor outersegment (OS) proteins (RHO, RP1, IFT139), aberrant light-driven translocation of alpha transducin and altered localization of glioma-associated oncogene family member 1 (GLI1). This mouse model exhibits key features of the retinal phenotype observed in patients with IFT172-associated blindness and can be used for in vivo testing of ciliopathy therapies.