Cathelicidin preserves intestinal barrier function in polymicrobial sepsis.

ObjectivesThe intestinal epithelium compartmentalizes the sterile bloodstream and the commensal bacteria in the gut. Accumulating evidence suggests that this barrier is impaired in sepsis, aggravating systemic inflammation. Previous studies reported that cathelicidin is differentially expressed in various tissues in sepsis. However, its role in sepsis-induced intestinal barrier dysfunction has not been investigated.DesignTo examine the role of cathelicidin in polymicrobial sepsis, cathelicidin wild-(Cnlp+/+) and knockout (Cnlp-/-) mice underwent cecal-ligation and puncture (CLP) followed by the assessment of septic mortality and morbidity as well as histological, biochemical, immunological, and transcriptomic analyses in the ileal tissues. We also evaluated the prophylactic and therapeutic efficacies of vitamin D3 (an inducer of endogenous cathelicidin) in the CLP-induced murine polymicrobial sepsis model.ResultsThe ileal expression of cathelicidin was increased by three-fold after CLP, peaking at 4 h. Knockout of Cnlp significantly increased 7-day mortality and was associated with a higher murine sepsis score. Alcian-blue staining revealed a reduced number of mucin-positive goblet cells, accompanied by reduced mucin expression. Increased number of apoptotic cells and cleavage of caspase-3 were observed. Cnlp deletion increased intestinal permeability to 4kD fluorescein-labeled dextran and reduced the expression of tight junction proteins claudin-1 and occludin. Notably, circulating bacterial DNA load increased more than two-fold. Transcriptome analysis revealed upregulation of cytokine/inflammatory pathway. Depletion of Cnlp induced more M1 macrophages and neutrophils compared with the wild-type mice after CLP. Mice pre-treated with cholecalciferol (an inactive form of vitamin D3) or treated with 1alpha, 25-dihydroxyvitamin D3 (an active form of VD3) had decreased 7-day mortality and significantly less severe symptoms. Intriguingly, the administration of cholecalciferol after CLP led to worsened 7-day mortality and the associated symptoms.ConclusionsEndogenous cathelicidin promotes intestinal barrier integrity accompanied by modulating the infiltration of neutrophils and macrophages in polymicrobial sepsis. Our data suggested that 1alpha, 25-dihydroxyvitamin D3 but not cholecalciferol is a potential therapeutic agent for treating sepsis

Heterozygous Mutation of Drosophila Opa1 Causes the Development of Multiple Organ Abnormalities in an Age-Dependent and Organ-Specific Manner

Optic Atrophy 1 (OPA1) is a ubiquitously expressed dynamin-like GTPase in the inner mitochondrial membrane. It plays important roles in mitochondrial fusion, apoptosis, reactive oxygen species (ROS) and ATP production. Mutations of OPA1 result in autosomal dominant optic atrophy (DOA). The molecular mechanisms by which link OPA1 mutations and DOA are not fully understood. Recently, we created a Drosophila model to study the pathogenesis of optic atrophy. Heterozygous mutation of Drosophila OPA1 (dOpa1) by P-element insertion results in no obvious morphological abnormalities, whereas homozygous mutation is embryonic lethal. In eye-specific somatic clones, homozygous mutation of dOpa1 causes rough (mispatterning) and glossy (decreased lens deposition) eye phenotypes in adult Drosophila. In humans, heterozygous mutations in OPA1 have been associated with mitochondrial dysfunction, which is predicted to affect multiple organs. In this study, we demonstrated that heterozygous dOpa1 mutation perturbs the visual function and an ERG profile of the Drosophila compound eye. We independently showed that antioxidants delayed the onset of mutant phenotypes in ERG and improved larval vision function in phototaxis assay. Furthermore, heterozygous dOpa1 mutation also caused decreased heart rate, increased heart arrhythmia, and poor tolerance to stress induced by electrical pacing. However, antioxidants had no effects on the dysfunctional heart of heterozygous dOpa1 mutants. Under stress, heterozygous dOpa1 mutations caused reduced escape response, suggesting abnormal function of the skeletal muscles. Our results suggest that heterozygous mutation of dOpa1 shows organ-specific pathogenesis and is associated with multiple organ abnormalities in an age-dependent and organ-specific manner

Essential Roles of the Tap42-Regulated Protein Phosphatase 2A (PP2A) Family in Wing Imaginal Disc Development of Drosophila melanogaster

Protein ser/thr phosphatase 2A family members (PP2A, PP4, and PP6) are implicated in the control of numerous biological processes, but our understanding of the in vivo function and regulation of these enzymes is limited. In this study, we investigated the role of Tap42, a common regulatory subunit for all three PP2A family members, in the development of Drosophila melanogaster wing imaginal discs. RNAi-mediated silencing of Tap42 using the binary Gal4/UAS system and two disc drivers, pnr- and ap-Gal4, not only decreased survival rates but also hampered the development of wing discs, resulting in a remarkable thorax cleft and defective wings in adults. Silencing of Tap42 also altered multiple signaling pathways (HH, JNK and DPP) and triggered apoptosis in wing imaginal discs. The Tap42RNAi-induced defects were the direct result of loss of regulation of Drosophila PP2A family members (MTS, PP4, and PPV), as enforced expression of wild type Tap42, but not a phosphatase binding defective Tap42 mutant, rescued fly survivorship and defects. The experimental platform described herein identifies crucial roles for Tap42•phosphatase complexes in governing imaginal disc and fly development

Global burden of 369 diseases and injuries in 204 countries and territories, 1990–2019: a systematic analysis for the Global Burden of Disease Study 2019

Background: In an era of shifting global agendas and expanded emphasis on non-communicable diseases and injuries along with communicable diseases, sound evidence on trends by cause at the national level is essential. The Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) provides a systematic scientific assessment of published, publicly available, and contributed data on incidence, prevalence, and mortality for a mutually exclusive and collectively exhaustive list of diseases and injuries. Methods: GBD estimates incidence, prevalence, mortality, years of life lost (YLLs), years lived with disability (YLDs), and disability-adjusted life-years (DALYs) due to 369 diseases and injuries, for two sexes, and for 204 countries and territories. Input data were extracted from censuses, household surveys, civil registration and vital statistics, disease registries, health service use, air pollution monitors, satellite imaging, disease notifications, and other sources. Cause-specific death rates and cause fractions were calculated using the Cause of Death Ensemble model and spatiotemporal Gaussian process regression. Cause-specific deaths were adjusted to match the total all-cause deaths calculated as part of the GBD population, fertility, and mortality estimates. Deaths were multiplied by standard life expectancy at each age to calculate YLLs. A Bayesian meta-regression modelling tool, DisMod-MR 2.1, was used to ensure consistency between incidence, prevalence, remission, excess mortality, and cause-specific mortality for most causes. Prevalence estimates were multiplied by disability weights for mutually exclusive sequelae of diseases and injuries to calculate YLDs. We considered results in the context of the Socio-demographic Index (SDI), a composite indicator of income per capita, years of schooling, and fertility rate in females younger than 25 years. Uncertainty intervals (UIs) were generated for every metric using the 25th and 975th ordered 1000 draw values of the posterior distribution. Findings: Global health has steadily improved over the past 30 years as measured by age-standardised DALY rates. After taking into account population growth and ageing, the absolute number of DALYs has remained stable. Since 2010, the pace of decline in global age-standardised DALY rates has accelerated in age groups younger than 50 years compared with the 1990–2010 time period, with the greatest annualised rate of decline occurring in the 0–9-year age group. Six infectious diseases were among the top ten causes of DALYs in children younger than 10 years in 2019: lower respiratory infections (ranked second), diarrhoeal diseases (third), malaria (fifth), meningitis (sixth), whooping cough (ninth), and sexually transmitted infections (which, in this age group, is fully accounted for by congenital syphilis; ranked tenth). In adolescents aged 10–24 years, three injury causes were among the top causes of DALYs: road injuries (ranked first), self-harm (third), and interpersonal violence (fifth). Five of the causes that were in the top ten for ages 10–24 years were also in the top ten in the 25–49-year age group: road injuries (ranked first), HIV/AIDS (second), low back pain (fourth), headache disorders (fifth), and depressive disorders (sixth). In 2019, ischaemic heart disease and stroke were the top-ranked causes of DALYs in both the 50–74-year and 75-years-and-older age groups. Since 1990, there has been a marked shift towards a greater proportion of burden due to YLDs from non-communicable diseases and injuries. In 2019, there were 11 countries where non-communicable disease and injury YLDs constituted more than half of all disease burden. Decreases in age-standardised DALY rates have accelerated over the past decade in countries at the lower end of the SDI range, while improvements have started to stagnate or even reverse in countries with higher SDI. Interpretation: As disability becomes an increasingly large component of disease burden and a larger component of health expenditure, greater research and developm nt investment is needed to identify new, more effective intervention strategies. With a rapidly ageing global population, the demands on health services to deal with disabling outcomes, which increase with age, will require policy makers to anticipate these changes. The mix of universal and more geographically specific influences on health reinforces the need for regular reporting on population health in detail and by underlying cause to help decision makers to identify success stories of disease control to emulate, as well as opportunities to improve. Funding: Bill & Melinda Gates Foundation. © 2020 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 licens

The effects of <i>mts^XE2258</i>, <i>Tap42^WT</i>, and <i>Tap42^ED</i> on the viability of <i>Tap42^RNAi</i> flie ^{a, b}.

<p>a. The actual surviving ratios of F1 progeny were quantified from the following crosses:</p><p>Cross 1: +/+; pnr-Gal4/TM3, Ser ♀ x UAS-Tap42^RNAi/CyO; +/+ ♂.</p><p>Cross 2: ap-Gal4/CyO ♀ x UAS-Tap42^RNAi/CyO ♂.</p><p>Cross 3: ap-Gal4/CyO ♀ x UAS-Tap42^RNAi, mts^XE2258/CyO ♂.</p><p>Cross 4: +/+; pnr-Gal4/TM3, Ser ♀ x UAS-Tap42^RNAi/CyO; UAS-Tap42^WT/MKRS ♂.</p><p>Cross 5: +/+; pnr-Gal4/TM3, Ser ♀ x UAS-Tap42^RNAi/CyO; UAS-Tap42^ED/MKRS ♂.</p><p>Cross 6: +/+ ♀ x mts^XE2258/CyO ♂.</p><p>b. Crosses were repeated at least three times and flies that enter eclosion were counted as survivors.</p

JNK and DPP signaling are altered in wing imaginal discs following depletion of Tap42.

<p>The activity and expression of BSK was monitored in wing imaginal discs using antibodies recognizing phospho-JNK or total JNK. The pattern of active JNK/BSK (green, A1-3) was not different between control <i>UAS-Tap42^RNAi</i> flies (A1) and flies co-expressing the <i>pnr</i> driver (A2). However, hyperphosphorylation of JNK/BSK was observed in the wing disc dorsal compartment (red arrows) along with hypophosphorylation of JNK/BSK in the ventral wing compartment when <i>Tap42^RNAi</i> was driven by <i>ap-Gal4</i> (A3). Total levels of JNK/BSK (green, B1-B3) did not change as a result of <i>Tap42</i> knockdown. <i>Dpp</i> gene expression (purple, C1-C3), as monitored by X-GAL staining of <i>dpp-LaZ</i>, in the scutellum and along the anterior/posterior boundary of the wing blade was similar in both control (C1) and <i>pnr</i>-<i>Gal4</i> driven <i>Tap42^RNAi</i> flies (C2). <i>ap-Gal4</i> driven <i>Tap42^RNAi</i> flies demonstrated decreased DPP signal in the scutellum (red arrow, C3) and expanded staining in the wing blade compartment (red dashed line, C3). Genotypes: (A1, B1, & C1) <i>UAS-Tap42^RNAi/+</i> as control. (A2, B2, & C2) <i>UAS-Tap42^RNAi/+; pnr-Gal4/+</i>. (A3, B3, & C3) <i>ap-Gal4/UAS-Tap42^RNAi; +/+</i>.</p

Tap42 interacts with all three PP2A members and is required for normal wing disc development.

<p>Panel A: FLAG immunoprecipitations (FLAG IPs) were performed from extracts of <i>Drosophila</i> S2 cells expressing HA₃-Mts, HA₃-PP4, or HA₃-PPV alone or together with wildtype (FLAG₃-Tap42^WT) or mutant Tap42 (FLAG₃-Tap42^ED). The FLAG immune complexes and corresponding cell extracts (lysates) were analyzed by Western blotting using the indicated epitope tag antibodies. Panel B: Adult flies expressing <i>Tap42^RNAi</i> in the <i>ap</i> domain displayed a marked thorax cleft (red arrow, B1) and shriveled wings (B5). Expression of <i>Tap42^WT</i> in this background completely rescued both thorax (B2) and wing defects (B6). However, introduction of the <i>Tap42^ED</i> mutant in this background failed to rescue the defects and the flies lacked the scutum (B3) and formed blistered wings (B7). Expression of <i>Tap42^ED</i> alone resulted in a mild defect around the scutum (B4) and the formation of a forked wing vein (B8). Genotypes: (B1 & B5) <i>ap-Gal4/UAS-Tap42^RNAi; +/+</i>. (B2 & B6) <i>ap-Gal4/UAS-Tap42^RNAi; +/UAS-Tap42^WT</i>. (B3 & B7) <i>ap-Gal4/UAS-Tap42^RNAi; +/UAS-Tap42^ED.</i> (A4 & B4) <i>ap-Gal4/+; +/UAS-Tap42^ED</i>.</p

Tap42 is expressed in imaginal discs and primarily localized in the peripodial epithelium (PE) region.

<p>Panel A: Wing (A1–A3), haltere/3^rd leg (A4–A6), 2^nd leg (A7–A9), and eye imaginal discs (A10–A12) isolated from 3^rd instar larvae were immunostained for Tap42 protein expression (green) and counter-stained with the nucleic acid dye TO-PRO3 (purple). <i>UAS-Tap42^RNAi</i> control flies exhibited abundant expression of <i>Tap42</i> in the PE region of these imaginal discs (A1, A4, A7, & A10). <i>Tap42^RNAi</i> expression with the <i>pnr</i> (A2, A5, A8, & A11) and <i>ap</i> (A3, A6, A9, & A12) drivers dramatically reduced <i>Tap42</i> expression to nearly undetectable levels. Of note, <i>ap-Gal4</i>-mediated silencing of <i>Tap42</i> also disrupted the morphological patterning of the wing disc, as revealed by TO-PRO3 staining (A3). Panel B: The localization of Tap42 in the PE region was confirmed by immunofluorescence histochemistry. Immunostaining of wing discs obtained from wild type flies revealed an overlap of Ubx (red) and Tap42 (green) expression (B1). An amplified view of the merged image highlights strong Tap42 expression around the presumptive medial edge (ME) cells of the PE, which localizes near the boundary of the PE and DP (B2). Some Tap42 expression was visualized in the disc proper (DP) cells. Wing discs were counter-stained with the nucleic acid dye TO-PRO3 (blue). Genotypes: (A1, A4, A7, & A10) <i>UAS-Tap42^RNAi/+</i> as control. (A2, A5, A8, & A11) <i>UAS-Tap42^RNAi/+; pnr-Gal4/+</i>. (A3, A6, A9, & A12) <i>ap-Gal4/UAS-Tap42^RNAi; +/+</i>. (B1 & B2) wild type <i>w¹¹¹⁸</i>.</p

Suppression of Tap42 expression in wing imaginal discs interrupts HH signaling, hampers mitosis, and triggers apoptosis.

<p>Panel A: Isolated wing imaginal discs were immunostained with antibodies recognizing Tap42 (green) and multiple components in the HH signaling pathway, including Ptc, Smo, and Ci (red). Control wing discs displayed strong Tap42 (A1) expression and the expected expression pattern for Ptc (B1), Smo (C1), and Ci (D1). Suppression of <i>Tap42</i> with the <i>pnr-Gal4</i> or ap<i>-Gal4</i> driver effectively reduced Tap42 levels in wing discs (A2 & A3). While the levels of the HH receptor Ptc were unaffected by Tap42 silencing (B3), the expression of other downstream components of HH signaling, Smo (C3) and Ci (D3), were abrogated. Suppression of Tap42 with the <i>pnr-Gal4</i> driver did not alter the expression pattern of HH signaling as shown in B2 (Ptc), C2 (Smo) and D2 (Ci). Genotypes: (A1, B1, C1, & D1) <i>UAS-Tap42^RNAi/+</i> as control. (A2, B2, C2, & D2) <i>UAS-Tap42^RNAi/+; pnr-Gal4/+</i>. (A3, B3, C3, & D3) <i>ap-Gal4/UAS-Tap42^RNAi; +/+</i>.</p