Macrophages exposed continuously to lipopolysaccharide and other agonists that act via toll-like receptors exhibit a sustained and additive activation state

BACKGROUND: Macrophages sense microorganisms through activation of members of the Toll-like receptor family, which initiate signals linked to transcription of many inflammation associated genes. In this paper we examine whether the signal from Toll-like receptors [TLRs] is sustained for as long as the ligand is present, and whether responses to different TLR agonists are additive. RESULTS: RAW264 macrophage cells were doubly-transfected with reporter genes in which the IL-12p40, ELAM or IL-6 promoter controls firefly luciferase, and the human IL-1β promoter drives renilla luciferase. The resultant stable lines provide robust assays of macrophage activation by TLR stimuli including LPS [TLR4], lipopeptide [TLR2], and bacterial DNA [TLR9], with each promoter demonstrating its own intrinsic characteristics. With each of the promoters, luciferase activity was induced over an 8 hr period, and thereafter reached a new steady state. Elevated expression required the continued presence of agonist. Sustained responses to different classes of agonist were perfectly additive. This pattern was confirmed by measuring inducible cytokine production in the same cells. While homodimerization of TLR4 mediates responses to LPS, TLR2 appears to require heterodimerization with another receptor such as TLR6. Transient expression of constitutively active forms of TLR4 or TLR2 plus TLR6 stimulated IL-12 promoter activity. The effect of LPS, a TLR4 agonist, was additive with that of TLR2/6 but not TLR4, whilst that of lipopeptide, a TLR2 agonist, was additive with TLR4 but not TLR2/6. Actions of bacterial DNA were additive with either TLR4 or TLR2/6. CONCLUSIONS: These findings indicate that maximal activation by any one TLR pathway does not preclude further activation by another, suggesting that common downstream regulatory components are not limiting. Upon exposure to a TLR agonist, macrophages enter a state of sustained activation in which they continuously sense the presence of a microbial challenge

Nonsteroidal Antiinflammatory Drug Use and Association With Incident Hypertension in Ankylosing Spondylitis.

ObjectiveNonsteroidal antiinflammatory drugs (NSAIDs) increase blood pressure and potentially cardiovascular burden, which may limit their use in ankylosing spondylitis (AS). Our objective was to determine the association of NSAID use with incident hypertension in a longitudinal AS cohort.MethodsAdults with AS were enrolled in a prospective cohort study of patient outcomes and examined every 4-6 months. Hypertension was defined by patient-reported hypertension; antihypertensive medication use; or, on 2 consecutive visits, systolic blood pressure ≥140 mm Hg or diastolic blood pressure ≥90 mm Hg. Continuous NSAID use was dichotomized based on the validated NSAID index. We assessed the association of NSAID use as a time-varying exposure with the incidence of hypertension using Cox proportional hazards models.ResultsOf the 1,282 patients in the cohort, 628 patients without baseline hypertension had at least 1 year of follow-up and were included in the analysis. Of these, 72% were male, the mean age at baseline was 39 ± 13 years, and 200 patients used NSAIDs continuously. On follow-up, 129 developed incident hypertension. After controlling for other variables, continuous NSAID use was associated with a hazard ratio of 1.12 for incident hypertension (95% confidence interval 1.04-1.20), compared to noncontinuous or no use. The association did not differ in subgroups defined by age, body mass index, biologic use, or disease activity.ConclusionIn our prospective, longitudinal AS cohort, continuous NSAID use was associated with a 12% increased risk for the development of incident hypertension, as compared to noncontinuous or no NSAID use

Statistical and trend analysis of annual maximum daily rainfall (AMDR) for Kuching City, Sarawak, Malaysia

Kuching city and its surrounding urban areas frequently experience extreme high annual maximum daily rainfall (AMDR), resulting in flash floods. This study aims to carry out statistical and trend analysis of extreme AMDR events for Kuching Airport rainfall station from 1975 to 2017. From the analysis, the AMDR records a high variability with a value of 36.9% while January has the highest occurrence of AMDR with 53.5% of the total data. Findings from the linear regression plot have shown that the AMDR has a slight decreasing trend over the past four decades though the trend was insignificant. Based on the drainage design capacity of Kuching city, AMDR of magnitude 180 mm was identified as a threshold. The frequency analysis results showed that the return period of flooding events with daily rainfall exceeding 180 mm was 2.69 years. The occurrence probability of the flood event at least once in 1, 2, 3, 4 and 5 years was 0.37, 0.60, 0.75, 0.84 and 0.90, respectively. This study contributed to understanding the magnitude and frequency of extreme high AMDR which could lead to flooding events in Kuching city

Chromosomal-level assembly of the Asian Seabass genome using long sequence reads and multi-layered scaffolding

We report here the ~670 Mb genome assembly of the Asian seabass (Lates calcarifer), a tropical marine teleost. We used long-read sequencing augmented by transcriptomics, optical and genetic mapping along with shared synteny from closely related fish species to derive a chromosome-level assembly with a contig N50 size over 1 Mb and scaffold N50 size over 25 Mb that span ~90% of the genome. The population structure of L. calcarifer species complex was analyzed by re-sequencing 61 individuals representing various regions across the species' native range. SNP analyses identified high levels of genetic diversity and confirmed earlier indications of a population stratification comprising three clades with signs of admixture apparent in the South-East Asian population. The quality of the Asian seabass genome assembly far exceeds that of any other fish species, and will serve as a new standard for fish genomics

Pyruvate kinase M2 activation may protect against the progression of diabetic glomerular pathology and mitochondrial dysfunction

Diabetic nephropathy (DN) is a major cause of end-stage renal disease, and therapeutic options for preventing its progression are limited. To identify novel therapeutic strategies, we studied protective factors for DN using proteomics on glomeruli from individuals with extreme duration of diabetes (≥ 50 years) without DN and those with histologic signs of DN. Enzymes in the glycolytic, sorbitol, methylglyoxal and mitochondrial pathways were elevated in individuals without DN. In particular, pyruvate kinase M2 (PKM2) expression and activity were upregulated. Mechanistically, we showed that hyperglycemia and diabetes decreased PKM2 tetramer formation and activity by sulfenylation in mouse glomeruli and cultured podocytes. Pkm-knockdown immortalized mouse podocytes had higher levels of toxic glucose metabolites, mitochondrial dysfunction and apoptosis. Podocyte-specific Pkm2-knockout (KO) mice with diabetes developed worse albuminuria and glomerular pathology. Conversely, we found that pharmacological activation of PKM2 by a small-molecule PKM2 activator, TEPP-46, reversed hyperglycemia-induced elevation in toxic glucose metabolites and mitochondrial dysfunction, partially by increasing glycolytic flux and PGC-1α mRNA in cultured podocytes. In intervention studies using DBA2/J and Nos3 (eNos) KO mouse models of diabetes, TEPP-46 treatment reversed metabolic abnormalities, mitochondrial dysfunction and kidney pathology. Thus, PKM2 activation may protect against DN by increasing glucose metabolic flux, inhibiting the production of toxic glucose metabolites and inducing mitochondrial biogenesis to restore mitochondrial function

The HicA toxin from Burkholderia pseudomallei has a role in persister cell formation

© 2014 The Authors Journal compilation. ©2014 Biochemical Society.This is an open access article that is freely available in ORE or from the publisher's website. Please cite the published version.Published by Portland Press on behalf of the Biochemical SocietyTA (toxin-antitoxin) systems are widely distributed amongst bacteria and are associated with the formation of antibiotic tolerant (persister) cells that may have involvement in chronic and recurrent disease. We show that overexpression of the Burkholderia pseudomallei HicA toxin causes growth arrest and increases the number of persister cells tolerant to ciprofloxacin or ceftazidime. Furthermore, our data show that persistence towards ciprofloxacin or ceftazidime can be differentially modulated depending on the level of induction of HicA expression. Deleting the hicAB locus from B. pseudomallei K96243 significantly reduced persister cell frequencies following exposure to ciprofloxacin, but not ceftazidime. The structure of HicA(H24A) was solved by NMR and forms a dsRBD-like (dsRNA-binding domain-like) fold, composed of a triple-stranded β-sheet, with two helices packed against one face. The surface of the protein is highly positively charged indicative of an RNA-binding protein and His24 and Gly22 were functionality important residues. This is the first study demonstrating a role for the HicAB system in bacterial persistence and the first structure of a HicA protein that has been experimentally characterized.Wellcome Trus

Inflammatory profiles across the spectrum of disease reveal a distinct role for GM-CSF in severe COVID-19

While it is now widely accepted that host inflammatory responses contribute to lung injury, the pathways that drive severity and distinguish coronavirus disease 2019 (COVID-19) from other viral lung diseases remain poorly characterized. We analyzed plasma samples from 471 hospitalized patients recruited through the prospective multicenter ISARIC4C study and 39 outpatients with mild disease, enabling extensive characterization of responses across a full spectrum of COVID-19 severity. Progressive elevation of levels of numerous inflammatory cytokines and chemokines (including IL-6, CXCL10, and GM-CSF) were associated with severity and accompanied by elevated markers of endothelial injury and thrombosis. Principal component and network analyses demonstrated central roles for IL-6 and GM-CSF in COVID-19 pathogenesis. Comparing these profiles to archived samples from patients with fatal influenza, IL-6 was equally elevated in both conditions whereas GM-CSF was prominent only in COVID-19. These findings further identify the key inflammatory, thrombotic, and vascular factors that characterize and distinguish severe and fatal COVID-19

A Systematic Review of the Efficacy and Toxicity of Brachytherapy Boost Combined with External Beam Radiotherapy for Nonmetastatic Prostate Cancer

CONTEXT: The optimum use of brachytherapy (BT) combined with external beam radiotherapy (EBRT) for localised/locally advanced prostate cancer (PCa) remains uncertain.OBJECTIVE: To perform a systematic review to determine the benefits and harms of EBRT-BT.EVIDENCE ACQUISITION: Ovid MEDLINE, Embase, and EBM Reviews-Cochrane Central Register of Controlled Trials databases were systematically searched for studies published between January 1, 2000 and June 7, 2022, according to the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) statement. Eligible studies compared low- or high-dose-rate EBRT-BT against EBRT ± androgen deprivation therapy (ADT) and/or radical prostatectomy (RP) ± postoperative radiotherapy (RP ± EBRT). The main outcomes were biochemical progression-free survival (bPFS), severe late genitourinary (GU)/gastrointestinal toxicity, metastasis-free survival (MFS), cancer-specific survival (CSS), and overall survival (OS), at/beyond 5 yr. Risk of bias was assessed and confounding assessment was performed. A meta-analysis was performed for randomised controlled trials (RCTs).EVIDENCE SYNTHESIS: Seventy-three studies were included (two RCTs, seven prospective studies, and 64 retrospective studies). Most studies included participants with intermediate-or high-risk PCa. Most studies, including both RCTs, used ADT with EBRT-BT. Generally, EBRT-BT was associated with improved bPFS compared with EBRT, but similar MFS, CSS, and OS. A meta-analysis of the two RCTs showed superior bPFS with EBRT-BT (estimated fixed-effect hazard ratio [HR] 0.54 [95% confidence interval {CI} 0.40-0.72], p &lt; 0.001), with absolute improvements in bPFS at 5-6 yr of 4.9-16%. However, no difference was seen for MFS (HR 0.84 [95% CI 0.53-1.28], p = 0.4) or OS (HR 0.87 [95% CI 0.63-1.19], p = 0.4). Fewer studies examined RP ± EBRT. There is an increased risk of severe late GU toxicity, especially with low-dose-rate EBRT-BT, with some evidence of increased prevalence of severe GU toxicity at 5-6 yr of 6.4-7% across the two RCTs.CONCLUSIONS: EBRT-BT can be considered for unfavourable intermediate/high-risk localised/locally advanced PCa in patients with good urinary function, although the strength of this recommendation based on the European Association of Urology guideline methodology is weak given that it is based on improvements in biochemical control.PATIENT SUMMARY: We found good evidence that radiotherapy combined with brachytherapy keeps prostate cancer controlled for longer, but it could lead to worse urinary side effects than radiotherapy without brachytherapy, and its impact on cancer spread and patient survival is less clear.</p

A Systematic Review of the Efficacy and Toxicity of Brachytherapy Boost Combined with External Beam Radiotherapy for Nonmetastatic Prostate Cancer

CONTEXT: The optimum use of brachytherapy (BT) combined with external beam radiotherapy (EBRT) for localised/locally advanced prostate cancer (PCa) remains uncertain.OBJECTIVE: To perform a systematic review to determine the benefits and harms of EBRT-BT.EVIDENCE ACQUISITION: Ovid MEDLINE, Embase, and EBM Reviews-Cochrane Central Register of Controlled Trials databases were systematically searched for studies published between January 1, 2000 and June 7, 2022, according to the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) statement. Eligible studies compared low- or high-dose-rate EBRT-BT against EBRT ± androgen deprivation therapy (ADT) and/or radical prostatectomy (RP) ± postoperative radiotherapy (RP ± EBRT). The main outcomes were biochemical progression-free survival (bPFS), severe late genitourinary (GU)/gastrointestinal toxicity, metastasis-free survival (MFS), cancer-specific survival (CSS), and overall survival (OS), at/beyond 5 yr. Risk of bias was assessed and confounding assessment was performed. A meta-analysis was performed for randomised controlled trials (RCTs).EVIDENCE SYNTHESIS: Seventy-three studies were included (two RCTs, seven prospective studies, and 64 retrospective studies). Most studies included participants with intermediate-or high-risk PCa. Most studies, including both RCTs, used ADT with EBRT-BT. Generally, EBRT-BT was associated with improved bPFS compared with EBRT, but similar MFS, CSS, and OS. A meta-analysis of the two RCTs showed superior bPFS with EBRT-BT (estimated fixed-effect hazard ratio [HR] 0.54 [95% confidence interval {CI} 0.40-0.72], p &lt; 0.001), with absolute improvements in bPFS at 5-6 yr of 4.9-16%. However, no difference was seen for MFS (HR 0.84 [95% CI 0.53-1.28], p = 0.4) or OS (HR 0.87 [95% CI 0.63-1.19], p = 0.4). Fewer studies examined RP ± EBRT. There is an increased risk of severe late GU toxicity, especially with low-dose-rate EBRT-BT, with some evidence of increased prevalence of severe GU toxicity at 5-6 yr of 6.4-7% across the two RCTs.CONCLUSIONS: EBRT-BT can be considered for unfavourable intermediate/high-risk localised/locally advanced PCa in patients with good urinary function, although the strength of this recommendation based on the European Association of Urology guideline methodology is weak given that it is based on improvements in biochemical control.PATIENT SUMMARY: We found good evidence that radiotherapy combined with brachytherapy keeps prostate cancer controlled for longer, but it could lead to worse urinary side effects than radiotherapy without brachytherapy, and its impact on cancer spread and patient survival is less clear.</p

Benefits and Risks of Primary Treatments for High-risk Localized and Locally Advanced Prostate Cancer : An International Multidisciplinary Systematic Review

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