Background There are minimal data on the differences in demographics, clinical presentations and outcomes for patients with different types and sub-types of influenza in the Middle East. Objectives To use population-based data from Iran to investigate factors associated with unfavorable disease outcome. Study Design Clinical data were compiled from the Iranian Ministry of Health for patients of all ages who fulfilled the severe acute respiratory infections (SARI) definition according to World Health Organization criteriatested for any reason and found to have and had laboratory proven influenza September 21, 2015 through March 20, 2018. Pulmonary, cardiac, renal, hematologic and neurologic complications were recorded. Results were compared by type, age, gender and health status. Multivariate analysis was used to analyze risk factors for complications and death. Results Of 11080 enrolled patients, 10046 (90.7%) were inpatients, 2254 (20.4%) were children, 8403 (75.8%) had influenza A, 2599 (23.5%) had influenza B, and 78 (0.7%) had unidentified types. Fever was less common in older patients (OR 0.99; 95% CI 0.98-0.99, p<0.001 and in those with comorbidity (OR 0.87; 95% CI 0.77-0.97, p=0.013). Although the rate of complications was lower with A(H1N1) pdm09 influenza than with A(H3N2) infection (12.8% versus 15.6%, p=0.001), the mortality rate was higher (7.0% versus 3.0%, p<0.001). Complications occurred more often during late versus early influenza season (OR 1.22; 95% CI 1.08-1.37, p=0.002). Patients with type B influenza (OR 0.85; 95% CI 0.74-0.98, p=0.025), or who presented with sore throat (OR 0.74; 95% CI 0.65-0.84, p<0.001) were less likely to develop complications. The risk of developing complications was increased in patients who had chronic heart disease (OR 1.51; 95% CI 1.29-1.76, p<0.001), chronic pulmonary disease (OR 1.62; 95% CI 1.37-1.91, p<0.001), diabetes (OR 1.24; 95% CI 1.03-1.50, p=022), or epilepsy (OR 1.55; 95% CI 1.17-2.05). Older age and male gender increased the risk of death but not of complications. Conclusions The clinical features, complications and outcomes of influenza vary by age and by viral type and sub-type. Comorbidites appear to be more important than age in predicting complications.

Background The performance of recently approved point-of-care (POC) HIV tests should be assessed using unprocessed specimens. Objective: To evaluate the sensitivity and specificity of four POC HIV tests using whole blood (WB) and two using oral fluid (OF) among persons recruited from health clinics in Seattle, Washington, during September 2015-September 2017. Studydesign Participants were tested with the POC tests, additional plasma and serum were collected for laboratory testing, and participant- reported use of antiretroviral therapy (ART) or pre-exposure prophylaxis (PrEP) was recorded. Participants testing negative on all tests could reenroll every 90 days. Specimens from persons previously diagnosed with HIV infection as well as from those who were newly diagnosed during the study were included in the sensitivity estimate. Sensitivity and specificity were calculated based on HIV status determined by laboratory testing. Results Of 1,256 visits, 179 were from persons with HIV infection; 120 of these were taking ART. Among 1,077 visits from participants not diagnosed with HIV, PrEP use was reported at 155 (14.4%) visits. Sensitivity was similar among POC WB tests (95.53%-97.21%; p>0.05). Among participants on ART, sensitivity was lower for the same test performed on OF compared to WB (p<0.003). Specificity was high for all tests (99.44%- 100.00%); we did not detect specificity differences with PrEP use. Conclusions These POC tests displayed relatively high sensitivity and specificity using unprocessed specimens, suggesting their effectiveness in identifying HIV infections whenever laboratory-based testing is not feasible. Nonetheless, clients with recent risk should retest to rule out the possibility of a false-negative result.

Previous studies of fetal death with maternal influenza have been inconsistent. We explored the effect of maternal influenza-like illness (ILI) in pregnancy on the risk of fetal death, distinguishing between diagnoses during regular influenza seasons and the 2009/2010 pandemic and between trimesters of ILI. We used birth records from the Medical Birth Registry of Norway to identify fetal deaths after the first trimester in singleton pregnancies (2006–2013). The Norwegian Directorate of Health provided dates of clinical influenza diagnoses by primary-health-care providers, whereas dates of laboratory-confirmed influenza A (H1N1) diagnoses were provided by the Norwegian Surveillance System for Communicable Diseases. We obtained dates and types of influenza vaccinations from the Norwegian Immunisation Registry. Cox proportional-hazards regression models were fitted to estimate hazard ratios (HRs) of fetal death, with associated 95% confidence intervals (CIs), comparing women with and without an ILI diagnosis in pregnancy. There were 2510 fetal deaths among 417,406 eligible pregnancies. ILI during regular seasons was not associated with increased risk of fetal death: adjusted HR = 0.90 (95% CI 0.64–1.27). In contrast, ILI during the pandemic was associated with substantially increased risk of fetal death, with an adjusted HR of 1.75 (95% CI 1.21–2.54). The risk was highest following first-trimester ILI (adjusted HR = 2.28 [95% CI 1.45–3.59]). ILI during the pandemic—but not during regular seasons—was associated with increased risk of fetal death in the second and third trimester. The estimated effect was strongest with ILI in first trimester.

Abstract Particulate matter from natural sources such as desert dust causes harmful effects for health. Asian dust (AD) increases the risk of acute myocardial infarction (AMI). However, little is known about the risk of myocardial infarction with nonobstructive coronary arteries (MINOCA), compared to myocardial infarction with coronary artery disease (MI-CAD). Using a time-stratified case-crossover design and conditional logistic regression models, the association between short-term exposure to AD whereby decreased visibility (< 10 km) observed at each monitoring station nearest to the hospitals was used for exposure measurements and admission for AMI in the spring was investigated using a nationwide administrative database between April 2012 and March 2016. According to presence of revascularization and coronary atherosclerosis, AMI patients (n = 30,435) were divided into 2 subtypes: MI-CAD (n = 27,202) or MINOCA (n = 3233). The single lag day-2 was used in AD exposure based on the lag effect analysis. The average level of meteorological variables and co-pollutants on the 3 days prior to the case/control days were used as covariates. The occurrence of AD events 2 days before the admission was associated with admission for MINOCA after adjustment for meteorological variables [odds ratio 1.65; 95% confidence interval (CI) 1.18–2.29], while the association was not observed in MI-CAD. The absolute risk difference of MINOCA admission was 1.79 (95% CI 1.21–2.38) per 100,000 person-year. These associations between AD exposure and the admission for MINOCA remained unchanged in two-pollutant models. This study provides evidence that short-term exposure to AD is associated with a higher risk of MINOCA, but not MI-CAD.

Polydnaviruses are obligate mutualists of parasitoid wasps and are divided into two genera, Bracovirus and Ichnovirus. Bracoviruses are thought to originate from a single integration of an ancestral nudivirus into the ancestor of microgastroid complex ∼100 million years ago. However, all the known nudiviruses are only distantly related to bracoviruses, and much remains obscure about the origin of bracoviruses. Here we employ a paleovirological method to screen endogenous nudivirus-like elements across arthropods. Interestingly, we identify many endogenous nudivirus-like elements within the genome of Eurytoma brunniventris, a species of the Chalcidoidea superfamily. Among them, we find 14 core gene sequences are likely to be derived from a betanudivirus (designated EbrENV-β), suggesting that betanudivirus has been circulating in parasitoid wasps. Phylogenomic analysis suggests that EbrENV-β is the known closest relative of bracoviruses. Synteny analyses show the order of core genes is not well conserved between EbrENV-β and nudiviruses, revealing the dynamic nature of the evolution of nudivirus genome structures. Our findings narrow down the evolutionary gap between bracoviruses and nudiviruses and provide novel insights into the origin and evolution of polydnaviruses.

MotivationAn intuitive graphical interface that allows statistical analyses and visualizations of extensive data without any knowledge of dedicated statistical software or programming. Implementation EpiMetal is a single-page web application written in JavaScript, to be used via a modern desktop web browser. General features Standard epidemiological analyses and self-organizing maps for data-driven metabolic profiling are included. Multiple extensive datasets with an arbitrary number of continuous and category variables can be integrated with the software. Any snapshot of the analyses can be saved and shared with others via a www-link. We demonstrate the usage of EpiMetal using pilot data with over 500 quantitative molecular measures for each sample as well as in two large-scale epidemiological cohorts (N >10 000). AvailabilityThe software usage exemplar and the pilot data are open access online at [http://EpiMetal.computationalmedicine.fi]. MIT licensed source code is available at the Github repository at [https://github.com/amergin/epimetal].

Zika virus (ZIKV) NS2B-NS3 protease is a validated antiviral target as it is essential for maturation of viral proteins. However, its negatively charged active site hinders the development of orthosteric small-molecule inhibitors. Fragment-based drug discovery (FBDD) is a powerful tool to generate novel chemical starting points against difficult drug targets. In this study, we screened a fragment compound library against the Zika protease using a primary thermal shift assay and identified twenty-two fragments which (bind to and) stabilize the protease. We then determined the X-ray crystal structures of two hits from different classes, all of which bind to the S1 pocket next to the protease active site. We confirmed that these two fragments bind to the protease without inducing significant conformational changes using solution NMR spectroscopy. These fragment scaffolds serve as the starting point for subsequent lead compound development.

The immunoproteasome contains three catalytic subunits (β1i, β2i and β5i) that are important modulators of immune cell homeostasis. A previous study showed a correlation between β5i and human atherosclerotic plaque instability; however, the causative role of β5i in atherosclerosis and the underlying mechanisms remain unknown. Here we explored this issue in apolipoprotein E (Apoe) knockout (eKO) mice with genetic deletion or pharmacological inhibition of β5i. We found that β5i expression was upregulated in lesional macrophages after an atherogenic diet (ATD). β5i/Apoe double KO (dKO) mice fed on the ATD had a significant decrease in both lesion area and necrotic core area, compared with eKO controls. Moreover, dKO mice had less caspase‐3+ apoptotic cell accumulation but enhanced efferocytosis of apoptotic cells and increased expression of Mer receptor tyrosine kinase (MERTK). Consistently, similar phenotypes were observed in eKO mice transplanted with dKO bone marrow or treated with β5i‐specific inhibitor PR‐957. Mechanistic studies in vitro revealed that β5i deletion reduced IκBα degradation and inhibited NF‐κB activation, promoting Mertk transcription and efferocytosis, thereby attenuating apoptotic cell accumulation. In conclusion, we demonstrate that β5i plays an important role in diet‐induced atherosclerosis by altering MERTK‐mediated efferocytosis. β5i might be a potential pharmaceutical target against atherosclerosis. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Psoriasis is a chronic inflammatory skin disease, in which dendritic cells (DCs) play a pivotal role by inducing Th1/Th17 immune responses; however, the regulation of DCs activation in psoriasis remains largely unknown. Previously we found that the level of soluble CD100 was increased in sera of psoriasis patients, and CD100 promoted the activation of inflammasome in keratinocytes. In the present study, CD100 knockout mice were utilized for generation of imiquimod (IMQ)‐induced psoriatic dermatitis, with the result that skin inflammation in the early, but not late, phase of the psoriatic dermatitis was significantly exacerbated compared to that in wild‐type controls. This was attributed mainly to the deficiency of CD100 in hematopoietic cells. Bone marrow‐derived DCs, but not T cells or keratinocytes, from CD100 knockout mice produced significantly increased levels of IL‐1β, IL‐36 and IL‐23 upon stimulation with IMQ in a Plexin‐B2‐dependent manner. Moreover, the surface level of Plexin‐B2 on DCs of psoriasis patients was lower than that of healthy individuals, and CD100 attenuated IMQ‐induced production of IL‐1β and IL‐36 from monocyte‐derived DCs of psoriasis patients. Our results uncovered a negative regulatory mechanism for DCs activation in psoriasis, which was mediated via CD100‐Plexin‐B2 in a cell type‐ and receptor‐ specific manner.

Horizontal pollen transmission by the raspberry bushy dwarf virus 1b deletion mutant (RBΔ1bstop), which is defective in virus virulence, was significantly decreased compared to wild-type raspberry bushy dwarf virus (wtRBDV). We assessed accumulation of viral genomic (g) RNAs in pollen grains from RBΔ1bstop-infected plants and found that the pollen grains had less viral gRNA than those from wtRBDV-infected plants. In addition, pollen grains from 1b-expressing transgenic plants (1b-plants) infected with RBΔ1bstop were more efficient in horizontal virus transmission to healthy plants after pollination than pollen from RBΔ1bstop-infected wild type plants. Moreover, viral gRNA accumulation in pollen grains from RBΔ1bstop-infected 1b-plants was higher than in pollen from RBΔ1bstop-infected wild type plants. We suggest that 1b increases the amount of viral gRNAs released from elongating pollen grains.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Kidney fibrosis is characterized by expansion and activation of platelet‐derived growth factor receptor‐β (PDGFR‐β)‐positive mesenchymal cells. To study the consequences of PDGFR‐β activation, we developed a model of primary renal fibrosis using transgenic mice with PDGFR‐β activation specifically in renal mesenchymal cells, driving their pathological proliferation and phenotypic switch toward myofibroblasts. This resulted in progressive mesangioproliferative glomerulonephritis, mesangial sclerosis, and interstitial fibrosis with progressive anemia due to loss of erythropoietin production by fibroblasts. Fibrosis induced secondary tubular epithelial injury at later stages, coinciding with microinflammation, and aggravated the progression of hypertensive and obstructive nephropathy. Inhibition of PDGFR activation reversed fibrosis more effectively in the tubulointerstitium compared to glomeruli. Gene expression signatures in mice with PDGFR‐β activation resembled those found in patients. In conclusion, PDGFR‐β activation alone is sufficient to induce progressive renal fibrosis and failure, mimicking key aspects of chronic kidney disease in humans. Our data provide direct proof that fibrosis per se can drive chronic organ damage and establish a model of primary fibrosis allowing specific studies targeting fibrosis progression and regression.

Pregnancy associated atypical hemolytic uremic syndrome (p-aHUS) is a disease with a triad of hemolytic anemia, thrombocytopenia and acute renal failure, which might be attributed to the uncontrolled complement activation. Herein, we sequenced a postpartum-aHUS patient and found the two missense variants of CD46, a novel mutation (c.403G>C, p.G135R) from her father and a once reported mutation (c.293C>T, p.T98I) without expressional and functional tests from her mother. The G135R mutation caused a significantly reduced membrane expression of CD46 in peripheral blood lymphocyte and renal cells. The T98I mutation caused a mild decrease membrane expression of CD46 in peripheral blood lymphocyte cells. Moreover, the expressed G135R protein was in precursor form, indicating that this mutant was retained intracellularly. The C3b binding ability of T98I mutant was slightly decreased while the C4b binding ability is not significantly changed. The cofactor ability of the two mutants for factor I in the degradation of C3b was demonstrated to be impaired. This study reported the first case of a four-generation postpartum-aHUS pedigree with isolated CD46 variants and the detailed disease progression, treatment, and prognosis provided more meaningful information for the understanding the disease.

Background Non-structural protein 1 (NS1) of dengue virus circulates in the serum of patients during the acute phase of the disease. Objectives To determine whether NS1 screening can serve in diagnosing primary and secondary infection and to evaluate its utility as a marker for predicting the severity of dengue in children. Study design Patients ≤15 years of age hospitalized for dengue between 2012-2018, with NS1 determination (Panbio, Australia) were included. Clinical y laboratorial characteristics were collected in a standardized data table for analysis of correlation between serotypes, primary or secondary condition of infection, severity, and presence of NS1. Results Of 709 children hospitalized for dengue with NS1 determination, 479 (67.5%) had the positive test. Of the 378 primary cases, 320 (85%) were NS1 (+). while among the 242 secondary cases only 103 (42.5%) were NS1 (+) (p < 0001). Of the 479 patients with NS1 (+), 344 (72%) were warnig-signed cases (WSC) and 94 (19%) were severe cases (SC), being these figures 62% and 34%, in the NS1 negative patients respectively (p < 0.001). There was no difference in the frequency of WSC or SC between patients with NS1 positive or negative test in secondary dengue; however, in primary dengue, the figures were 68% vs 32% (p < 0.001), and 87% vs 12% (p < 0.001), respectively. Conclusions The presence of NS1 positive test is associated with the condition of infection (primary or secondary) and exhibited an increased risk of developing forms with warning signs or severe dengue in primary cases, but not in secondary cases.

Cytomegalovirus (CMV) drug resistance mutation maps are updated with recent information for polymerase inhibitors, the terminase inhibitor letermovir and the UL97 kinase inhibitor maribavir. Newly mapped mutations and their phenotypes provide more detail on cross-resistance properties and suggest the need to expand the CMV gene regions covered in diagnostic testing. Next-generation deep sequencing technology offers a more sensitive, higher resolution view of emerging antiviral resistance and is recommended for use in clinical trials. Issues of standardization and diagnostic utility in comparison with traditional Sanger sequencing remain unresolved. Quality control is important for the accurate and reproducible detection of mutant viral populations in clinical specimens.

Human cytomegalovirus (HCMV) infections are widespread among the human population. Infection is persistent and mostly asymptomatic, except in immunocompromised individuals, particularly transplant patients, where significant morbidity and mortality can occur. Currently approved drugs for treating HCMV-related disease [including ganciclovir (GCV), valganciclovir (VGCV), cidofovir (CDV) and foscarnet (FOS)] all target the viral DNA polymerase and suffer from dose-limiting toxicity and resistance issues. The most recently approved drug, letermovir (LMV), was approved only for prophylaxis in adult HCMV-seropositive stem cell transplant recipients. Although LMV is highly potent, high-grade resistance mutations in the terminase gene were shown to readily emerge in vitro and in treated patients. Therefore, there is a need for new drugs that can be used for combinatorial therapeutic and/or prophylactic regimens to counteract the emergence of resistant mutants. Filociclovir (FCV), also known as cyclopropavir or MBX-400, is a methylenecyclopropane nucleoside analog, which has successfully completed Phase I safety studies, and is now entering Phase II clinical efficacy studies for the treatment of HCMV-related disease in transplant patients. FCV is 10-fold more active than GCV against HCMV in vitro, and has activity against all human herpesviruses except HSV-1 and HSV-2. Recently, FCV was also shown to be highly potent against human adenoviruses. This activity spectrum suggests that FCV could be used to treat/prevent infection with several viruses that pose significant risk to transplant patients. The active triphosphate form of FCV (FCV-TP) reaches higher peak levels than GCV-TP in HCMV-infected cells, and exhibits about 10-fold higher affinity to HCMV DNA polymerase UL54. Furthermore, FCV was shown to retain activity against a panel of GCV-resistant HCMV isolates, suggesting that it could be a useful alternative therapy for treating patients infected with some GCV-resistant HCMV strains. This review summarizes the early discovery work of FCV and highlights the recent advances in the continued development of this clinical candidate.

Currently, therapies to treat chronic hepatitis B (CHB) infection are based on the use of interferon-α or nucleos(t)ide analogs (NAs) to prevent viral DNA synthesis by inhibiting the reverse transcriptase activity of the hepatitis B virus (HBV) polymerase (Pol). However, these therapies are not curative; thus, the development of novel anti-HBV agents is needed. In accordance with this unmet medical need, we devised a new target- and cell-based, high-throughput screening assay to identify novel small molecules that block the initial interaction of the HBV Pol with its replication template the viral pregenomic RNA (pgRNA). We screened approximately 110,000 small molecules for the ability to prevent HBV Pol recognition of the pgRNA 5′ epsilon (ε) stem-loop structure, identifying (Z)-2-(allylamino)-4-amino-N′-cyanothiazole-5-carboximidamide (AACC). Viral nucleocapsid-captured quantitative RT-PCR and Western blot results revealed that AACC significantly decreased encapsidated pgRNA levels and blocked capsid assembly without affecting core protein expression in stable HBV-replicating cells. As a result, both intra- and extracellular accumulation of viral DNA was strongly reduced. AACC treatment of HepG2-sodium taurocholate transporting polypeptide (NTCP) cells and primary human hepatocytes infected with cell culture- or patient-derived HBV isolates showed both time- and dose-dependent inhibition of infectious viral progeny and rcDNA production. Furthermore, AACC showed cross-genotypic activity against genotypes B, C, and D. Of note, AACC inhibited the viral replication of lamivudine and a capsid inhibitor-resistant HBV, and showed synergistic effects with NAs and a capsid inhibitor. In conclusion, we identified a novel class of compounds specifically targeting the ε-Pol interaction and thereby preventing the encapsidation of pgRNAs into viral capsids. This promising new HBV inhibitor class potently inhibits HBV amplification with distinct characteristics from existing NAs and other drugs currently under development, promising to add value to existing therapies for CHB.

Nipah virus (NiV) is a highly pathogenic zoonotic paramyxovirus that continues to cause outbreaks in humans characterized by high mortality and significant clinical sequelae in survivors. Currently, no therapeutics are approved for use in humans against NiV infection. Here, we report that 4ʹ-chloromethyl-2ʹ-deoxy-2ʹ-fluorocytidine (ALS-8112) inhibits NiV. ALS-8112 is the parent nucleoside of lumicitabine, which has been evaluated in phase I and II clinical trials to treat pediatric and adult respiratory syncytial virus infection. In this study, we tested ALS-8112 against NiV and other major human respiratory pneumo- and paramyxoviruses in 2 human lung epithelial cell lines, and demonstrated the ability of ALS-8112 to reduce infectious wild-type NiV yield by over 6 orders of magnitude with no apparent cytotoxicity. However, further cytotoxicity testing in primary cells and bone marrow progenitor cells indicated cytotoxicity at higher concentrations of ALS-8112. Our results warrant the evaluation of lumicitabine against NiV infection in relevant animal models.

The H3 subtype avian influenza virus (AIV) poses a threat to both animal and human health. In this study, phylogenetic analysis showed that the H3 AIVs had various genomic constellations and extensive reassortments, increasing genetic diversity and the emergence of new pathogenic viruses that might infect human beings. Molecular analysis demonstrated that the major molecular markers linked to drug resistance were identified in M genes of three studied viruses, and there might be wide range of resistant virus infections in poultry in the future. Although all the H3 viruses preferentially bound to the avian-type receptor, the growth kinetics experiments showed that the selected H3 viruses were capable of efficient replication in mammalian cells, suggesting a potential cross-species transmission of H3 viruses. Overall, our results emphasize the need for continued surveillance of H3 outbreaks and may also help us improve knowledge on H3 AIVs prevention and control.

Recent epidemiological studies have shown varying associations between coffee consumption and bladder cancer (BC). This research aims to elucidate the association between coffee consumption and BC risk by bringing together worldwide cohort studies on this topic. Coffee consumption in relation to BC risk was examined by pooling individual data from 12 cohort studies, comprising of 2601 cases out of 501,604 participants. Pooled multivariate hazard ratios (HRs), with corresponding 95% confidence intervals (CIs), were obtained using multilevel Weibull regression models. Furthermore, dose–response relationships were examined using generalized least squares regression models. The association between coffee consumption and BC risk showed interaction with sex (P-interaction < 0.001) and smoking (P-interaction = 0.001). Therefore, analyses were stratified by sex and smoking. After adjustment for potential confounders, an increased BC risk was shown for high (> 500 ml/day, equivalent to > 4 cups/day) coffee consumption compared to never consumers among male smokers (current smokers: HR = 1.75, 95% CI 1.27–2.42, P-trend = 0.002; former smokers: HR = 1.44, 95% CI 1.12–1.85, P-trend = 0.001). In addition, dose–response analyses, in male smokers also showed an increased BC risk for coffee consumption of more than 500 ml/day (4 cups/day), with the risk of one cup (125 ml) increment as 1.07 (95% CI 1.06–1.08). This research suggests that positive associations between coffee consumption and BC among male smokers but not never smokers and females. The inconsistent results between sexes and the absence of an association in never smokers indicate that the associations found among male smokers is unlikely to be causal and is possibly caused by residual confounding of smoking.

Screening to detect colorectal cancer (CRC) in an early or premalignant state is an effective method to reduce CRC mortality rates. Current stool‐based screening tests, e.g. fecal immunochemical test (FIT), have a suboptimal sensitivity for colorectal adenomas and difficulty distinguishing adenomas at high risk of progressing to cancer from those at lower risk. We aimed to identify stool protein biomarker panels that can be used for the early detection of high‐risk adenomas and CRC. Proteomics data (LC–MS/MS) were collected on stool samples from adenoma (n = 71) and CRC patients (n = 81) as well as controls (n = 129). Colorectal adenoma tissue samples were characterized by low‐coverage whole‐genome sequencing to determine their risk of progression based on specific DNA copy number changes. Proteomics data were used for logistic regression modeling to establish protein biomarker panels. In total, 15 of the adenomas (15.8%) were defined as high risk of progressing to cancer. A protein panel, consisting of haptoglobin (Hp), LAMP1, SYNE2, and ANXA6, was identified for the detection of high‐risk adenomas (sensitivity of 53% at specificity of 95%). Two panels, one consisting of Hp and LRG1 and one of Hp, LRG1, RBP4, and FN1, were identified for high‐risk adenomas and CRCs detection (sensitivity of 66% and 62%, respectively, at specificity of 95%). Validation of Hp as a biomarker for high‐risk adenomas and CRCs was performed using an antibody‐based assay in FIT samples from a subset of individuals from the discovery series (n = 158) and an independent validation series (n = 795). Hp protein was significantly more abundant in high‐risk adenoma FIT samples compared to controls in the discovery (p = 0.036) and the validation series (p = 9e‐5). We conclude that Hp, LAMP1, SYNE2, LRG1, RBP4, FN1, and ANXA6 may be of value as stool biomarkers for early detection of high‐risk adenomas and CRCs. © 2019 Authors. Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

CDGSH iron–sulfur domain‐containing protein 2 (Cisd2), a protein that declines in an age‐dependent manner, mediates lifespan in mammals. Cisd2 deficiency causes accelerated aging and shortened lifespan, whereas persistent expression of Cisd2 promotes longevity in mice. Alzheimer's disease (AD) is the most prevalent form of senile dementia and is without an effective therapeutic strategy. We investigated whether Cisd2 upregulation is able to ameliorate amyloid β (Aβ) toxicity and prevent neuronal loss using an AD mouse model. Our study makes three major discoveries. First, using the AD mouse model (APP/PS1 double transgenic mice), the dosage of Cisd2 appears to modulate the severity of AD phenotypes. Cisd2 overexpression (∼two‐fold) significantly promoted survival and alleviated the pathological defects associated with AD. Conversely, Cisd2 deficiency accelerated AD pathogenesis. Secondly, Cisd2 overexpression protected against Aβ‐mediated mitochondrial damage and attenuated loss of neurons and neuronal progenitor cells. Finally, an increase in Cisd2 shifted the expression profiles of a panel of genes that are dysregulated by AD toward the patterns observed in wild‐type mice. These findings highlight Cisd2‐based therapies as a potential disease‐modifying strategy for AD. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Arterial media calcification refers to ectopic mineralization in the arterial wall and favors arterial stiffness and cardiovascular events. Patients with chronic kidney disease (CKD), diabetes, or osteoporosis are highly vulnerable to the development of arterial media calcifications. Tissue non‐specific alkaline phosphatase (TNAP) is upregulated in calcified arteries and plays a key role in the degradation of the calcification inhibitor pyrophosphate into inorganic phosphate ions. A recent study published in The Journal of Pathology showed that an oral dosage of 10 or 30 mg/kg/day SBI‐425, a selective TNAP inhibitor, inhibited the development of arterial media calcification in mice with CKD, without affecting bone mineralization. Their results indicated that SBI‐425 is an effective and safe treatment for arterial media calcification. However, additional studies regarding the effect of TNAP‐inhibitor SBI‐425 on the progression and even the reversion of pre‐existing pathological arterial media calcifications without affecting physiological bone mineralization are deserved. Furthermore, investigating the extent to which SBI‐425 inhibits arterial calcification in a non‐CKD context would be of particular interest to treat this comorbidity in diabetes and osteoporosis patients. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Bone marrow‐derived monocyte–macrophages promote healing of injured tissue cooperatively with vasculogenic hematopoietic stem/progenitor cells. However, diabetes dysregulates hematopoiesis and attenuates bone marrow‐derived tissue‐reparative responses. In a recent issue of The Journal of Pathology, Barman et al extensively characterized myelopoietic responses in bone marrow following skin wounding in a type 2 model of diabetes. The study demonstrated that accumulation of monocyte–macrophages in the peripheral tissues is increased due to diabetic myelopoiesis that would oppose the reparative process following tissue injury. Interestingly, in this model, pathological myelopoiesis is independent of IL‐1β. The potential prophylactic and therapeutic implications of these data are discussed in terms of paracrine signaling, macrophage polarization, and hematopoietic stem cell mobilization/retention. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Endometrial carcinoma (EC) molecular classification based on four molecular subclasses identified in The Cancer Genome Atlas (TCGA) has gained relevance in recent years due to its prognostic utility and potential to predict benefit from adjuvant treatment. While most ECs can be classified based on a single classifier (POLE exonuclease domain mutations – POLEmut, MMR deficiency – MMRd, p53 abnormal – p53abn), a small but clinically relevant group of tumours harbour more than one molecular classifying feature and are referred to as ‘multiple‐classifier’ ECs. We aimed to describe the clinicopathological and molecular features of multiple‐classifier ECs with abnormal p53 (p53abn). Within a cohort of 3518 molecularly profiled ECs, 107 (3%) tumours displayed p53abn in addition to another classifier(s), including 64 with MMRd (MMRd–p53abn), 31 with POLEmut (POLEmut–p53abn), and 12 with all three aberrations (MMRd–POLEmut–p53abn). MMRd–p53abn ECs and POLEmut–p53abn ECs were mostly grade 3 endometrioid ECs, early stage, and frequently showed morphological features characteristic of MMRd or POLEmut ECs. 18/28 (60%) MMRd–p53abn ECs and 7/15 (46.7%) POLEmut–p53abn ECs showed subclonal p53 overexpression, suggesting that TP53 mutation was a secondary event acquired during tumour progression. Hierarchical clustering of TCGA ECs by single nucleotide variant (SNV) type and somatic copy number alterations (SCNAs) revealed that MMRd–p53abn tumours mostly clustered with single‐classifier MMRd tumours (20/23) rather than single‐classifier p53abn tumours (3/23), while POLEmut–p53abn tumours mostly clustered with single‐classifier POLEmut tumours (12/13) and seldom with single‐classifier p53abn tumours (1/13) (both p ≤ 0.001, chi‐squared test). Finally, the clinical outcome of patients with MMRd–p53abn and POLEmut–p53abn ECs [stage I 5‐year recurrence‐free survival (RFS) of 92.2% and 94.1%, respectively] was significantly different from single‐classifier p53abn EC (stage I RFS 70.8%, p = 0.024 and p = 0.050, respectively). Our results support the classification of MMRd–p53abn EC as MMRd and POLEmut–p53abn EC as POLEmut. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Klebsiella pneumoniae is a gram‐negative bacterium that is increasingly difficult to treat due to the emergence of multidrug resistant strains. In a recent article, Ding et al demonstrate that prekallikrein depletion in mice followed by intranasal instillation of K. pneumoniae leads to a reduced bacterial burden and prolonged host survival, together with evidence of reduced distant organ damage. These effects are apparently independent of the role of prekallikrein in the contact system, and are associated with transcriptional changes relevant to innate immunity in the lung, established prior to infection. This study highlights the importance of further investigating the role of prekallikrein and other contact cascade components in host defence to counter K. pneumoniae (and perhaps other pathogens), with an overall aim of identifying potential therapeutic targets relevant to pulmonary infection with such resistant pathogens.

Previous studies showed that red blood cell distribution width (RDW) can be used as a prognostic and diagnostic index in various non-hematological diseases, including severe infections and sepsis. Here, we provide a narrative review to summarize the findings of available studies investigating the relationship between RDW and sepsis. Current evidence supports that increased RDW on admission, both in adults and neonates, is associated with unfavorable outcomes on the short- and long-term. In patients with suspected sepsis, RDW has modest value for predicting positive blood culture. Accordingly, its diagnostic value for sepsis is limited. Dynamic changes of RDW are also associated with outcome of sepsis. Taken together, these results suggest that RDW could be used as a prognostic index in septic patients.

Atherosclerosis, a complex multifactorial disease, is the leading cause of acute cardiovascular events. Substantial evidence confirms that chronic stress plays a pivot role in the occurrence and development of atherosclerosis, but the specific mechanism remains unclear. Autophagy serves as a safeguard mechanism for sustaining cellular homeostasis via eliminating unnecessary or/and harmful components, and damaged organelles in response to various stress. An increasing number of studies indicate that autophagy plays vital roles in the development of atherosclerosis. Therefore, understanding the role of chronic stress in the regulation of autophagy may provide new insight into prevention and treatment atherosclerotic disease, especially with respect to emerging targeted therapy. In present review, we focus on changes in autophagic function under chronic stress and its relationship to atherosclerosis.

Interleukin-1β (IL-1β) is a vital cytokine that plays an important role in regulating immune responses to infectious challenges and sterile insults. In addition, two endogenous inhibitors of functional receptor binding, IL-1 receptor antagonist (IL-1Ra), complete the family. To gain biological activity, IL-1β requires processing by the protease caspase-1 and activation of inflammasomes. Numerous clinical association studies and experimental approaches have implicated members of the IL-1 family, their receptors, or components of the processing machinery in the underlying processes of cardiovascular diseases. Here, we summarize the current state of knowledge regarding the pro-inflammatory and disease-modulating role of the IL-1 family in aneurysm. We discuss clinical evidence, signalling pathway, and mechanism of action and last, lend a perspective on currently developing therapeutic strategies involving IL-1β in aneurysm.

Bladder cancer (BC) is the ninth most common malignant disease and ranks fourteenth in cancer mortality worldwide. Moreover, among cancers, the incidence and mortality of BC in males increased to the 6th and 9th place, respectively. The overall survival (OS) declines dramatically as the cancer progresses, especially when urothelial cells transition from noninvasive to invasive. It is well known that epithelial cells can acquire invasive properties and a propensity to metastasize through the epithelial-to-mesenchymal transition (EMT) process in tumourigenesis and progression. However, the potential molecular mechanisms and key pathways are still unclear. As the sequencing technology advances, long non-coding RNAs (lncRNAs) have been proven to play an important role in regulating biological processes and cellular pathways. Here, we reviewed important lncRNAs, such as H19, UCA1 and MALAT1, that participate in the malignant phenotype of BC and regulate EMT signalling networks in the invasion-metastasis cascade during BC development. We further discuss MALAT1, PCAT-1 and SPRY4-IT1, and also urine and blood exosomal H19 and PTENP as potential noninvasive biomarkers. Moreover, antisense oligonucleotides (ASOs) and a double-stranded DNA plasmid (BC-819) have been designed for use in preclinical cancer models and clinical trials in patients. Therefore, the results of investigations have gradually prompted the utility of lncRNAs.

No approved therapy exists for cancer‐associated cachexia. The colon‐26 mouse model of cancer cachexia mimics recent late‐stage clinical failures of anabolic anti‐cachexia therapy and was unresponsive to anabolic doses of diverse androgens, including the selective androgen receptor modulator (SARM) GTx‐024. The histone deacetylase inhibitor (HDACi) AR‐42 exhibited anti‐cachectic activity in this model. We explored combined SARM/AR‐42 therapy as an improved anti‐cachectic treatment paradigm. A reduced dose of AR‐42 provided limited anti‐cachectic benefits, but, in combination with GTx‐024, significantly improved body weight, hindlimb muscle mass, and grip strength versus controls. AR‐42 suppressed the IL‐6/GP130/STAT3 signaling axis in muscle without impacting circulating cytokines. GTx‐024‐mediated β‐catenin target gene regulation was apparent in cachectic mice only when combined with AR‐42. Our data suggest cachectic signaling in this model involves catabolic signaling insensitive to anabolic GTx‐024 therapy and a blockade of GTx‐024‐mediated anabolic signaling. AR‐42 mitigates catabolic gene activation and restores anabolic responsiveness to GTx‐024. Combining GTx‐024, a clinically established anabolic therapy, with AR‐42, a clinically evaluated HDACi, represents a promising approach to improve anabolic response in cachectic patients.

Diabetic retinopathy (DR) is a common complication of diabetes and leads to blindness. Anti‐VEGF is a primary treatment for DR. Its therapeutic effect is limited in non‐ or poor responders despite frequent injections. By performing a comprehensive analysis of the semaphorins family, we identified the increased expression of Sema4D during oxygen‐induced retinopathy (OIR) and streptozotocin (STZ)‐induced retinopathy. The levels of soluble Sema4D (sSema4D) were significantly increased in the aqueous fluid of DR patients and correlated negatively with the success of anti‐VEGF therapy during clinical follow‐up. We found that Sema4D/PlexinB1 induced endothelial cell dysfunction via mDIA1, which was mediated through Src‐dependent VE‐cadherin dysfunction. Furthermore, genetic disruption of Sema4D/PlexinB1 or intravitreal injection of anti‐Sema4D antibody reduced pericyte loss and vascular leakage in STZ model as well as alleviated neovascularization in OIR model. Moreover, anti‐Sema4D had a therapeutic advantage over anti‐VEGF on pericyte dysfunction. Anti‐Sema4D and anti‐VEGF also conferred a synergistic therapeutic effect in two DR models. Thus, this study indicates an alternative therapeutic strategy with anti‐Sema4D to complement or improve the current treatment of DR.

During obesity, macrophages infiltrate the breast tissue leading to low‐grade chronic inflammation, a factor considered responsible for the higher risk of breast cancer associated with obesity. Here, we formally demonstrate that breast epithelial cells acquire malignant properties when exposed to medium conditioned by macrophages derived from human healthy donors. These effects were mediated by the breast cancer oncogene IKKε and its downstream target—the serine biosynthesis pathway as demonstrated by genetic or pharmacological tools. Furthermore, amlexanox, an FDA‐approved drug targeting IKKε and its homologue TBK1, delayed in vivo tumour formation in a combined genetic mouse model of breast cancer and high‐fat diet‐induced obesity/inflammation. Finally, in human breast cancer tissues, we validated the link between inflammation–IKKε and alteration of cellular metabolism. Altogether, we identified a pathway connecting obesity‐driven inflammation to breast cancer and a potential therapeutic strategy to reduce the risk of breast cancer associated with obesity.

Somatic copy number alterations play a critical role in oncogenesis. Loss of chromosomal regions containing tumor suppressors can lead to collateral deletion of passenger genes. This can be exploited therapeutically if synthetic lethal partners of such passenger genes are known and represent druggable targets. Here, we report that VPS4B gene, encoding an ATPase involved in ESCRT‐dependent membrane remodeling, is such a passenger gene frequently deleted in many cancer types, notably in colorectal cancer (CRC). We observed downregulation of VPS4B mRNA and protein levels from CRC patient samples. We identified VPS4A paralog as a synthetic lethal interactor for VPS4B in vitro and in mouse xenografts. Depleting both proteins profoundly altered the cellular transcriptome and induced cell death accompanied by the release of immunomodulatory molecules that mediate inflammatory and anti‐tumor responses. Our results identify a pair of novel druggable targets for personalized oncology and provide a rationale to develop VPS4 inhibitors for precision therapy of VPS4B‐deficient cancers.

BackgroundE-values are a recently introduced approach to evaluate confounding in observational studies. We aimed to empirically assess the current use of E-values in published literature. MethodsWe conducted a systematic literature search for all publications, published up till the end of 2018, which cited at least one of two inceptive E-value papers and presented E-values for original data. For these case publications we identified control publications, matched by journal and issue, where the authors had not calculated E-values. ResultsIn total, 87 papers presented 516 E-values. Of the 87 papers, 14 concluded that residual confounding likely threatens at least some of the main conclusions. Seven of these 14 named potential uncontrolled confounders. 19 of 87 papers related E-value magnitudes to expected strengths of field-specific confounders. The median E-value was 1.88, 1.82, and 2.02 for the 43, 348, and 125 E-values where confounding was felt likely to affect the results, unlikely to affect the results, or not commented upon, respectively. The 69 case-control publication pairs dealt with effect sizes of similar magnitude. Of 69 control publications, 52 did not comment on unmeasured confounding and 44/69 case publications concluded that confounding was unlikely to affect study conclusions. ConclusionsFew papers using E-values conclude that confounding threatens their results, and their E-values overlap in magnitude with those of papers acknowledging susceptibility to confounding. Facile automation in calculating E-values may compound the already poor handling of confounding. E-values should not be a substitute for careful consideration of potential sources of unmeasured confounding. If used, they should be interpreted in the context of expected confounding in specific fields.

BackgroundIncreasing active transport is proposed as a means to address both health and environmental issues. However, the associations between specific modes, such as cycling, walking and public transport, and health outcomes remain unclear. We examined the association between mode of travel to work and mortality. MethodsCohort studies of the entire New Zealand working population were created using 1996, 2001 and 2006 censuses linked to mortality data. Mode of travel to work was that reported on census day, and causes of death examined were ischaemic heart disease and injury. Main analyses were Poisson regression models adjusted for socio-demographics. Sensitivity analyses included: additional adjustment for smoking in the 1996 and 2006 cohorts, and bias analysis about non-differential misclassification of cycling vs car use. ResultsWalking (5%) and cycling (3%) to work were uncommon. Compared with people reporting using motor vehicles to travel to work, those cycling had a reduced all-cause mortality (ACM) in the socio-demographic adjusted models RR 0.87 (0.77–0.98). Those walking (0.97, 0.90–1.04) and taking public transport (0.96, 0.88–1.05) had no substantive difference in ACM. No mode of transport was associated with detectable statistically significant reductions in cause-specific mortality. Sensitivity analyses found weaker associations when adjusting for smoking and stronger associations correcting for likely non-differential misclassification of cycling. ConclusionsThis large cohort study supports an association between cycling to work and reduced ACM, but found no association for walking or public-transport use and imprecise cause-specific mortality patterns.

Most epidemiological studies examine how risk factors relate to average difference in outcomes (linear regression) or odds of a binary outcome (logistic regression); they do not explicitly examine whether risk factors are associated differentially across the distribution of the health outcome investigated. This paper documents a phenomenon found repeatedly in the minority of epidemiological studies which do this (via quantile regression): associations between a range of established risk factors and body mass index (BMI) are progressively stronger in the upper ends of the BMI distribution. In this paper, we document this finding and provide illustrative evidence of it in the 1958 British birth cohort study. Associations of low childhood socio-economic position, high maternal weight, low childhood general cognition and adult physical inactivity with higher BMI are larger at the upper end of the BMI distribution, on both absolute and relative scales. For example, effect estimates for socio-economic position and childhood cognition were around three times larger at the 90th compared with 10th quantile, while effect estimates for physical inactivity were increasingly larger from the 50th to 90th quantiles, yet null at lower quantiles. We provide potential explanations for these findings and discuss implications. Risk factors may have larger causal effects among those in worse health, and these effects may not be discovered when health is only examined in average terms. In such scenarios, population-based approaches to intervention may have larger benefits than anticipated when assuming equivalent benefit across the population. Further research is needed to understand why effect estimates differ across the BMI outcome distribution and to investigate whether differential effects exist for other physical and mental health outcomes.

BackgroundSouth Africa is at the epicentre of the HIV pandemic, with the world's highest number of new infections and the largest treatment programme. Using metrics proposed by the Joint United Nations Programme on AIDS (UNAIDS), we evaluate progress toward epidemic control and highlight areas for intervention in a hyperendemic South African setting. MethodsThe Africa Health Research Institute (AHRI) maintains a comprehensive population-based surveillance system in the Hlabisa sub-district of KwaZulu-Natal. Between 2005 and 2017, we tested 39 735 participants (aged 15–49 years) for HIV and followed 22 758 HIV-negative and 13 460 HIV-positive participants to identify new infections and all-cause AIDS-related deaths, respectively. Using these data, we estimated the percentage reduction in incidence, the absolute incidence rate, the incidence-mortality ratio and the incidence-prevalence ratio over place and time. ResultsWe observed a 62% reduction in the number of new infections among men between 2012 and 2017 and a 34% reduction among women between 2014 and 2017. Among men, the incidence-mortality ratio peaked at 4.1 in 2013 and declined to 3.1 in 2017, and among women it fell from a high of 6.4 in 2014 to 4.3 in 2017. Between 2012 and 2017, the female-incidence/male-prevalence ratio declined from 0.24 to 0.13 and the male-incidence/female-prevalence ratio from 0.05 to 0.02. ConclusionsUsing data from a population-based cohort study, we report impressive progress toward HIV epidemic control in a severely affected South African setting. However, overall progress is off track for 2020 targets set by the UNAIDS. Spatial estimates of the metrics, which demonstrate remarkable heterogeneity over place and time, indicate areas that could benefit from additional or optimized HIV prevention services.

Neuropeptides are important signalling molecules in the brain and alterations in their expression levels have been linked to neurological disorders such as Parkinson's disease. It is challenging to map neuropeptide changes across and within brain regions because of their low in vivo concentrations and complex post-translational processing. Consequently, the role of neuropeptides in Parkinson's disease is not well understood. Thus, we have developed and validated a method to image multiple neuropeptides simultaneously in both rat and primate brain tissue sections by matrix-assisted laser desorption-ionization mass spectrometry imaging at high lateral resolution. Using a unilateral 6-hydroxydopamine rat model of Parkinson's disease, we imaged changes in enkephalins, dynorphins, tachykinins and neurotensin associated with the dopaminergic denervation and L-DOPA treatment in multiple brain regions. L-DOPA administration significantly affected neuropeptides in the globus pallidus, while neuropeptides in the caudate-putamen were mostly affected by dopamine depletion. Using high lateral resolution imaging, we observed an increase of neurotensin in the dorsal sub-region of the globus pallidus after dopamine depletion. This study highlights the capacity of mass spectrometry imaging to elucidate the dynamics of neuropeptide signalling during Parkinson's disease and its treatment.

This review aims to assemble many years of research and clinical experience in the fields of neurodevelopment and neuroscience to present an up-to-date understanding of the clinical presentation, molecular and brain pathology associated with Fragile X syndrome, a neurodevelopmental condition that develops with the full mutation of the FMR1 gene, located in the q27.3 loci of the X chromosome, and Fragile X-associated tremor/ataxia syndrome a neurodegenerative disease experienced by aging premutation carriers of the FMR1 gene. It is important to understand that these two syndromes have a very distinct clinical and pathological presentation while sharing the same origin: the mutation of the FMR1 gene; revealing the complexity of expansion genetics.

Structural and molecular myelination deficits represent early pathological features of Huntington disease (HD). Recent evidence from germ-free (GF) animals suggests a role for microbiota-gut-brain bidirectional communication in the regulation of myelination. In this study, we aimed to investigate the impact of microbiota on myelin plasticity and oligodendroglial population dynamics in the mixed-sex BACHD mouse model of HD. Ultrastructural analysis of myelin in the corpus callosum revealed alterations of myelin thickness in BACHD GF compared to specific-pathogen free (SPF) mice, whereas no differences were observed between wild-type (WT) groups. In contrast, myelin compaction was altered in all groups when compared to WT SPF animals. Levels of myelin-related proteins were generally reduced, and the number of mature oligodendrocytes was decreased in the prefrontal cortex under GF compared to SPF conditions, regardless of genotype. Minor differences in commensal bacteria at the family and genera levels were found in the gut microbiota of BACHD and WT animals housed in standard living conditions. Our findings indicate complex effects of a germ-free status on myelin-related characteristics, and highlight the adaptive properties of myelination as a result of environmental manipulation.

Rho GTPases play a central role in neuronal survival; however, the antagonistic relationship between Rac and Rho in the regulation of motor neuron survival remains poorly defined. In the current study, we demonstrate that treatment with NSC23766, a selective inhibitor of the Rac-specific guanine nucleotide exchange factors, Tiam1 and Trio, is sufficient to induce the death of embryonic stem cell (ESC)-derived motor neurons. The mode of cell death is primarily apoptotic and is characterized by caspase-3 activation, de-phosphorylation of ERK5 and AKT, and nuclear translocation of the BH3-only protein Bad. As opposed to the inhibition of Rac, motor neuron cell death is also induced by constitutive activation of Rho, via a mechanism that depends on Rho kinase (ROCK) activity. Investigation of Rac and Rho in the G93A mutant, human Cu, Zn-superoxide dismutase (hSOD1) mouse model of amyotrophic lateral sclerosis (ALS), revealed that active Rac1-GTP is markedly decreased in spinal cord motor neurons of transgenic mice at disease onset and end-stage, when compared to age-matched wild type (WT) littermates. Furthermore, although there is no significant change in active RhoA-GTP, total RhoB displays a striking redistribution from motor neuron nuclei in WT mouse spinal cord to motor neuron axons in end-stage G93A mutant hSOD1 mice. Collectively, these data suggest that the intricate balance between pro-survival Rac signaling and pro-apoptotic Rho/ROCK signaling is critical for motor neuron survival and therefore, disruption in the balance of their activities and/or localization may contribute to the death of motor neurons in ALS.

Food restriction has been widely associated with beneficial effects on brain aging and age-related neurodegenerative diseases such as Alzheimer's disease. However, previous studies on the effects of food restriction on aging- or pathology-related cognitive decline are controversial, emphasizing the importance of the type, onset and duration of food restriction. In the present study, we assessed the effects of preventive every-other-day (EOD) feeding regimen on neurodegenerative phenotype in 5XFAD transgenic mice, a commonly used mouse model of Alzheimer's disease. EOD feeding regimen was introduced to transgenic female mice at the age of 2 months and the effects on amyloid-β (Aβ) accumulation, gliosis, synaptic plasticity, and blood-brain barrier breakdown were analyzed in cortical tissue of 6-month-old animals. Surprisingly, significant increase of inflammation in the cortex of 5XFAD fed EOD mice was observed, reflected by the expression of microglial and astrocytic markers. This increase in reactivity and/or proliferation of glial cells was accompanied by an increase in proinflammatory cytokine TNF-α, p38 MAPK and EAAT2, and a decrease in GAD67. NMDA receptor subunit 2B, related to glutamate excitotoxicity, was increased in the cortex of 5XFAD-EOD mice indicating additional alterations in glutamatergic signaling. Furthermore, 4 months of EOD feeding regimen had led to synaptic plasticity proteins reduction and neuronal injury in 5XFAD mice. However, EOD feeding regimen did not affect Aβ load and blood-brain barrier permeability in the cortex of 5XFAD mice. Our results demonstrate that EOD feeding regimen exacerbates Alzheimer's disease-like neurodegenerative and neuroinflammatory changes irrespective of Aβ pathology in 5XFAD mice, suggesting that caution should be paid when using food restrictions in the prodromal phase of this neurodegenerative disease.

The toxic conformer of amyloid β-protein (Aβ) ending at 42 (Aβ42), which contains a unique turn conformation at amino acid residue positions 22 and 23 and tends to form oligomers that are neurotoxic, was reported to play a critical role in the pathomechanisms of Alzheimer's disease (AD), in which diabetes mellitus (DM)-like mechanisms are also suggested to be operative. It remains to be established whether the attenuation of insulin signaling is involved in an increase of toxic Aβ42 conformer levels. The present study investigated the association between impaired insulin metabolism and formation of toxic Aβ42 conformers in the brains of an AD mouse model. In particular, we studied whether insulin deficiency or resistance affected the formation of toxic Aβ42 conformers in vivo. We induced insulin deficiency and resistance in 3xTg-AD mice, a mouse AD model harboring two familial AD-mutant APP (KM670/671NL) and PS1 (M146 V) genes and a mutant TAU (P301L) gene, by streptozotocin (STZ) injection and a high fructose diet (HFuD), respectively. Cognitive impairment was significantly worsened by STZ injection but not by HFuD. Dot blot analysis revealed significant increases in total Aβ42 levels and the ratio of toxic Aβ42 conformer/total Aβ42 in STZ-treated mice compared with control and HFuD-fed mice. Immunostaining showed the accumulation of toxic Aβ42 conformers and hyper-phosphorylated tau protein (p-tau), which was more prominent in the cortical and hippocampal neurons of STZ-treated mice compared with HFuD-fed and control mice. HFuD-fed mice showed only a mild-to-moderate increase of these proteins compared with controls. Toxic Aβ42 conformers were co-localized with p-tau oligomers (Pearson's correlation coefficient = 0.62) in the hippocampus, indicating their co-aggregation. Toxic Aβ42 conformer levels were inversely correlated with pancreatic insulin secretion capacity as shown by fasting immunoreactive insulin levels in STZ-treated mice (correlation coefficient = −0.5879, p = .04441), but not HFuD-fed mice, suggesting a decrease in serum insulin levels correlates with toxic Aβ42 conformer formation. Levels of p-Akt and phosphorylated glycogen synthase kinase-3β measured by a homogeneous time-resolved fluorescence assay were significantly lower in STZ-treated mice than in HFuD-fed mice, suggesting a greater inhibition of brain insulin signaling by STZ than HFuD, although both levels were significantly decreased in these groups compared with controls. Iba1-positive and NOS2-positive areas in the cortex and hippocampus were significantly increased in STZ-treated mice and to a lesser extent in HFuD-fed mice compared with controls. These findings suggest that insulin deficiency rather than insulin resistance and the resultant impairment of brain insulin signaling facilitates the formation of toxic Aβ42 conformer and its co-aggregation with p-tau oligomers, and that insulin deficiency is an important pathogenic factor in the progression of AD.