Dehydrozaluzanin C- derivative protects septic mice by alleviating over-activated inflammatory response and promoting the phagocytosis of macrophages.

Host-directed therapy (HDT) is a new adjuvant strategy that interfere with host cell factors that are required by a pathogen for replication or persistence. In this study, we assessed the effect of dehydrozaluzanin C-derivative (DHZD), a modified compound from dehydrozaluzanin C (DHZC), as a potential HDT agent for severe infection. LPS-induced septic mouse model and Carbapenem resistant Klebsiella pneumoniae (CRKP) infection mouse model was used for testing in vivo. RAW264.7 cells, mouse primary macrophages, and DCs were used for in vitro experiments. Dexamethasone (DXM) was used as a positive control agent. DHZD ameliorated tissue damage (lung, kidney, and liver) and excessive inflammatory response induced by LPS or CRKP infection in mice. Also, DHZD improved the hypothermic symptoms of acute peritonitis induced by CRKP, inhibited heat-killed CRKP (HK-CRKP)-induced inflammatory response in macrophages, and upregulated the proportions of phagocytic cell types in lungs. In vitro data suggested that DHZD decreases LPS-stimulated expression of IL-6, TNF-α and MCP-1 via PI3K/Akt/p70S6K signaling pathway in macrophages. Interestingly, the combined treatment group of DXM and DHZD had a higher survival rate and lower level of IL-6 than those of the DXM-treated group; the combination of DHZD and DXM played a synergistic role in decreasing IL-6 secretion in sera. Moreover, the phagocytic receptor CD36 was increased by DHZD in macrophages, which was accompanied by increased bacterial phagocytosis in a clathrin- and actin-dependent manner. This data suggests that DHZD may be a potential drug candidate for treating bacterial infections.

BMAL1 modulates smooth muscle cells phenotypic switch towards fibroblast-like cells and stabilizes atherosclerotic plaques by upregulating YAP1.

BACKGROUND: Ischemic heart diseases and ischemic stroke are closely related to circadian clock and unstable atherosclerotic plaques. Vascular smooth muscle cells (VSMCs) can stabilize or destabilize an atherosclerotic lesion through phenotypic switch. BMAL1 is not only an indispensable core component in circadian clock but also an important regulator in atherosclerosis and VSMCs proliferation. However, little is known about the modulation mechanisms of BMAL1 in VSMCs phenotypic switch and atherosclerotic plaque stability. METHODS: We integrated histological analysis of human plaques, in vivo experiments of VSMC-specific Bmal1-/- mice, in vitro experiments, and gene set enrichment analysis (GSEA) of public datasets of human plaques to explore the function of BMAL1 in VSMCs phonotypic switch and plaque stability. FINDINGS: Comparing to human unstable plaques, BMAL1 was higher in stable plaques, accompanied by elevated YAP1 and fibroblast maker FSP1 which were positively correlated with BMAL1. In response to Methyl-ß-cyclodextrin-cholesterol, oxidized-low-density-lipoprotein and platelet-derived-growth-factor-BB, VSMCs embarked on phenotypic switch and upregulated BMAL, YAP1 and FSP1. Besides, BMAL1 overexpression promoted VSMCs phonotypic switch towards fibroblast-like cells by transcriptionally upregulating the expression of YAP1. BMAL1 or YAP1 knock-down inhibited VSMCs phonotypic switch and downregulated FSP1. Furthermore, VSMC-specific Bmal1-/- mice exhibited VSMCs with lower YAP1 and FSP1 levels, and more vulnerable plaques with less collagen content. In addition, BMAL1 suppressed the migration of VSMCs. The GSEA results of public datasets were consistent with our laboratory findings. INTERPRETATION: Our results highlight the importance of BMAL1 as a major regulator in VSMCs phenotypic switch towards fibroblast-like cells which stabilize an atherosclerotic plaque.

Identification of potential therapeutic targets for atherosclerosis by analysing the gene signature related to different immune cells and immune regulators in atheromatous plaques.

BACKGROUND: Atherosclerosis is a chronic inflammatory disease that affects multiple arteries. Numerous studies have shown the inherent immune diversity in atheromatous plaques and suggest that the dysfunction of different immune cells plays an important role in atherosclerosis. However, few comprehensive bioinformatics analyses have investigated the potential coordinators that might orchestrate different immune cells to exacerbate atherosclerosis. METHODS: Immune infiltration of 69 atheromatous plaques from different arterial beds in GSE100927 were explored by single-sample-gene-set enrichment analysis (presented as ssGSEA scores), ESTIMATE algorithm (presented as immune scores) and CIBERSORT algorithm (presented as relative fractions of 22 types of immune cells) to divide these plaques into ImmuneScoreL cluster (of low immune infiltration) and ImmuneScoreH cluster (of high immune infiltration). Subsequently, comprehensive bioinformatics analyses including differentially-expressed-genes (DEGs) analysis, protein-protein interaction networks analysis, hub genes analysis, Gene-Ontology-terms and KEGG pathway enrichment analysis, gene set enrichment analysis, analysis of expression profiles of immune-related genes, correlation analysis between DEGs and hub genes and immune cells were conducted. GSE28829 was analysed to cross-validate the results in GSE100927. RESULTS: Immune-related pathways, including interferon-related pathways and PD-1 signalling, were highly enriched in the ImmuneScoreH cluster. HLA-related (except for HLA-DRB6) and immune checkpoint genes (IDO1, PDCD-1, CD274(PD-L1), CD47), RORC, IFNGR1, STAT1 and JAK2 were upregulated in the ImmuneScoreH cluster, whereas FTO, CRY1, RORB, and PER1 were downregulated. Atheromatous plaques in the ImmuneScoreH cluster had higher proportions of M0 macrophages and gamma delta T cells but lower proportions of plasma cells and monocytes (p < 0.05). CAPG, CECR1, IL18, IGSF6, FBP1, HLA-DPA1 and MMP7 were commonly related to these immune cells. In addition, the advanced-stage carotid plaques in GSE28829 exhibited higher immune infiltration than early-stage carotid plaques. CONCLUSIONS: Atheromatous plaques with higher immune scores were likely at a more clinically advanced stage. The progression of atherosclerosis might be related to CAPG, IGSF6, IL18, CECR1, FBP1, MMP7, FTO, CRY1, RORB, RORC, PER1, HLA-DPA1 and immune-related pathways (IFN-γ pathway and PD-1 signalling pathway). These genes and pathways might play important roles in regulating immune cells such as M0 macrophages, gamma delta T cells, plasma cells and monocytes and might serve as potential therapeutic targets for atherosclerosis.

Interactions Between Targeted Ultrasound Contrast Agents With Anti-Human Interleukin 8 Monoclonal Antibody and Activated Endothelial Cells.

The study aimed to explore the role of interleukin 8 (IL-8) in atherosclerotic plaques and develop a new method for the evaluation of endothelial function by assessing the interactions between the injured endothelial cells and the targeted ultrasound agent that carried anti-human IL-8 monoclonal antibody. Anti-human IL-8 monoclonal antibodies were associated to the shells of SonoVue microbubbles by covalent conjugation technology. The specific interaction between the microbubbles and the normal or injured endothelial cells was observed using an inverted microscope. The microbubble adherence was quantified by calculating the ratio of the adherent microbubbles to endothelial cells. The results were compared with the control microbubbles. There were rare adherences of control microbubbles to the normal or injured endothelial cells, whereas the targeted microbubbles could adhere to endothelial cells well. Importantly, compared with the normal endothelial cells, a significantly higher number of targeted microbubbles bound to the injured endothelial cells. The ultrasound agents with anti-human IL-8 monoclonal antibody can specifically bind to the injured endothelial cell, which provides a new insight to the noninvasive detection of endothelial dysfunction using ultrasound imaging techniques.

Assessment of fibrosis during the development of fatty liver in rabbits using real-time shear-wave elastography.

Nonalcoholic and alcoholic rabbit models of fatty liver were established by feeding on high-fat diet and alcohol, respectively, and fatty liver stiffness at different pathological stages was assessed with real-time shear-wave elastography (SWE), so as to investigate the fibrosis process during the development of fatty liver. The fatty liver stiffness of rabbit in nonalcoholic and alcoholic groups was higher than that in the control group, and that in alcohol group was higher than that in the nonalcoholic group (P<0.01). The elasticity modulus of liver in fatty liver rabbits of nonalcoholic and alcoholic groups showed a positive correlation with progression of liver fibrosis (P<0.01). Real-time SWE, as a noninvasive diagnostic method, can objectively reflect the liver stiffness change and progression of liver fibrosis during the development of fatty liver.

Effects of photoperiod and temperature on diapause induction in Conogethes punctiferalis (Lepidoptera: Pyralidae).

The yellow peach moth, Conogethes punctiferalis (Guenée), a multivoltine species that overwinters as diapausing larvae, is one of the most serious insect pests on maize in China. Effect of photoperiod and temperature on larval diapause was examined under empirical laboratory conditions. Short-day treatments caused larval diapause at 25°C, and the critical photoperiod was between 12 and 13 h (or 12 h 51 min) light per day. No sensitive instar was identified for diapause induction under alternated short- (L : D 11 : 13 h) and long-day (L : D 14 : 10 h) treatments at different larval stages. However, accumulative treatment of three instars and 10 d under short-day treatment was required for the induction of 50% larval diapause. All larvae entered diapause at 20°C, whereas less than 3% did so at 30°C, irrespective of the long- or short-day treatment. Furthermore, under the short-day treatment, more than 90% of larvae went into diapause with temperatures ≤ 25°C, but less than 17% did so at 28°C. In contrast, under the long-day treatment, less than 19% of larvae went into diapause with temperatures ≥ 23°C. The forward shift (5°C) of critical temperature under the long-day regime demonstrated the compensatory effect of temperature and photoperiod on diapause induction. In conclusion, C. punctiferalis had a temperature-dependent type I photoperiodic diapause response; there was no sensitive instar for diapause determination, but the photoperiodic accumulation time countermeasures both of the short-day cycles and the number of instars exposed, and the photoperiodic diapause response, was a temperature-compensated phenomenon.

Protein disulfide isomerase interacts with tau protein and inhibits its fibrillization.

BACKGROUND: Tau protein is implicated in the pathogenesis of neurodegenerative disorders such as tauopathies including Alzheimer disease, and Tau fibrillization is thought to be related to neuronal toxicity. Physiological inhibitors of Tau fibrillization hold promise for developing new strategies for treatment of Alzheimer disease. Because protein disulfide isomerase (PDI) is both an enzyme and a chaperone, and implicated in neuroprotection against Alzheimer disease, we want to know whether PDI can prevent Tau fibrillization. In this study, we have investigated the interaction between PDI and Tau protein and the effect of PDI on Tau fibrillization. METHODOLOGY/PRINCIPAL FINDINGS: As evidenced by co-immunoprecipitation and confocal laser scanning microscopy, human PDI interacts and co-locates with some endogenous human Tau on the endoplasmic reticulum of undifferentiated SH-SY5Y neuroblastoma cells. The results from isothermal titration calorimetry show that one full-length human PDI binds to one full-length human Tau (or human Tau fragment Tau244-372) monomer with moderate, micromolar affinity at physiological pH and near physiological ionic strength. As revealed by thioflavin T binding assays, Sarkosyl-insoluble SDS-PAGE, and transmission electron microscopy, full-length human PDI remarkably inhibits both steps of nucleation and elongation of Tau244-372 fibrillization in a concentration-dependent manner. Furthermore, we find that two molecules of the a-domain of human PDI interact with one Tau244-372 molecule with sub-micromolar affinity, and inhibit both steps of nucleation and elongation of Tau244-372 fibrillization more strongly than full-length human PDI. CONCLUSIONS/SIGNIFICANCE: We demonstrate for the first time that human PDI binds to Tau protein mainly through its thioredoxin-like catalytic domain a, forming a 1∶1 complex and preventing Tau misfolding. Our findings suggest that PDI could act as a physiological inhibitor of Tau fibrillization, and have applications for developing novel strategies for treatment and early diagnosis of Alzheimer disease.

Both the C-terminal polylysine region and the farnesylation of K-RasB are important for its specific interaction with calmodulin.

BACKGROUND: Ras protein, as one of intracellular signal switches, plays various roles in several cell activities such as differentiation and proliferation. There is considerable evidence showing that calmodulin (CaM) binds to K-RasB and dissociates K-RasB from membrane and that the inactivation of CaM is able to induce K-RasB activation. However, the mechanism for the interaction of CaM with K-RasB is not well understood. METHODOLOGY/PRINCIPAL FINDINGS: Here, by applying fluorescence spectroscopy and isothermal titration calorimetry, we have obtained thermodynamic parameters for the interaction between these two proteins and identified the important elements of K-RasB for its interaction with Ca(2+)/CaM. One K-RasB molecule interacts with one CaM molecule in a GTP dependent manner with moderate, micromolar affinity at physiological pH and physiologic ionic strength. Mutation in the polybasic domain of K-Ras decreases the binding affinity. By using a chimera in which the C-terminal polylysine region of K-RasB has been replaced with that of H-Ras and vice versa, we find that at physiological pH, H-Ras-(KKKKKK) and Ca(2+)/CaM formed a 1:1 complex with an equilibrium association constant around 10(5) M(-1), whereas no binding reaction of K-RasB-(DESGPC) with Ca(2+)/CaM is detected. Furthermore, the interaction of K-RasB with Ca(2+)/CaM is found to be enhanced by the farnesylation of K-RasB. CONCLUSIONS/SIGNIFICANCE: We demonstrate that the polylysine region of K-RasB not only contributes importantly to the interaction of K-RasB with Ca(2+)/CaM, but also defines its isoform specific interaction with Ca(2+)/CaM. The farnesylation of K-RasB is also important for its specific interaction with Ca(2+)/CaM. Information obtained here can enhance our understanding of how CaM interacts with K-RasB in physiological environments.

[Characteristics of operational performance and membrane fouling in a sidestream membrane sequencing batch reactor with aerobic granule].

The characteristics of operational performance and membrane fouling were investigated using synthetic wastewater as feed in a sidestream membrane sequencing batch reactor (MSBR) system. The experimental results showed that the average removal efficiencies of COD, TN, TP and NH4+ -N were 90%, 60%-80%, 80% and 95% respectively under the influent COD of 200 mg x L(-1) to 1200 mg x L(-1) during more than 150 days' operation. It was possible to achieve a complete granulation of sludge. In 70th day, sludge particles around 350 microm were detected more than 90%. From then, sludge grew up continuously and finally reached to 394 microm in diameter. With the formation of granular sludge in MSBR, the cleaning time of membrane system was prolonged to 65 days, which was larger than three times compared with flocculent sludge MSBR. It was also much better than conventional MBR. These results clearly demonstrated that the formation of granular sludge was good for improving the resistance to organic loading shock and retaining the membrane fouling of the system, and maintaining the stable operation for a long time.

[Characteristics of membrane fouling and its control in membrane sequencing batch reactor process].

Characteristics of membrane fouling and its control were investigated using synthetic wastewater as feed in an anaerobic/aerobic membrane sequencing batch reactor (MSBR) for 300 days operational period. The experimental results showed that the sludge was in flocculent form, and the range of SVI was 64.6-110.6 mL x g(-1) during the initial 75 days operation. Membrane fouling occurred in a quick exponential growth, and the average growth rate of TMP was 0.309 kPa x d(-1). Membrane resistance was in the range of 0.393 x 10(11) - 1.298 x 10(11) m(-1) x d(-1) and specific membrane flux decreased from 4.4 L x (m2 x h x kPa) (-1) to 0.52 L x (m2 x h x kPa)(-1). The critical membrane flux was 20 L x (m2 x h)(-1) at 75th day. In 75-120 d operation, MSBR condition was regulated, and aerobic granular sludge in reactor appeared. SVI decreased steadily, and finally maintained approximately 40 mL x g(-1) from 170th day on. The sludge particle grew much gradually, and size distribution was mainly in diameter 500-1000 microm at 220th day. Membrane fouling developed very slowly. The average accumulating rate of TMP was only 0.062 kPa x d(-1) membrane resistance varied from 0.291 x 10(11) m(-1) x d(-1) to 0.404 x 10(11) m(-1) x d(-1), specific membrane flux decreased from 4.4 L x (m2 x h x kPa)(-1) to 1.4 L x (m2 x h x kPa) (-1) and critical membrane flux was 40 L x (m2 x h)(-1) at 220th day. These data clearly demonstrated that the formation of aerobic granular sludge was beneficial to cease the growth rate of membrane fouling. Specific membrane flux was the biggest at aeration strength of 100 m3 x (m2 x h)(-1) while membrane fouling rate was the lowest at aeration strength of 69 m3 x (m2 x h)(-1).

[Investigation of nitrogen removal performance with membrane sequencing batch reactor process].

Nitrogen removal performance was investigated using synthetic wastewater as feed without sludge discharge in an anaerobic/aerobic membrane sequencing batch reactor (MSBR) during 300 days operation. The results showed that MLSS in reactor was retained up to about 18 g x L(-1), sludge size larger than 100 microm was 96%, and aerobic granular sludge was developed. The bacterial community observation of AOB and NOB by FISH-CLSM for sludge revealed that they were existed in larger numbers. When influent NH(4+) -N concentration was about 50 mg x L(-1), effluent NH(4+) -N concentration was lower than to 1 mg x L(-1), and nitrification could complete in 180-210 min. There was a good correlation between nitrification reaction and aeration strength. When aeration strength was 100 m3 x (m2 x h)(-1), NH(4+) -N degradation rate 24.25 mg x (L x h)(-1), and nitrification reaction in MSBR was stable. The main factor determining nitrogen removal of the system was denitrification rate, which was optimal at aeration 69 m3 x (m2 x h)(-1), when the nitrification rate of NO(3-) -N 10.98 mg x (L x h)(-1), effluent NO(3-) -N 4.4 mg x L(-1), and NO(3-) -N in the beginning of anaerobic phase 3.5 mg x L(-1). The denitrification performance was not benefited by excessive aeration or deficient aeration. Bigger volumetric exchange ratio was helpful for nitrogen removal and the system treatment capacity. The C/N ratio 2 was suitable to good denitrification rate, while there was a NO(2-) -N accumulation if C/N ratio was larger than 2.

[Sequential extraction experiments applied to study chemical mobility of fluorine in rocks].

Sequential extraction experiments were used to study the chemical mobility of fluorine in rocks. The results show that there are quite big differences in chemical mobility of fluorine in rocks of different types. Fluorine in carbonate rock is very active, in which the proportion of leachable fluorine is generally more than 75%. Fluorine in black rocks of Lower Cambrian is closely related to their different metamorphosed grades, in which fluorine in black carbonaceous slate with higher metamorphosed grade mostly has lower leachability than black shale and black siliceous rock. Generally speaking, the leachable percentage of fluorine is high in phosphorite rocks and low in phyllite. The leachable fluorine in diabase is in direct proportion to its fluorine concentration. There are some differences in chemical mobility of fluorine in stone coal of different ages. Fluorine in stone coal of Silurian has higher leachability than stone coal of Cambrian.

[Treatment of cleft palate with secretory otitis media].

OBJECTIVE: To gain a clear idea of the function of middle ear and hearing in cleft palate with secretory otitis media after pressure equalization tube insertion. METHODS: 53 cleft palate cases with secretory otitis media including 62 ears were divided into treatment and control groups. They were examined by tympanogram and hearing threshold. RESULTS: Hearing threshold in treatment group after PE tube insertion respectively was 23.3 dB and 23.4 dB in the near future and at a specified future date. It was normal. But there were still 64% tympanogram B and C. CONCLUSIONS: PE tube insertion could solve hearing problem, but could not improve the function of middle ear.

The prevalence of connexin 26 ( GJB2) mutations in the Chinese population.

Mutations in GJB2, encoding gap junction beta 2 protein (connexin 26), are responsible for the commonest form of non-syndromic recessive deafness in many populations. It has been reported recently that the most common 35delG mutation in GJB2 is exceptionally low in Japanese and Korean populations, but another deletion, 235delC, is relatively frequent. Since the Chinese constitute approximately one fifth of the global population, the frequency of GJB2 mutations in the population has important implications for understanding worldwide causes of genetic deafness. To determine whether GJB2 mutations are an important cause of deafness in Chinese, we conducted mutation screening for GJB2 in 118 deaf Chinese probands, including 60 from simplex and 58 from multiplex families with non-syndromic deafness, and 150 normal hearing Chinese controls. Four mutations, including 235delC, 299-300delAT, V37I, and 35delG, were found in the patients. Thirty-nine percent of the probands had a GJB2mutation. Of the 118 probands, 19 carried two definitely pathogenic mutations: three among the 58 multiplex cases (5.2%) and 16 among the 60 simplex cases (26.7%). Twenty-seven probands (22.9%) were found to carry only single GJB2 mutations. None of them had mutations in exon 1 of GJB2 and or the 342-kb deletion of GJB6. The 235delC mutation was the most prevalent mutation (20.3% of alleles), accounting for 81% of the pathologic alleles in multiplex cases and 67% in simplex cases. Analysis of the affected haplotypes in the patients with the homozygous 235delC mutation yielded evidence for a single origin of the mutation. The carrier frequency of the 235delC mutation in control subjects with normal hearing was 1.3%. The 35delG mutation was only noted as a heterozygous change in two simplex cases (1.2% of alleles). These results indicated that mutations in GJB2 are a major cause of inherited and sporadic congenital deafness in the Chinese population. The 235delC mutation, rather than 35delG, is the most common mutation found in the Chinese deaf population. Our data support the view that specific combinations of GJB2 mutation exist in different populations.