Preparation, characterization, pharmacokinetics and ulcerative colitis treatment of hyperoside-loaded mixed micelles.

At present, ulcerative colitis (UC) has become a global disease due to its high incidence. Hyperoside (HYP) is a naturally occurring flavonoid compound with many pharmacological effects. This study aimed to develop HYP-loaded mixed micelles (HYP-M) to improve oral bioavailability of HYP and to evaluate its therapeutic effect on UC. The prepared HYP-M exhibited stable physical and chemical properties, smaller particle size (PS) (21.48 ± 1.37 nm), good polydispersity index (PDI = 0.178 ± 0.013), negative Zeta potential (ZP) (- 20.00 ± 0.48 mV) and high entrapment rate (EE) (89.59 ± 2.03%). In vitro release and in vivo pharmacokinetic results showed that HYP-M significantly increased the releasing rate of HYP, wherein its oral bioavailability was 4.15 times higher than that of free HYP. In addition, HYP-M was more effective in the treatment of UC than free HYP. In conclusion, HYP-M could serve as a novel approach to improve bioavailability and increase anti-UC activity of HYP.

Methanol-water mixture evaporation-induced self-assembly of ZIF-8 particles.

We report a strategy for the self-assembly of zeolitic imidazolate framework-8 (ZIF-8) particles induced by the evaporation of a methanol-water mixture. This strategy effectively suppresses the coffee ring effect, facilitates the rapid assembly of ZIF-8 particles, and improves the orientation and optical properties of self-assembled superstructures.

The impact of wearing facemask on COPD patients: A protocol of a systematic review and meta-analysis.

INTRODUCTION: Chronic obstructive pulmonary disease (COPD) is a common, irreversible but preventable disease characterized by persistent respiratory symptoms. The mortality rate of COPD is predicted to reach 5.4 million by the year 2060. Despite its heavy burden on healthcare expenditure worldwide, only 15% of cases are medically identified. The potential benefits of facemask-wearing for COPD patients remain a topic of debate. METHODS: We will conduct a systematic review of all randomized trials and non-randomized controlled trials to evaluate the impact of facemasks on COPD patients. Our review will be based on literature obtained through a comprehensive search strategy across multiple electronic databases, including the Cochrane Library, Embase, PubMed, Web of Science, the Chinese Biomedical Database (SinoMed), and China National Knowledge Infrastructure (CNKI), with no restrictions on language or date of publication. Two independent researchers will extract and assess all relevant data using pre-designed data extraction forms. The included studies will be assessed using the Cochrane RoB2 tool and the suggested risk of bias criteria proposed by the Effective Practice and Organization of Care reviews group of the Cochrane collaboration. The quality of evidence will be assessed using the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach. We will use Review Manager 5.4 software for statistical analysis. DISCUSSION: In the context of COVID-19, it is important for COPD patients to wear facemasks. This study aims to conduct a comprehensive and systematic assessment of the impact of facemasks on the physiology and activity of COPD patients. TRIAL REGISTRATION: PROSPERO registration number CRD42022326265.

Colorimetric Aerogel Gas Sensor with High Sensitivity and Stability.

The detection of formic acid vapor in the usage environment is extremely important for human health and safety. The utilization of metal-organic frameworks (MOFs) for the detection of gaseous molecules is an attractive strategy. However, the rational design and construction of MOF-based gas sensors with high sensitivity and mechanical stability remain a significant challenge. In this study, a simple approach is reported to fabricate colorimetric aerogel sensors assembled from MOF particles via ice template-assisted methods. As the aerogel sensor with staggered lamellae structures significantly provides a high air-volume intake of flowing gas, it generates a sufficient probability of contact reactions for highly mobile target molecules. Additionally, it enhances the mechanical stability by providing stress resistance between the staggered lamellae structures. Compared to conventional film sensors for the detection of formic acid molecules, aerogel sensors exhibit an 8-fold lower limit of detection, 15-fold better sensitivity at low concentrations, 34-fold faster response time, and higher stability. This approach shows great potential for rapid and real-time detection of target molecules as well as superior performance in the structural construction of various gas-sensitive materials.

Fine control for the preparation of ceria nanorods (111).

The morphologies and exposed surfaces of ceria nanocrystals are important factors in determining their performance. In order to establish a structure-property relationship for ceria nanomaterials, it is essential to have materials with well-defined morphologies and specific exposed facets. This is also crucial for acquiring high resolution 17O solid-state NMR spectra. In this study, we explore the synthesis conditions for preparing CeO2 nanorods with exposed (111) facets. The effects of alkali concentration, hydrothermal temperature and time, cerium source and oxidation agent are investigated and optimal synthesis conditions are found. The resulting CeO2 nanorods show very narrow 17O NMR peaks for the oxygen ions in the first, second and third layers, providing a foundation for future research on mechanisms involving ceria materials using 17O solid-state NMR spectroscopy.

In-vitro and in-vivo evaluation and anti-colitis activity of esculetin-loaded nanostructured lipid carrier decorated with DSPE-MPEG2000.

OBJECTIVE: Encapsulation of esculetin into DSPE-MPEG2000 carrier was performed to improve its water solubility and oral bioavailability, as well as enhance its anti-inflammatory effect on a mouse model of ulcerative colitis that was induced with dextran sulphate sodium (DSS). METHODS: We determined the in-vitro and in-vivo high-performance liquid chromatographic (HPLC) analysis method of esculetin; Esculetin-loaded nanostructure lipid carrier (Esc-NLC) was prepared using a thin-film dispersion method, wherein a particle size analyser was used to measure the particle size (PS) and zeta potential (ZP) of the Esc-NLC, while a transmission electron microscope (TEM) was employed to observe its morphology. Also, HPLC was used to measure its drug loading (DL), encapsulation efficiency (EE) and the in-vitro release of the preparation, as well as investigate the pharmacokinetic parameters. In addition, its anti-colitis effect was evaluated via histopathological examination of HE-stained sections and detection of the concentrations of tumour necrosis factor-alpha (TNF-α), interleukin (IL)-1 beta (ß), and IL-6 in serum with ELISA kits. RESULTS: The PS of Esc-NLC was 102.29 ± 0.63 nm with relative standard deviation (RSD) of 1.08% (with poly-dispersity index-PDI of 0.197 ± 0.023), while the ZP was -15.67 ± 1.39 mV with RSD of 1.24%. Solubility of esculetin was improved coupled with prolonged release time. Its pharmacokinetic parameters were compared with that of free esculetin, wherein the maximum concentration of the drug in plasma was increased by 5.5 times. Of note, bioavailability of the drug was increased by 1.7 times, while the half-life was prolonged by 2.4 times. In the anti-colitis efficacy experiment, the mice in Esc and Esc-NLC groups exhibited significantly reduced levels of TNF-α, IL-1ß, and IL-6 in their sera comparable to the DSS group. Colon histopathological examination revealed that mice with ulcerative colitis in both Esc and Esc-NLC groups displayed improved inflammation, amid the Esc-NLC groups having the best prophylactic treatment effect. CONCLUSION: Esc-NLC could ameliorate DSS-induced ulcerative colitis by improving bioavailability, prolonging drug release time and regulating cytokine release. This observation confirmed the potential of Esc-NLC to reduce inflammation in ulcerative colitis, albeit the need for follow-up research to verify the application of this strategy to clinical treatment of ulcerative colitis.

Vitexin loaded mixed polymeric micelles: preparation, optimization, evaluation and anti-osteoporotic effect.

In this regard, we developed vitexin (Vi)-loaded D-ɑ-tocopherol polyethylene glycol succinate, polyvinylpyrrolidone K30 and sodium cholate mixed micelles (Vi-MMs) mainly for improving oral bioavailability and enhancing anti-osteoporotic effect of Vi. Thin layer dispersion method was employed to prepare Vi-MMs, and then the optimal prescription was optimized by the orthogonal design-response surface method, wherein encapsulation efficiency (EE) was used as optimizing index. The physical properties of Vi-MMs such as appearance morphology, particle size, and zeta potential were also characterized. We further analyzed thein-vitrorelease of Vi and Vi-MMs in three media and investigated the pharmacokinetics of Vi and Vi-MMs in rats. Anti-osteoporotic activity of Vi and Vi-MMs was assessed by establishing a zebrafish osteoporosis model with prednisone. Drug loading, EE, particle size and zeta potential of the optimized Vi-MMs were 8.58 ± 0.13%, 93.86 ± 1.79%, 20.41 ± 0.64 nm and -10 ± 0.56 mV, respectively. The optimized Vi-MMs were shaped spherically as exhibited by transmission electron microscopic technique, with evident core shell nano-structure, well dispersed. In all three media, the release rate of Vi-MMs was significantly higher than that of free Vi. The oral bioavailability of Vi-MMs was increased by 5.6-fold compared to free Vi. In addition, alleviation of prednisone induced osteoporosis in zebrafish by Vi-MMs further demonstrated good anti-osteoporotic effect. In summary, Vi-MMs exhibited enhanced bioavailability and anti-osteoporotic effect, which is expected to be potential nanocarrier for Vi applications in drug development.

Preparation, Physical Characterization, Pharmacokinetics and Anti-Hyperglycemic Activity of Esculetin-Loaded Mixed Micelles.

Despite its low water solubility, esculetin (EC) have been described to demonstrate various health benefits. Thus, we sought to develop esculetin-loaded mixed micelles (EC-M) delivery system to purposively improve biological availability and anti-hyperglycemia activity of EC. Thin-film hydration method was employed to fabricate EC-M, amid characterization with transmission electron microscopic analysis (TEM), coupled with physical properties such as particle size (PS), poly-dispersity index (PDI), zeta-potential (ZP) and stability testing. We analyzed in-vitro release and studied EC-M pharmacokinetics in rats. The hyperglycemic mice model was established with streptozotocin (STZ) to evaluate anti-hyperglycemic activity of EC-M. The PS, PDI and ZP of EC-M were 47.97 ± 0.41 nm, 0.189 ± 0.005 and -25.55 ± 0.28 mV, respectively. The release rate of EC-M increased comparable to free EC in the three media. The oral biological availability and half-life of EC-M increased respectively by 3.06 and 1.45 folds compared to free EC. Besides, we observed 46.21% decrease in blood glucose of mice in EC-M group comparable to the model control, wherein, the anti-hyperglycemic effect of EC-M was better compared to free EC. Conclusively, EC-M may ideally serve as a novel approach to enhance biological availability and increased anti-hyperglycemic activity of EC.

The impact of facemask on patients with COPD: A systematic review and meta-analysis.

Background: Since the emergence of COVID-19, mandatory facemask wearing has been implemented around the world to prevent viral transmission, however, the impact of wearing facemasks on patients with COPD was unclear. Methods: The current study undertakes a systematic review and meta-analysis of a comprehensive literature retrieval from six databases, based on the pre-determined eligibility criteria, irrespective of language. The risk of bias was assessed using an established instrument. We primarily focused on analyzing ETCO2, SpO2, and heart and respiratory rates, and also considered the impacts on physiological and exercise performance. A descriptive summary of the data and possible meta-analysis was performed. Forest plots were generated to pool estimates based on each of the study outcomes. Results: Of the 3,751 publications considered, six publications were selected for a systematic review and two publications were included for meta-analysis, however, the quality of these six studies was relatively low overall. In the case of inactivity, the facemask wearing COPD cohort had higher respiratory rates than that of the non-facemask wearing cohort (MD = 1.00 and 95% CI 0.47-1.53, P < 0.05). There was no significant difference in ETCO2 (MD = 0.10 and 95% CI -1.57-1.78, P > 0.05) and heart rate (MD = 0.40 and 95% CI -3.59-4.39, P > 0.05) nor SpO2 (MD = -0.40 and 95% CI -0.84-0.04, P > 0.05) between the COPD patients with and without facemasks. Furthermore, it was observed that the only significant differences between the COPD patients with and without facemasks undertaking different activities were FEV1 (%) (MD = 3.84 and 95% CI 0.14-7.54, P < 0.05), FEV1/FVC (%) (MD = 3.25 and 95% CI 0.71-5.79, P < 0.05), and blood lactate (MD = -0.90 and 95% CI -1.73 to -0.07, P < 0.05). Conclusion: Wearing facemasks decreased the exercise performance of patients with COPD, however, it had minimal impact on physiological indexes. Further investigations will be performed on the high-quality data from randomized control studies. Systematic review registration: https://www.crd.york.ac.uk/PROSPERO/display_record.php?RecordID=326265, identifier: CRD42022326265.

Preparation, in vitro and in vivo evaluation of pinocembrin-loaded TPGS modified liposomes with enhanced bioavailability and antihyperglycemic activity.

PURPOSE: To prepare polyethylene glycol succinate-vitamin E modified pinocembrin (PCB)-loaded liposomes (PCBT-liposomes) and evaluate PCBT-liposomal pharmacokinetics and antihyperglycemic activity. SIGNIFICANCE: The novel PCBT-liposomes demonstrated a promising application prospect as a nano drug carrier for future research. METHODS: Thin film dispersion was used to prepare PCBT-liposomes. We measured a series of characterization, followed by in vitro cumulative release, in vivo pharmacokinetic study, and antihyperglycemic activity evaluation. RESULTS: PCBT-liposomes displayed spherical and bilayered nanoparticles with mean particle size (roughly 92 nm), negative zeta potential (about -26.650 mV), high drug encapsulation efficiency (87.32 ± 1.34%) and good storage (at 4 or 25 °C) stability during 48 h after hydration. The cumulative release rate of PCBT-liposomes was markedly higher than free PCB in four different pH media. In vivo investigation showed that PCBT-liposomes could obviously improve oral bioavailability of PCB by 1.96 times, whereas the Cmax, MRT0-t, and T1/2 of PCBT-liposomes were roughly 1.700 ± 0.139 µg·mL-1, 12.695 ± 1.647 h, and 14.244 h, respectively. In terms of biochemical analysis, aspartate amino-transferase (AST), alanine amino-transferase (ALT), interleukin-1 (IL-1), and tumor necrosis factor-α (TNF-α) concentrations in serum of diabetic mice were respectively decreased 28.28%, 17.23%, 17.77%, and 8.08% after PCBT-liposomal treatment. CONCLUSION: These results show PCBT-liposomal preparation as an excellent nano-carrier which has the potential to improve water solubility, bioavailability, and antihyperglycemic activity of PCB, amid broadening the application of PCB in the clinical settings.

Preparation of Pinocembrin-Loaded F127/MPEG-PDLLA Polymer Micelles and Anti-Osteoporotic Activity.

Pinocembrin (PCB) is 5,7-dihydroxyl flavanone and has multiple pharmacological activities, namely, anti-inflammation, anti-osteoporotic, and so on. However, low water solubility and bioavailability have hindered its application. Herein, we aimed to increase its bioavailability through preparation of F127/MPEG-PDLLA polymer micelles (PCB-M). We characterized the micelles through appropriate attributes such as analysis of particle size (PS), polydispersity (PDI), transmission electron microscopic (TEM) image, stability test, and evaluation of in vitro release of drug. After physical characterization, the respective PS, PDI, and entrapment efficiency (EE) of PCB-M were estimated to be 27.63 ± 0.17 nm, 0.055 ± 0.02, and 90.53 ± 0.01%. Fluorescence probe method was employed to measure critical micelle concentration (CMC) of PCB-M, we observed CMC was low, thereby suggesting that PCB-M had good stability. In vitro release analysis indicated that the rate of cumulative PCB release from PCB-M was greater than 90% in each medium compared with free PCB, which was less than 40%, thus pointing to a significantly improved solubility of PCB. In vivo pharmacokinetic results showed that oral biological availability of PCB-M increased 5.3 folds comparable to free PCB. The effects of PCB on osteoblasts and ALP activities were investigated; subsequently, zebrafish osteoporotic model was established with prednisolone to study the anti-osteoporotic effects of PCB and PCB-M. The results showed that PCB improved osteoporosis with PCB-M being more effective than free PCB. Finally, PCB-M can be used as a promising method to improve the solubility of PCB, while the bioavailability and anti-osteoporotic effect of PCB could be improved, thus laying a foundation for clinical use in the future.

Hyperoside-loaded TPGs/mPEG-PDLLA self-assembled polymeric micelles: preparation, characterization and in vitro/in vivo evaluation.

Hyperoside (Hyp) self-assembled polymeric micelles (Hyp-PMs) were purposely developed to enhance aqueous solubility, in vivo availability and anti-oxidative effect of Hyp. In preparing Hyp-PMs, we employed the thin film dispersion method with the micelles consisting of TPGs and mPEG2000-PDLLA3000. The particle size, polydispersity index and zeta potential of Hyp-PMs were 67.42 ± 1.44 nm, 0.229 ± 0.015 and -18.67 ± 0.576 mV, respectively, coupled with high encapsulation efficiency （EE）of 90.63 ± 1.45% and drug loading (DL) of 6.97 ± 1.56%. Furthermore, the value of critical micelle concentration (CMC) was quite low, which indicated good stability and improved self-assembly ability of Hyp-PMs. Also, trend of in vitro Hyp release from Hyp-PMs demonstrated enhanced solubility of Hyp. Similarly, in comparison with free Hyp, oral bioavailability of Hyp-PMs was improved (about 8 folds) whilst half-life of Hyp-PMs was extended (about 3 folds). In vitro anti-oxidative effect showed obvious strong scavenging DPPH capability of Hyp-PMs, which may be attributed to its smaller size and better solubility. Altogether, Hyp-PMs may serve as a possible strategy to potentially enhance aqueous solubility, bioavailability and anti-oxidative effect of Hyp, which may play a key role in Hyp application in the pharmaceutical industries.

Multimodule characterization of immune subgroups in intrahepatic cholangiocarcinoma reveals distinct therapeutic vulnerabilities.

BACKGROUND: Immune microenvironment is well recognized as a critical regulator across cancer types, despite its complex roles in different disease conditions. Intrahepatic cholangiocarcinoma (iCCA) is characterized by a tumor-reactive milieu, emphasizing a deep insight into its immunogenomic profile to provide prognostic and therapeutic implications. METHODS: We performed genomic, transcriptomic, and proteomic characterization of 255 paired iCCA and adjacent liver tissues. We validated our findings through H&E staining (n=177), multiplex immunostaining (n=188), single-cell RNA sequencing (scRNA-seq) (n=10), in vitro functional studies, and in vivo transposon-based mouse models. RESULTS: Integrated multimodule data identified three immune subgroups with distinct clinical, genetic, and molecular features, designated as IG1 (immune-suppressive, 25.1%), IG2 (immune-exclusion, 42.7%), and IG3 (immune-activated, 32.2%). IG1 was characterized by excessive infiltration of neutrophils and immature dendritic cells (DCs). The hallmark of IG2 was the relatively higher tumor-proliferative activity and tumor purity. IG3 exhibited an enrichment of adaptive immune cells, natural killer cells, and activated DCs. These immune subgroups were significantly associated with prognosis and validated in two independent cohorts. Tumors with KRAS mutations were enriched in IG1 and associated with myeloid inflammation-dominated immunosuppression. Although tumor mutation burden was relatively higher in IG2, loss of heterozygosity in human leucocyte antigen and defects in antigen presentation undermined the recognition of neoantigens, contributing to immune-exclusion behavior. Pathological analysis confirmed that tumor-infiltrating lymphocytes and tertiary lymphoid structures were both predominant in IG3. Hepatitis B virus (HBV)-related samples tended to be under-represented in IG1, and scRNA-seq analyses implied that HBV infection indeed alleviated myeloid inflammation and reinvigorated antitumor immunity. CONCLUSIONS: Our study elucidates that the immunogenomic traits of iCCA are intrinsically heterogeneous among patients, posing great challenge and opportunity for the application of personalized immunotherapy.

Pinocembrin polymeric micellar drug delivery system: preparation, characterisation and anti-hyperuricemic activity evaluation.

Aim: Hydrophobic pinocembrin (PCB) was incorporated into a new nano-drug delivery system to enhance solubility, bioavailability and anti-hyperuricemic activity of the drug.Methods: We fabricated PCB loaded polymeric micelles (PCB-FPM) by thin film dispersion method and appropriately determined their physical characteristics. The oral relative bioavailability and anti-hyperuricemic activity of PCB-FPM and free PCB were observed.Results: The optimum particle size of the micelles was 19.90 ± 0.93 nm. PCB-FPM exhibited great stability within 18 days, coupled with lower cytotoxicity and higher biocompatibility. Moreover, the percent cumulative release of PCB-FPM was much higher than free PCB in the dissolution media. The oral bioavailability of PCB-FPM was increased by 2.61 times compared with free PCB. Uric acid (UA) level of rats was reduced in PCB-FPM group (200 mg/kg) by 78.82% comparable to the model control.Conclusion: PCB-FPM may become an ideal strategy to increase oral in-vivo availability and anti-hyperuricemic activity of PCB.

Toxicity of avermectin to Eriocheir sinensis and the isolation of a avermectin-degrading bacterium, Ochrobactrum sp. AVM-2.

Avermectin is widely used in the prevention and treatment of parasites diseases in aquaculture. However, the residual avermectin has a serious impact on the growth and quality of aquatic animals including Eriocheir sinensis. This study shows that the LC50 of avermectin to E. sinensis for 24, 48, 72 and 96 h was 21.88, 13.40, 9.11 and 7.10 mg/L, respectively. After avermectin stress, the activities of superoxide dismutase (SOD), catalase (CAT) and phenol oxidase (PO) in the hepatopancreas of E. sinensis increased and reached the peak on the 6th day. The content of malondialdehyde (MDA) accumulated with the increase of exposure time and concentration of avermectin. After 15 days of avermectin exposure, hepatopancreas was damaged seriously. These results indicated that avermectin had toxicity to E. sinensis. In order to solve the pollution problem caused by residual avermectin, a degrading bacterium AVM-2 was separated from the sediment of E. sinensis breeding pond. The strain was confirmed to be Ochrobactrum sp by morphology observation, physiological and biochemical identification and 16 S rDNA sequences analysis. When the pH value was 7, the temperature was 30 â„ƒ, the concentration of substrate was low, the quantity of inoculation was high, Ochrobactrum sp. AVM-2 had better degradation effect on avermectin. When the addition of Ochrobactrum sp. AVM-2 was 2.34 × 108 CFU/L, the residual avermectin in muscle and hepatopancreatine significantly decreased, and the degradation rate was about 66%. In summary, Ochrobactrum sp. AVM-2 could be used to solve the residual problem of avermectin and ensure the food safety of E. sinensis.

A colloidal gold test strip assay for the detection of African swine fever virus based on two monoclonal antibodies against P30.

African swine fever (ASF), caused by African swine fever virus (ASFV), was first reported in Kenya in 1921, but an effective vaccine or antiviral drug is still not available for ASFV control. Rapid and effective diagnostics are key steps in managing ASF. We generated two monoclonal antibodies (MAbs) against the ASFV phosphoprotein P30 and designated these as 3H7A7 and 6H9A10. Epitope mapping revealed that MAb 3H7A7 and 6H9A10 recognized aa 144-154 and aa 12-18 of P30, respectively. A signal-amplified sandwich colloidal gold test strip for rapid detection of ASFV was developed based using these MAbs. Sensitivity and specificity analysis showed that the detection limit of the strip was 2.16 ng of P30. The strip only reacted with ASFV and did not react with other common porcine viruses. In detection tests using 153 clinical field samples including sera, plasma, anticoagulant-treated blood, and tissue, the strip had 95.42% concordance with real-time PCR. The new MAbs specific for P30 and the rapid colloidal gold test strip helped to reveal novel B cell epitopes in P30 and provide an efficient diagnostic test for on-site clinical detection of ASF.

Fusion expression of the two soluble viral receptors of porcine reproductive and respiratory syndrome virus with a single adeno-associated virus vector.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an economically important pathogen affecting global swine industry. Our recent study has shown that the first four Ig-like domains of sialoadhesin (Sn4D) and the scavenger receptor cysteine-rich domains 5-9 (SRCR59) of CD163 can act as the soluble viral receptors (SVRs) of PRRSV. Co-injection with the two SVR-expressing recombinant adenovirus (rAd) vectors can protect pigs from the lethal challenge with three PRRSV strains. However, the in vivo expression of the two SVRs persists for only two weeks and thus their long-term anti-PRRSV effects remain to be improved. In this study, we fused the two SVRs with a flexible linker or self-cleaving peptide and expressed them with a single recombinant adeno-associated virus (rAAV) vector. The two rAAVs, namely rAAV-Sn4D-SRCR59-Fc and rAAV-SRCR59-Fc/Sn4D-Fc, were generated by using baculovirus-insect cell system. Western blotting analysis showed that the two SVR fusions were efficiently expressed in and secreted from the rAAV-transduced cells. Viral infection blocking assay showed that PRRSV titers in porcine alveolar macrophage (PAM) cells were reduced by 1.6-2.7 log10 after co-cultivation with rAAV-Sn4D-SRCR59-Fc-transduced cells or by 1.9-3.2 log10 after co-cultivation with rAAV-SRCR59-Fc/Sn4D-Fc-transduced cells. After single-dose injection of mice with the rAAV vectors, the expression of two SVR fusions persisted for at least 35 days, which was significantly longer than SRCR59-Fc expression in rAd-SRCR59-Fc-injected mice. Among the two SVR fusions expressed, both expression level and anti-PRRSV activity of SRCR59-Fc/Sn4D-Fc were higher than that of Sn4D-SRCR59-Fc. Therefore, rAAV-SRCR59-Fc/Sn4D-Fc generated can be developed as a novel anti-PRRSV reagent.

JNK selective inhibitor, IQ-1S, protects the mice against lipopolysaccharides-induced sepsis.

Sepsis is a severe systemic inflammatory response induced by infection. Innate immunity recognizes pathogen components such as lipopolysaccharides (LPS), and mediates the polarization of immune cells and the release of cytokines. However, this process is also crucial for triggering sepsis and septic shock. To investigate the potential therapeutic function of 11H-indeno [1,2-b] quinoxalin-11-one oxime (IQ-1S) to sepsis, LPS plus d-galactosamine was used to establish a sepsis mouse model. Flow cytometry was performed to catalyze T cells and macrophages in mouse spleen. ELISA assay and qRT-PCR assay were performed to estimate the expression levels of cytokines and related genes including TNF-α, IL-6, IL-1ß, Nos2, Arg and Mrc. The protein levels of NF-κB, AP1, NF-Y, p-JNK2, JNK2, p-p38, p38, p-IκBα, IκBα, p-IKKß and IKKß were evaluated by Western blot assay. IQ-1S treatment significantly reduced mortality and lung inflammation in sepsis mice. IQ-1S treatment decreased the levels of inflammatory cytokines in sepsis mice. Polarization of M1 macrophages was suppressed by IQ-1S in vitro. IQ-1S significantly inhibited the activation of the JNK signaling pathway and reduced the phosphorylation level of JNK2 in sepsis mice. IQ-1S protected the mice against LPS-induced sepsis through inhibiting JNK signaling pathway.

Assessment of neutrophil gelatinase-associated lipocalin as an early biomarker for canine renal ischemia-reperfusion injury.

BACKGROUND: The pathological mechanism of ischemia/reperfusion acute kidney injury (I/R-AKI) differs from other forms of AKI. Neutrophil gelatinase-associated lipocalin (NGAL) is a sensitive biomarker for early diagnosis of AKI, but its utility for diagnosis of canine I/R-AKI remains to be evaluated. The aims of this study were to establish an I/R-AKI model in dogs and to evaluate the diagnostic value of NGAL for canine I/R-AKI. METHODS: We randomly divided 12 beagle dogs into a sham and an I/R group. Artery and vein of the left kidneys of I/R group were cross-clamped for 60 min followed by reperfusion. The kidney samples were analyzed for histopathological lesions. Serum and urinary samples were analyzed for blood urea nitrogen (BUN), serum creatinine (sCr), serum NGAL (sNGAL), urinary creatinine (uCr), and urinary NGAL (uNGAL). Their detection sensitivities and specificities were compared using a receiver operating characteristics (ROC) method. The expression of NGAL in the renal tissues was analyzed by quantitative RT-PCR (qRT-PCR) and immunohistochemical (IHC) analysis. RESULTS: After I/R, histopathological analysis showed typical AKI lesions in the dog kidneys of the I/R group, but not in the sham group. Compared to that of the sham group, BUN and sCr of the I/R group rose to significant high levels from 24 h after I/R. Both uNGAL and sNGAL rose rapidly from 2 h, reached to the peak levels at 12 h, and then receded to the pre-operation levels by 72 h after I/R. The uNGAL/uCr ratio (uNCR) rose rapidly from 2 h and remained at variably high levels from 6 to 60 h after I/R. The ROC analysis showed that detection sensitivities of uNCR, uNGAL, and sNGAL were significantly (P<0.0001) higher than that of sCr, without significant difference in specificity. The cut-off values of sNGAL, uNGAL and uNCR were 14,642 pg/mL, 6,773 pg/mL, and 6,701 pg/mg, respectively. Both qRT-PCR and IHC analyses confirmed the dynamic expression of NGAL in the dog kidneys with ischemic acute kidney injury (I-AKI). CONCLUSIONS: There is potential for NGAL to be used as a sensitive biomarker for early diagnosis of canine I-AKI.

An ShK-domain serine protease of Eriocheir sinensis regulates the PO activity to resist Spiroplasma eriocheiris infection.

A novel serine protease contains two ShK-domain was found from the Chinese mitten crab Eriocheir sinensis (EsShK-SP). The full-length EsShK-SP cDNA is 1927 bp and contains a 1260-bp open reading frame encoding a protein of 420 amino acids, including a signal peptide, two ShK domain, and Tryp-SPC domain. Quantitative real-time PCR showed that EsShK-SP was expressed mainly in the hemocytes, gills, intestine, and nerve, but weakly in heart, muscle, and hepatopancreas. After infected with Spiroplasma eriocheiris, the expression of EsShK-SP was significantly up-regulated from 1 d to 9 d. The Tryp-SPC domain was ligated with pGEX-4T-1 vector and prokaryotic expressed to obtain recombinant protein rSPC. When rSPC and S. eriocheiris stimulated the hemocytes of E. sinensis, the PO activity was significantly up-regulated. The subcellular localization revealed that recombinant EsShK-SP was mainly located in the cytoplasm of Drosophila S2 cells. Both absolute real-time PCR and confocal laser scanning microscope results showed that over-expression of EsShK-SP in S2 cells could decrease the copy number of S. eriocheiris. Meanwhile, the over-expression of EsShK-SP also increased the PO activity and cell viability of S2 cells. After EsShK-SP RNA interference using dsRNA, the expression levels of proPO and activity of PO decreased significantly from 48 h to 96 h. The knockdown of EsShK-SP by RNAi resulted in the copy number of S. eriocheiris in the EsShK-SP silenced group was significantly increased compared to the control groups during S. eriocheiris infection. Meanwhile, the survival rate of crabs decreased in the EsShK-SP-dsRNA group. The above results indicated that EsShK-SP plays an important immune role during E. sinensis against S. eriocheiris through regulation of the proPO system.