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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. [ABSTRACT FROM AUTHOR]

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 numberCRD42022326265. [ABSTRACT FROM AUTHOR]

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). 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 (β), and IL-6 in serum with ELISA kits. 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-1β, 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. 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. [ABSTRACT FROM AUTHOR]

To prepare polyethylene glycol succinate-vitamin E modified pinocembrin (PCB)-loaded liposomes (PCBT-liposomes) and evaluate PCBT-liposomal pharmacokinetics and antihyperglycemic activity. The novel PCBT-liposomes demonstrated a promising application prospect as a nano drug carrier for future research. 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. 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. 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

A cyclometalated ruthenium (II) complex 1 [(Ru (Phen)2(Pbznh)]+ PF6− (Phen = 1,10‐phenanthroline and Pbznh = N‐(4‐(pyridine‐2‐yl)benzylidene) nicotinohydrazide) with nicotinohydrazide as a functional group was synthesized and characterized. Changes of its absorption spectra and color induced by Cu2+ and Fe3+ were systematic investigated. The results demonstrated that complex 1 could be served as a colorimetric probe to fast, selective and sensitive detection of Cu2+ and Fe3+ both in acetonitrile and filter paper based strips. Upon addition of Cu2+ and Fe3+ to solution of probe 1, solution color changed from pink to colorless and light yellow respectively, and their corresponding detection limit were calculated to be 3.26 × 10−8 M and 3.12 × 10−7 M. Moreover, color of test papers with 1 changed from pink to colorless/yellow when Cu2+/Fe3+ were dropwise added. Therefore, it can be used as a desirable 'naked‐eye' indicator candidate for Cu2+ and Fe3+. [ABSTRACT FROM AUTHOR]

BackgroundVarious online rumors have led to inappropriate behaviors among the public in response to the COVID-19 epidemic in China. These rumors adversely affect people’s physical and mental health. Therefore, a better understanding of the relationship between public emotions and rumors during the epidemic may help generate useful strategies for guiding public emotions and dispelling rumors. ObjectiveThis study aimed to explore whether public emotions are related to the dissemination of online rumors in the context of COVID-19. MethodsWe used the web-crawling tool Scrapy to gather data published by People’s Daily on Sina Weibo, a popular social media platform in China, after January 8, 2020. Netizens’ comments under each Weibo post were collected. Nearly 1 million comments thus collected were divided into 5 categories: happiness, sadness, anger, fear, and neutral, based on the underlying emotional information identified and extracted from the comments by using a manual identification process. Data on rumors spread online were collected through Tencent’s Jiaozhen platform. Time-lagged cross-correlation analyses were performed to examine the relationship between public emotions and rumors. ResultsOur results indicated that the angrier the public felt, the more rumors there would likely be (r=0.48, P

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 LC 50 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 ℃, 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. • Avermectin was toxic to Eriocheir sinensis. • A avermectin degrading strain was selected and named Ochrobactrum sp. AVM-2. • Ochrobactrum sp. AVM-2 can effectively reduce the residues of avermectin. [ABSTRACT FROM AUTHOR]

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-1β, Nos2, Arg and Mrc. The protein levels of NF-κB, AP1, NF-Y, p-JNK2, JNK2, p-p38, p38, p-IκBα, IκBα, p-IKKβ and IKKβ 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. [ABSTRACT FROM AUTHOR]

With the module of assimilating AMSU-A (Advanced Microwave Sounding Unit-A) and AIRS (Atmospheric Infrared Sounder) data in the WRFDA (Weather Research and Forecasting Model Data Assimilation) system, the impacts of joint assimilation of the radiance observations from two satellites on the simulation of typhoon Chan-hom (2015) are addressed. For comparison, experiments with the assimilation of solely GTS (Global Telecommunications System) data, AMSU-A data, or AIRS data are also performed. The results show that, compared to other experiments, the analysis field after assimilating multiple radiance data is closer to the observation. The simulated steering flow in its forecast field is conductive to the northeast twist of the typhoon. In addition, the simulated rainband and the FSS (fraction skill score) calculated from the experiment with assimilating multiple radiance data are better. In the deterministic forecast, better performance is obtained from the simulation with multiple radiance data in the forecast of track, MSLP (minimum sea level pressure), and MSW (maximum surface wind). [ABSTRACT FROM AUTHOR]

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. • A novel serine proteases contain two ShK-domains from Eriocheir sinensis , EsShK-SP, was reported first time so far. • The expression of EsShK-SP was significantly up-regulated after Spiroplasma eriocheiris infection. • The over-expression of EsShK-SP increased the proPO transcription and PO activity to resist S. eriocheiris infection. • EsShK-SP RNAi decreased the proPO transcription and PO activity to promote S. eriocheiris infection. [ABSTRACT FROM AUTHOR]

• We compare ELP-VLP vaccine efficacy with two commercial PCV2 vaccines. • We demonstrate that ELP-VLP induces stronger VN antibody and IFN-γ responses. • We show that ELP-VLP is more effective against PCV2 infection. Porcine circovirus type 2 (PCV2) is an economically important swine pathogen and vaccination is the primary tool for the disease control. Previously, we developed a more cost-effective PCV2 virus-like particle (VLP) vaccine by using ELPylation technology. In the present study, we compared the ELPylated VLP (ELP-VLP) PCV2 vaccine efficacy with commercial inactivated Yuanlijia vaccine and VLP-based Circoflex vaccine by experimental challenge. After one dose of vaccination with the three different vaccines, ELP-VLP vaccine group showed significantly (p < 0.05) stronger virus neutralizing antibody and interferon-γ responses than the two commercial vaccine groups. All vaccinated pigs showed significant (p < 0.05) improvement in average daily weight gain (ADWG) before challenge. After challenge with PCV2, however, only ELP-VLP-vaccinated pigs showed significant (p < 0.05) improvement in ADWG. All vaccinated pigs showed significant (p < 0.05) reductions in PCV2 loads in the blood, nasal secretion and lymph nodes, ELP-VLP-vaccinated pigs in particular. In addition, vaccination with ELP-VLP vaccine provided stronger protection against pulmonary and lymphoid pathologies than that with the two commercial vaccines. Therefore, ELP-VLP vaccine is more effective to control PCV2 infection than the two commercial vaccines based on clinical, immunological, virological and pathological evaluations. [ABSTRACT FROM AUTHOR]

Abstract Recombinant interferon-α (rIFN-α) has been widely used for treating viral infections. However, the clinical efficacy of unmodified rIFN-α is limited due to small molecular size and rapid clearance from circulation. In this study we developed a novel strategy for half-life extension of porcine IFN-α (PoIFN-α) by fusion to the immunoglobulin (Ig)-binding C2 domain of streptococcal protein G (SPG). The coding sequences for PoIFN-α6 and SPG C2 domain, with a tobacco etch virus (TEV) protease recognition sequence introduced at the 5-end, were cloned into an elastin-like polypeptide (ELP) fusion expression vector and expressed as an ELP-PoIFNα-C2 fusion protein. After optimization of the conditions for soluble protein expression and purification, the fusion protein was purified to more than 90% purity by two rounds of inverse transition cycling (ITC) in the presence of 0.5% Triton X-100. After cleavage with self-aggregating peptide ELK-16-tagged tobacco etch virus protease, the protease was removed by quick centrifugation and PoIFNα-C2 protein was recovered by an additional round of ITC with 98% purity. Western blotting analysis showed that PoIFNα-C2 protein had the specific affinity for pig IgG binding. The antiviral assay showed that PoIFNα-C2 protein had potent antiviral activities against vesicular stomatitis virus and porcine pseudorabies virus. After single intravenous or subcutaneous injection into rats, PoIFNα-C2 protein showed 16- or 4-fold increase in serum half-life with significantly improved bioavailability. Highlights • We develop a novel strategy for half-life extension of PoIFN-α by fusing to SPG domain C2. • We optimize the conditions for soluble ELP-PoIFNα-C2 protein expression and purification. • We cleave the ELP tag with active TEV protease aggregate. • We demonstrate significantly improved half-life of PoIFNα-C2 protein in rats. [ABSTRACT FROM AUTHOR]

Porcine reproductive and respiratory syndrome (PRRS) is one of the most important swine diseases affecting pig industry worldwide. Sialoadehesin (Sn) and CD163 are the two specific receptors for PRRSV infection of porcine alveolar macrophages. Our previous study showed that the soluble Sn receptor Sn4D-Fc and soluble CD163 receptor SRCR59-Fc expressed by the two recombinant adenoviral (rAd) vectors have an additive anti-PRRSV effect in vitro . In the present study, rAd-Sn4D-Fc and rAd-SRCR59-Fc were inoculated into pigs, and the efficient expression of Sn4D-Fc and SRCR59-Fc proteins was detected by ELISA. Then, PRRSV-naïve pigs were inoculated with rAd-Sn4D-Fc and/or rAd-SRCR59-Fc before contagious infection with different PRRSV strains. Among the three rAd inoculation groups, simultaneous inoculation with the two rAd vectors provided the best protection against highly pathogenic JXA1 strain PRRSV, followed by rAd-SRCR59-Fc inoculation and rAd-Sn4D-Fc inoculation. Clinical observation and quantitative RT-PCR analyses showed that all of the double rAd-inoculated pigs (n = 9) survived from the contagious infection with highly pathogenic JXA1, JS07 or SH1705 strain PRRSV with significantly alleviated clinical scores, viremia, fecal viral emission and tissue virus loads. These data suggest that rAd-Sn4D-Fc and rAd-SRCR59-Fc can be developed further as the universal therapeutic vaccine to facilitate PRRSV eradication. [ABSTRACT FROM AUTHOR]

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 the in-vitro release 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., (© 2023 IOP Publishing Ltd.)

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.

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., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

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., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/atm-20-6314). The authors have no conflicts of interest to declare., (2020 Annals of Translational Medicine. All rights reserved.)

Toll-like receptor (TLR) ligands have emerged as the attractive adjuvant for subunit vaccines. However, selection of TLR ligands needs to be rationally chosen on the basis of antigen and adjuvant properties. In the present study, we expressed the Ag473 lipoprotein from Neisseria meningitides, flagellin FlaB from Vibrio vulnificus and heat shock protein 70 from Mycobacterium tuberculosis (mHsp70) in Escherichia coli as single proteins and fusion proteins with VP2 protein of infectious bursal disease virus (IBDV). Both cellular and humoral adjuvanticities of the three TLR ligands were compared by immunization of mice in two different ways. Among the three co-administered TLR ligands, recombinant Ag473 lipoprotein exhibited the highest cellular and humoral adjuvanticities, including promotion of IL-4, IL-12, IFN-γ and IBDV VP2-specific antibody production. Among the three genetically fused TLR ligands, fusion with Ag473 D1 domain exhibited the highest cellular and humoral adjuvanticities. Overall, the adjuvanticities of genetically fused TRL ligands were significantly higher than that of co-administered TLR ligands. Fusion with Ag473 D1 domain exhibited superior adjuvanticity among the three TLR ligands delivered in two different ways., (© 2020 The Author(s).)