Clinical, experimental and pathophysiological effects of Yaq-001: a non-absorbable, gut-restricted adsorbent in models and patients with cirrhosis.

OBJECTIVE: Targeting bacterial translocation in cirrhosis is limited to antibiotics with risk of antimicrobial resistance. This study explored the therapeutic potential of a non-absorbable, gut-restricted, engineered carbon bead adsorbent, Yaq-001 in models of cirrhosis and acute-on-chronic liver failure (ACLF) and, its safety and tolerability in a clinical trial in cirrhosis. DESIGN: Performance of Yaq-001 was evaluated in vitro. Two-rat models of cirrhosis and ACLF, (4 weeks, bile duct ligation with or without lipopolysaccharide), receiving Yaq-001 for 2 weeks; and two-mouse models of cirrhosis (6-week and 12-week carbon tetrachloride (CCl4)) receiving Yaq-001 for 6 weeks were studied. Organ and immune function, gut permeability, transcriptomics, microbiome composition and metabolomics were analysed. The effect of faecal water on gut permeability from animal models was evaluated on intestinal organoids. A multicentre, double-blind, randomised, placebo-controlled clinical trial in 28 patients with cirrhosis, administered 4 gr/day Yaq-001 for 3 months was performed. RESULTS: Yaq-001 exhibited rapid adsorption kinetics for endotoxin. In vivo, Yaq-001 reduced liver injury, progression of fibrosis, portal hypertension, renal dysfunction and mortality of ACLF animals significantly. Significant impact on severity of endotoxaemia, hyperammonaemia, liver cell death, systemic inflammation and organ transcriptomics with variable modulation of inflammation, cell death and senescence in the liver, kidneys, brain and colon was observed. Yaq-001 reduced gut permeability in the organoids and impacted positively on the microbiome composition and metabolism. Yaq-001 regulated as a device met its primary endpoint of safety and tolerability in the clinical trial. CONCLUSIONS: This study provides strong preclinical rationale and safety in patients with cirrhosis to allow clinical translation. TRIAL REGISTRATION NUMBER: NCT03202498.

A portable CRISPR-Cas12a triggered photothermal biosensor for sensitive and visual detection of Staphylococcus aureus and Listeria monocytogenes.

The detection of foodborne pathogens is crucial for ensuring the maintenance of food safety. In the present study, a portable CRISPR-Cas12a triggered photothermal biosensor integrating branch hybrid chain reaction (bHCR) and DNA metallization strategy for sensitive and visual detection of foodborne pathogens was proposed. The sheared probes were utilized to block the locker probes, which enabled preventing the assembly of bHCR in the absence of target bacteria, while target bacteria can activate the cleavage of sheared probes through CRISPR-Cas12a. Therefore, the locker probes functioned as initiating chains, triggering the formation of the branching double-stranded DNA consisting of H1, H2, and H3. The silver particles, which were in situ deposited on the DNA structure, functioned as a signal factor for conducting photothermal detection. Staphylococcus aureus and Listeria monocytogenes were selected as the foodborne pathogens to verify the analytical performance of this CRISPR-Cas12a triggered photothermal sensor platform. The sensor exhibited a sensitive detection with a low detection limit of 1 CFU/mL, while the concentration ranged from 100 to 108 CFU/mL. Furthermore, this method could efficiently detect target bacteria in multiple food samples. The findings demonstrate that this strategy can serve as a valuable reference for the development of a portable platform enabling quantitative analysis, visualization, and highly sensitive detection of foodborne bacteria.

Comparison of Reversed-Phase and Mixed-Mode SPE for Enrichment and Clean-Up of Surrogate Peptides in CysC Quantified by LC-MS/MS.

Accurate quantification of low-abundance protein Cystatin-C (CysC) in serum by liquid chromatography tandem mass spectrometry (LC-MS/MS) method is very difficult. After sample processing and tryptic digestion, the matrix of CysC surrogate peptides is very complexity, and the concentrations of them are very low, so solid-phase extraction (SPE) must be used to make the surrogate peptides purification and enrichment. In this paper, we used C18 reversed-phase SPE (RP-SPE) and mixed-mode SPE as SPE cartridges. We quantitatively assessed and compared the CysC surrogate peptides recoveries and matrix effects by different SPE cartridges. The sequence of CysC surrogate peptide is ALDFAVGEYNK, and sequence-specific subions y6 (VGEYNK+, m/z 709.3) and y9 (DFAVGEYNK+, m/z 1042.4) were selected for quantification of CysC, because the two fragment ions showed the highest sensitivity. In neat solution, the highest recoveries were similarly for y9 and y6 when used RP-SPE and mixed-mode SPE. However, in serum matrix, the recoveries were significantly higher when used mixed-mode SPE than RP-SPE, which was caused by matrix effects. Results showed that both RP-SPE and mixed-mode SPE were resulted in ion enhancement for CysC surrogate peptides quantification by LC-MS/MS, but mixed-mode SPE reduced more matrix effects. So mixed-mode SPE was more suitable SPE type for purification and enrichment of CysC surrogate peptides.

[Effect of electroacupuncture combined with intradermal needling on simple obesity and serum intestinal lymphatic function-related factors].

OBJECTIVE: To assess the efficacy of the combined treatment with electroacupuncture (EA) and intradermal needling on simple obesity and explore its underlying effect mechanism. METHODS: A total number of 116 patients with simple obesity were randomized into an observation group (58 cases, 3 cases dropped off and 2 cases removed) and a control group (58 cases, 4 cases dropped off and 1 cases removed). Patients in the control group received EA at Zhongwan (CV 12), Quchi (LI 11), Zusanli (ST 36), Pishu (BL 20), Weishu (BL 21), etc., for 30 min each time. On the base of the intervention as the control group, the patients in the observation group received the intradermal needling at Tianshu (ST 25), Daheng (SP 15), Zusanli (ST 36), Shangjuxu (ST 37), Quchi (LI 11), Pishu (BL 20) and Weishu (BL 21). In each group, the intervention was given once every two days, 3 times a week, consecutively for 3 months. Before and after treatment, the obesity indexes (body mass [BW], body mass index [BMI], body fat percentage [F%], adiposity [A] and waist circumference [WC]), the serum intestinal lymphatic function-related factors (vascular endothelial growth factor C [VEGF-C], delta-like ligand 4 [DLL4], adrenomedullin [ADM]), blood lipid (total cholesterol [TC], triglyceride [TG] and low density lipoprotein-cholesterol [LDL-C]), and fasting plasma glucose (FPG), fasting insulin (FINS) and insulin resistance index (HOMA-IR) were observed in the patients of both groups; and the efficacy was assessed. RESULTS: The effective rate was 88.7% (47/53) in the observation group, higher than 71.7% (38/53) in the control group (P<0.05). After treatment, except FPG in the control group, BW, BMI, F%, A, WC, and the concentrations of serum VEGF-C, DLL4 and ADM, as well as TC, TG, LDL-C, FBG, FINS and HOMA-IR were all reduced compared with those before treatment in both groups (P<0.05). The reduction ranges of BW, BMI, F%, A, WC, and the concentrations of serum VEGF-C, DLL4 and ADM, and TC, LDL-C, FINS and HOMA-IR in the observation group were all larger than those in the control group (P<0.05). CONCLUSION: Electroacupuncture combined with intradermal needling can reduce body weight and lipid, and improve insulin resistance in treatment of simple obesity, which is achieved probably through inhibiting lymphangiogenesis and promoting lymphatic endothelial permeability.

Isotope-dilution liquid chromatography-tandem mass spectrometry for quantification of human retinol binding protein 4 in serum.

Serum retinol binding protein 4 levels play critical roles in the early diagnosis and therapeutic monitoring of diabetic kidney disease. In this paper, an liquid chromatography-tandem mass spectrometry approach for the absolute quantification of serum RBP4 with high sensitivity and specificity was presented. Following series of procedures including denaturation, reduction, alkylation and trypsin digestion, the signature peptides of RBP4 were separated on the HPLC system by gradient elution. Quantitative analysis was achieved by a triple quadrupole mass spectrometer under multiple reaction monitoring in the positive ion (ESI+) mode. The calibration range was from 6.25 to 125 mg/L (R2 > 0.993), the lower limit of quantification was 2.50 mg/L, and the lower limit of detection reached 0.0150 mg/L. In the study, the accuracy ranged from 94.6% to 107%, and the relative standard deviation of intra-assay and inter-assay imprecision was less than 5%. The method was demonstrated to realize sensitive and reliable absolute quantification of serum RBP4 conforming to guidelines for bioanalytical method validation.

Establishment of a direct quantitative method for measurement of microRNA-224 in serum by UHPLC/MS/MS.

MicroRNAs (miRNAs) are known as potential noninvasive biomarkers for cancer diagnosis. Previous studies have been reported that miR-224 is upregulated in hepatocellular carcinoma (HCC) tissues and sera samples. However, current available methods of miRNA detection typically require pre-enrichment, amplification and labeling steps, and mostly they are semi-quantitative. Herein, we developed an isotope dilution mass spectrometry approach to convert the signal of miR-224 into the mass response of a reporter peptide. Specifically, the newly formed DNA-peptide probe was hybridized with miR-224, which was biotinylated and attached to streptavidin agarose in advance. After through trypsinization, solid phase extraction and blow drying, it used a UHPLC/MS/MS-based quasi-targeted proteomics assay for miR-224 quantification and determines the peak area or peak height ratio of labeled and non-labeled analytes to which an isotope label was added to estimate the concentration of the analyte in the sample. Moreover, this method showed good linearity, precision, accuracy and recovery in the calibration range. In addition, it also demonstrated good performance in comparison with qRT-PCR. Taken together, this study may offer a novel direct quantitative method for serum miRNA analysis applied in clinical practice.

Absolute quantification of urinary neutrophil gelatinase-associated lipocalin by ultra-high-performance liquid chromatography/tandem mass spectrometry and the diagnostic efficacy of acute kidney injury.

RATIONALE: To establish an absolute quantification method for neutrophil gelatinase-associated lipocalin (NGAL) by ultra-high-performance liquid chromatography tandem positive ion electrospray ionization mass spectrometry (UHPLC/MS/MS) and evaluate its diagnostic efficacy for acute kidney injury (AKI). METHODS: Three target peptides of NGA were prescreened by Skyline software, and two of them could be detected in tryptic peptides of NGAL recombinant protein and human urinary NGAL (uNGAL). Peptide (WYVVGLAGNAILR) was then selected as surrogate peptide. The corresponding isotope-labeled peptide as the internal standard was next synthesized. Quantification of uNGAL was based on equations of linear regression, and method validation was then conducted. The diagnostic efficacy of uNGAL for AKI was also evaluated using receiver operating characteristic curve (ROC) analysis. Lastly, the UHPLC/MS/MS and the particle-enhanced turbidimetric immunoassay (PETIA) methods for uNGAL quantification were compared. RESULTS: For the y9 and y10 product ions, the linear regression equations were y = 2.5519x-4.6955 (R2 = 0.994, P<.01) and y = 2.4619x-4.3 (R2 =0.993, P<.01), respectively, and both of the linear ranges were from 0.5 to 15 mg/L. The limits of detection and quantification were 0.037 mg/L and 0.081 mg/L, respectively. The recoveries were from 97.32% to 107.28% at different uNGAL levels, and the within- and between-day CVs for uNGAL quantification were from 0.22% to 7.65% and from 0.66% to 5.97%, respectively. The carryover rates of uNGAL were in the range of 0.70%-0.99%. The area under the ROC curve (AUC) of uNGAL was 0.96 (P<0.01), and the sensitivity and specificity of uNGAL for AKI diagnosis were 90.0% and 92.5%, respectively. In addition, the UHPLC/MS/MS and PETIA methods showed good agreement for uNGAL quantification (y = 0.7112x-0.0139, P = 0.34). CONCLUSIONS: The UHPLC/MS/MS method for uNGAL quantification has a wide linear range, high sensitivity, precision, and recovery, and low carryover rates, and uNGAL detected by this method had high sensitivity and specificity for AKI diagnosis.

An LC-MS/MS assay with a label-free internal standard for quantitation of serum cystatin C.

Cystatin C is considered as an alternative to the evaluation of glomerular filtration rate. In this study, we highlighted an LC-MS/MS approach for the absolute quantitation of serum cystatin C based on label-free internal standards. A tryptic peptide (ALDFAVGEYNK) was selected as the surrogate whilst analogue (ALDFAVGEYQK) served as an internal standard. After denaturation, reduction, alkylation, digestion and concentration, the target peptides were separated on an LC column and monitored under MRM. The calibration range was from 0.25 mg/L to 15 mg/L with LLOQ of 0.05 mg/L and LOD of 0.03 mg/L, respectively. The certified reference material (ERM-DA471) was determined at 5.12 mg/L with bias of 6.57%. The recovery was between 89.68% and 92.43%. The RSD of intra- and inter-assay imprecision were both <10%. Good stability was also observed. The assay also demonstrated that the quantification of native cystatin C in human serum could be achieved using label-free internal standards. The assay was robust, cheap and sensitive.

Quantification of cystatin-C in human serum by stable isotope dilution liquid chromatography electrospray ionization tandem mass spectrometry.

Cystatin C (CysC)-based estimated glomerular filtration rate is an excellent marker for early diagnosis of Chronic Kidney Disease (CKD). Particle-enhanced nephelometric immunoassay (PENIA) and particle-enhanced turbidimetric immunoassay (PETIA) are commonly used methods for CysC quantification in clinical testing. However, both of them lack of specificity which mass spectrometry offers. In this paper, an isotope dilution liquid chromatography tandem mass spectrometry (ID-LC-MS/MS) method was established for quantification of CysC in human serum. After CysC denaturation, reduction and alkylation of cysteine residues, trypsin was added for proteolytic digestion of CysC. Specific peptide ALDFAVGEYNK was selected as surrogates for intact CysC protein, then quantified CysC by stable isotope-labelled internal standard peptide A[13C615N]LDFAVGEYNK based on calibration curve method. The calibration curves were y=0.1565x-1.6715 (R2=0.988) and y=1.8785x-2.2497 (R2=0.991) for y9 and y6, respectively. The linear range was 0.1-10mg/L and 0.5-15mg/L for y9 and y6, respectively. The limit of quantification (LOQ) was 0.052mg/L. At different concentrations of CysC, the recoveries were in the range of 80.5%-93.7%, the intraday precisions were in the range of 0.3%-2.2%, and the inter-day precisions were in the range of 0.2%-2.8%. The results show that ID-LC-MS/MS and PETIA methods have higher consistency (y=0.8021x+0.6611, p=0.14), and the mean difference of the two methods was -0.29mg/L, and 95% of the individual difference values were in the range of -0.91mg/L-0.33mg/L.

Evaluation of a modified lateral flow immunoassay for detection of high-sensitivity cardiac troponin I and myoglobin.

We prospectively evaluated the use of lateral flow immunoassay (LFIA) test modified with nanoparticles for combined detection of high-sensitivity cardiac troponin I (hs-cTnI) and myoglobin with the aim of excluding acute myocardial infarction (AMI). Specimens from 173 patients with symptoms suggestive of AMI were collected to measure hs-cTnI and myoglobin using an electrochemiluminescence immunoassay (ECLI) and the LFIA test modified with nanoparticles, and a comparison was performed between the modified method and a commercial LFIA test for detection of the two proteins. The accuracy of the modified LFIA test was also evaluated. Consistent agreement was observed in the quantitative comparison of 173 clinical samples using the modified LFIA and ECLI, and the modified method was more sensitive than the commercial LFIA test. The accuracy of the modified LFIA was <12% for both hs-cTnI and myoglobin. Thus, the new approach has great potential to improve LFIAs test, demonstrating its usefulness for simple screening applications and for sensitivity and quantitative immunoassays for diagnosis ofAMI.

Effect of HSP65 on the expression of adhesion molecules in mice heart endothelial cells.

This study aims to research the effect of HSP65 on the expression of adhesion molecules in activated mice heart endothelial cells (MHECs), which were from myocardial tissue of newborn animals. We used different concentrations of LPS as potent inducers to stimulate MHECs, adhesion molecule expression in vitro, including intercellular adhesion molecule-1 (ICAM-1), vascular adhesion molecule-1 (VCAM-1), E-, and P-selectins, then compared the mRNA and protein levels of adhesion molecules expression with or without HSP65 treatment at different levels. The optimal concentration of LPS to induce MHECs adhesion molecule expression is 100 ng/ml; HSP65 treatment significantly reduced the mRNA and protein levels of MHECs' ICAM-1, VCAM-1, E-, and P-selectins expression (p < 0.05), and the optimal concentration of HSP65 in inhibiting MHECs activation is 0.8 ng. HSP65 has the inhibitory effect on adhesion molecules expression in activated MHECs.

Overexpression of PPARγ can down-regulate Skp2 expression in MDA-MB-231 breast tumor cells.

Skp2 is frequent amplified and overexpressed in breast cancer, making it a potential molecular target for cancer therapy. The objective of this study was to examine the effect of PPARγ overexpression on Skp2 expression in breast cancer cell lines. First, we investigated the role of PPARγ and Skp2 in human breast cancer progression. Immunohistochemical analysis of 70 specimens on formalin-fixed paraffin sections was performed. Furthermore in vitro, Western blot analysis was used to study the relationship between PPARγ and Skp2. We found that the expression of PPARγ and Skp2 expression was inverse correlation whether in vivo or in vitro. In addition, PPARγ overexpression can down-regulate the expression of Skp2 mRNA and protein in breast cancer cells. PPARγ overexpression decreased breast cancer cell proliferation and induced spontaneous apoptosis even in the absence of exogenous ligand. These PPARγ-overexpressing cells were dramatically more sensitive to PPARγ ligand-induced apoptosis compared with parental or Myc-control transfected cells. Overexpressing of Skp2 partially reversed PPARγ's pro-apoptotic and anti-proliferative abilities. These results suggested that PPARγ's pro-apoptotic and anti-proliferative abilities appear to be triggered at least in part by the modulation of Skp2.

PPARγ agonist pioglitazone inhibits microglia inflammation by blocking p38 mitogen-activated protein kinase signaling pathways.

OBJECTIVE: The aim of this paper was to investigate the inhibitory effect of peroxisome proliferator-activated receptor-gamma (PPARγ) agonist pioglitazone on microglia inflammation induced by lipopolysaccharide (LPS). MATERIALS AND METHODS: Highly aggressively proliferating immortalized cells were used from a rat microglial cell line. Expression of PPARγ, inducible NO synthase (iNOS), the p42/44 extracellular signal-regulated kinase (ERK) MAPKs, c-Jun NH2-terminal kinases (JNKs) and p38 MAPK were determined by Western blot analysis. The protein levels of tumor necrosis factor α (TNF-α), interleukin-6 (IL-6), and interleukin-1ß (IL-1ß) were determined by enzyme-linked immunosorbent assay. The production of nitric oxide (NO) was determined by a Nitric Oxide Assay Kit. The subcellular localization of PPARγ was studied by immunofluorescence microscopy analysis and nuclear-cytosolic fractionation technology, respectively. The transcriptional activity of PPARγ was detected by PPRE-Luciferase transcription assay. RESULTS: Pioglitazone effectively inhibited NO, iNOS, TNF-α, IL-6, IL-1ß production in LPS-stimulated microglial cells. Additionally, pioglitazone suppressed PPARγ loss; enhanced transcriptional activity of PPARγ; and inhibited nucleus-export of PPARγ in microglia induced by LPS. And p38 MAPK inhibitor SB203580 had the similarity effects with pioglitazone. Signal transduction studies indicated that pioglitazone blocked the phosphorylation of p38 MAPK challenged by LPS. CONCLUSION: The results show that pioglitazone can inhibit LPS-stimulated microglia inflammation by blocking p38 MAPK signaling pathway.

Involvement of Src-suppressed C kinase substrate in experimental autoimmune encephalomyelitis: a link between release of astrocyte proinflammatory factor and oligodendrocyte apoptosis.

Src-suppressed C kinase substrate (SSeCKS) is involved in inflammation in the central nervous system (CNS), and plays a role in control of cell signaling and cytoskeletal arrangement. However, the expression and function of SSeCKS and its function in multiple sclerosis (MS) and its common animal model, experimental autoimmune encephalomyelitis (EAE) remained to be elucidated. In the present study, we first reported that SSeCKS was remarkably increased in astrocytes of EAE rats in vivo. TNF-alpha and NO were significantly induced in astrocytes stimulated with LPS/IFN-gamma in vitro, which was blocked in astrocytes transfected with SSeCKS siRNA. These results indicated that SSeCKS played a role in the production of TNF-alpha and NO in astrocytes with inflammatory stimulation. As excessive release of TNF-alpha and NO were major mediators in autoimmune diseases and correlated with oligodendrocyte cell death, we further investigated whether SSeCKS participated in oligodendrocyte apoptosis. Conditioned media (CM) from astrocytes treated with LPS/IFN-gamma decreased oligodendrocyte cell viability, while siRNA targeted to SSeCKS in astrocytes inhibited oligodendrocyte cell death. The results from antibody neutralization and NO inhibition suggested that the oligodendrocyte apoptosis may be due to the production of astrocyte-derived proinflammatory factors (TNF-alpha and NO). These findings revealed that there was a pathogenic interaction between SSeCKS expression in astrocytes and oligodendrocyte apoptosis. Understanding the mechanism of SSeCKS in the pathogenesis of EAE may contribute to the development of new therapeutic strategies against EAE and MS.

Peroxisome proliferator-activated receptor-gamma agonists suppress iNOS expression induced by LPS in rat primary Schwann cells.

In bacterial-induced peripheral nervous system (PNS) inflammation, Schwann cells (SCs) are activated, producing inducible nitric oxide synthase (iNOS), contributed to the pathogenesis of demyelinating disease, such as multiple sclerosis. Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) has been shown to play a protective role in cellular inflammatory responses. Here we showed that LPS-induced iNOS biosynthesis was in a concentration and time-dependent manner. In LPS-treated primary SCs, retreatment with PPAR-gamma agonist remitted the increase of iNOS, p38 phosphorylation and TLR4, MyD88, augmented the expression of PPAR-gamma and localization in nuclear. Coadministration of GW 9662 reversed the effect of PPAR-gamma agonists. These results suggest that PPAR-gamma agonists, 15d-PGJ(2) and pioglitazone, had the anti-inflammatory effects.