The association between activation of the ERK1/2-NF-κB signaling pathway by TIMP2 expression and chronic renal allograft dysfunction in the CRAD rat model.

PURPOSE: The tissue inhibitor of metalloproteinase 2 (TIMP2), a natural inhibitor of matrix metalloproteinase (MMP), regulates inflammation, fibrosis, and cell proliferation. Chronic renal allograft dysfunction (CRAD) is a primary factor affecting the long-term survival of renal allografts. We assessed whether up-regulation of TIMP2 expression may affect the ERK1/2-NF-κB signaling pathway and CRAD development. METHODS: Lewis rats received orthotopic F344 kidney allografts to establish the classical CRAD model. The treatment group was injected with a lentivirus encoding a TIMP2-targeting small hairpin (sh)RNA (LTS) at 5 × 108 TU/ml monthly after kidney transplantation. A second CRAD group was injected with a lentivirus TIMP2-control vector (LTC). After 12 weeks, blood, urine, and kidney tissue were harvested to evaluate renal function and pathological examinations. Hematoxylin and eosin staining, Masson staining, and Periodic acid-Schiff staining were performed for renal histopathological evaluation according to the Banff criteria. TIMP2, phospho (p)-ERK1/2, p-p65 (NF-κB) expression levels were measured via immunohistochemical and Western blot analyses. RESULTS: Compared to the F344 and Lewis control groups, the expression of TIMP2, p-ERK1/2, and p-p65 were significantly higher in the CRAD and CRAD+LTC renal tissues (p < 0.05). There were also increased levels of serum creatinine, nitrogen, and 24 h urinary protein in these two groups (p < 0.05). Typical histopathological changes of CRAD were observed in the CRAD and CRAD+LTC groups. Administration of LTS effectively decreased the expression of TIMP2, p-ERK1/2, and p-P65, and reduced interstitial fibrosis and macrophage infiltration in the treatment group (p < 0.05). Additionally, MCP1 and ICAM-1, which are downstream cytokines of the NF-κB pathway, were also inhibited in the renal rat kidney from the LTS group (p < 0.05). Furthermore, renal function was well preserved in the LTS group compared to the CRAD group and CRAD+LTC group. CONCLUSION: A decrease of TIMP2 can alleviate the progression of inflammation in CRAD via inhibition of the ERK1/2-NF-κB signaling pathway.

A frameshift variant in the SIRPB1 gene confers susceptibility to Crohn's disease in a Chinese population.

Background: Crohn's disease (CD), a chronic gastrointestinal inflammatory disease, is increasing in China. With a focus on Han Chinese families with CD, the aim of this study was to find genetic variations that increase CD susceptibility by genome sequencing, genetic association, expression, and functional research. Materials and methods: We performed family-based genome sequencing (WGS) analysis on 24 patients with CD from 12 families and then filtered shared potential causal variants by incorporating association results from meta-analyses of CD GWAS and immunology genes and in silico variant effect prediction algorithms. Replication analyses were performed in an independent cohort including 381 patients with CD and 381 control subjects. Results: There were 92 genetic variants significantly associated with CD in Chinese individuals. Among them, 61 candidate loci were validated in replication analyses. As a result, patients carrying a rare frameshift variant (c.1143_1144insG; p. Leu381_Leu382fs) in gene SIRPB1 had significantly higher risk to develop CD (p = 0.03, OR 4.59, 95% CI 0.98-21.36, 81.82% vs. 49.53%). The frameshift variation induced tyrosine phosphorylation of Syk, Akt, and Jak2, elevated the expression of SIRPB1 at the mRNA and protein levels, activated DAP12, and controlled the activation of NF-κB in macrophages. Additionally, it promoted the synthesis of the pro-inflammatory cytokines IL-1, TNF-, and IL-6. Conclusion: Our results suggest that the rare gain-of-function frameshift variant in SIRPB1 is associated in Han Chinese patients with CD. The functional mechanism of SIRPB1 and its downstream inflammatory pathways was preliminarily explored in CD.

A single nucleotide polymorphism-based formula to predict the risk of propofol TCI concentration being over 4 µg mL-1 at the time of loss of consciousness.

We aim to develop a formula based on single nucleotide polymorphisms (SNPs) to predict whether the propofol target-controlled infusion (TCI) concentration would be over 4 µg mL-1 at the time of loss of consciousness (LOC). We recruited 184 patients undergoing thyroid or breast surgeries with propofol anaesthesia. A total of 48 SNPs of CYP2B6, CYP2C9, UGT1A9, HNF4A, ABCB1, ABCC4, ABCG2, GABRA2, GABRA4, GABRB1, GABRB3, GABRG2, GABBR2, GAD1, SLC1A3, BDNF, and NRXN1, previously associated with propofol metabolic and pharmacology pathway, were genotyped. The formula was developed in the training cohort using the least absolute shrinkage and selection operator logistic regression model, and then validated in the testing cohort. The SNPs, GABBR2 rs1167768, GABBR2 rs1571927, NRXN1 rs601010, BDNF rs2049046, GABRA4 rs1512135, UGT1A9 rs11692021, GABBR2 rs2808536, HNF4A rs1884613, GABRB3 rs2017247, and CYP2B6 rs3181842 were selected to construct the SNP-based formula, which was used to calculate the risk score for over 4 µg mL-1 TCI concentration of propofol at the time of LOC. Patients in the high-risk group were more likely to require a propofol concentration higher than 4 µg mL-1 and presented a longer LOC latency. The SNP-based formula may significantly improve the safety and effectiveness of propofol-induced anaesthesia.

Genome-Wide Analysis of the Rab Gene Family in Melilotus albus Reveals Their Role in Salt Tolerance.

Melilotus albus is a high-quality forage, due to its high protein content, and aboveground biomass and salt tolerance. Rab (Ras-related protein in the brain) proteins are the largest GTPase family which play a key role in intracellular membrane transport, and many Rab genes have been identified in eukaryotes. The growth and distribution of M. albus are severely hampered by soil salinization. However, little is known about candidate genes for salt tolerance in M. albus. In this study, 27 Rab family genes were identified for the first time from M. albus, and divided into eight groups (Groups A-H). The number of introns in MaRabs ranged from one to seven, with most genes containing one intron. In addition, most MaRab proteins showed similarities in motif composition. Phylogenetic analysis and structural-domain comparison indicated that Rab family genes were highly conserved in M. albus. Members of the MaRab gene family were distributed across all eight chromosomes, with the largest distribution on chromosome 1. Prediction of the protein interaction network showed that 24 Rab proteins exhibited protein-protein interactions. Analysis of the promoter cis-acting elements showed that MaRab-gene family members are extensively involved in abiotic stress responses. RNA-seq data analysis of the MaRab-gene-expression patterns suggested that the Rab gene family possesses differentially expressed members in five organs and under salt stress, drought stress, and ABA (Abscisic Acid) treatment. Differentially expressed genes under drought stress, salt stress and ABA stress were validated by quantitative real-time PCR. Furthermore, heterologous expression in yeast was used to characterize the functions of MaRab1 and MaRab17, which were upregulated in reaction to salt stress. In summary, this study provided valuable information for further research into the molecular mechanism of the response of M. albus to saline stress, as well as the possibility of developing cultivars with high salt-resistance characteristics.

Multi-alleles predict primary non-response to infliximab therapy in Crohn's disease.

BACKGROUND: Infliximab (IFX) is the first-line treatment for patients with Crohn's disease (CD) and is noted for its relatively high cost. The therapeutic efficacy of IFX has noticeable individual differences. Known single-gene polymorphisms (SNPs) are inadequate for predicting non-response to IFX. In this study, we aimed to identify new genetic factors associated with IFX-therapy failure and to predict non-response to IFX by developing a multivariate predictive model. METHODS: In this retrospective study, we collected and analysed the data of Chinese patients with CD who received IFX therapy at one hospital between June 2013 and June 2019. Primary non-response (PNR) and non-durable response (NDR) were evaluated using a simple endoscopic score for CD (SES-CD). A total of 125 SNPs within 44 genes were genotyped. A multivariate logistic-regression model was established to predict non-response to IFX. An area-under-the-receiver-operating-characteristics curve (AUROC) was applied to evaluate the predictive model performance. RESULTS: Forty-two of 206 (20.4%) patients experienced PNR and 15 of 159 (9.4%) patients experienced NDR. Nine SNPs were associated with PNR (P < 0.05). A PNR predictive model was established, incorporating 2-week high-sensitivity C-reactive protein (hs-CRP), rs61886887, rs61740234, rs357291, rs2269330, and rs111504845, and the AUROC on training and testing data sets were 0.818 (P < 0.001) and 0.888 (P < 0.001), respectively. At week 14, hs-CRP levels ≥ 2.25 mg/L were significantly associated with NDR (AUROC = 0.815, P < 0.001). PNR-associated SNPs were not mutually associated with NDR, suggesting distinct mechanisms between PNR and NDR. CONCLUSION: Genetic polymorphisms are significantly associated with response to IFX among Chinese CD patients.

Randomised clinical trial: dose optimising strategy by NUDT15 genotyping reduces leucopenia during thiopurine treatment of Crohn's disease.

INTRODUCTION: Thiopurine S-methyltransferase (TPTM) is a well known biomarker for thiopurine-induced leucopenia, which has limited value in Asia. Instead, NUDT15 C415T is a promising predictor in Asia. AIMS: To explore whether an optimised strategy based on NUDT15 C415T genotypes affects thiopurine-induced leucopenia, as well as efficacy in Chinese patients with Crohn's disease. METHODS: Patients with Crohn's disease and indications for thiopurines were included from two hospitals in China. They were randomly assigned to either the intervention or the control group. In the intervention group, those with genotype CC received a standard dose (control group), those with CT genotype received 50% of the standard dose, those with TT genotype received alternative drugs. The primary endpoint was thiopurine-induced leucopenia (<3.5 × 109 /L). Secondary outcomes were the incidence of other adverse events and the efficacy for maintaining steroid-free remission at week 36. RESULTS: The rate of thiopurine-induced leucopenia was lower in the intervention group (n = 52) than in the control group (n = 66) (23.7% vs 32.4%, P = 0.049, RR = 0.73, 95% CI 0.53-1.00). In CT subgroup, the incidence of leucopenia in the intervention group (n = 10) was significantly lower than in the control group (n = 28) (31.3% vs 65.1%, RR = 0.48, 95% CI 0.28-0.84). Neither other adverse events nor treatment efficacy was significantly different between the two groups during follow-up. CONCLUSIONS: Among Chinese patients with Crohn's disease, dose optimisation by NUDT15 C415T reduced the rate of thiopurine-induced leucopenia, without significant influence on efficacy. Using 50% dose reduction for heterozygotes, and alternative drugs for homozygotes, are practicable strategies. Clinical trial number: NCT02929706.

TGF-ß1 Signaling: Immune Dynamics of Chronic Kidney Diseases.

Chronic kidney disease (CKD) is a major cause of morbidity and mortality worldwide, imposing a great burden on the healthcare system. Regrettably, effective CKD therapeutic strategies are yet available due to their elusive pathogenic mechanisms. CKD is featured by progressive inflammation and fibrosis associated with immune cell dysfunction, leading to the formation of an inflammatory microenvironment, which ultimately exacerbating renal fibrosis. Transforming growth factor ß1 (TGF-ß1) is an indispensable immunoregulator promoting CKD progression by controlling the activation, proliferation, and apoptosis of immunocytes via both canonical and non-canonical pathways. More importantly, recent studies have uncovered a new mechanism of TGF-ß1 for de novo generation of myofibroblast via macrophage-myofibroblast transition (MMT). This review will update the versatile roles of TGF-ß signaling in the dynamics of renal immunity, a better understanding may facilitate the discovery of novel therapeutic strategies against CKD.

Skeletal screening IMPC/KOMP using µCT and computer automated cryohistology: Application to the Efna4 KO mouse line.

The IMPC/KOMP program provides the opportunity to screen mice harboring well defined gene-inactivation mutations in a uniform genetic background. The program performs a global tissue phenotyping survey that includes skeletal x-rays and bone density measurements. Because of the relative insensitivity of the two screening tests for detecting variance in bone architecture, we initiated a secondary screen based on µCT and a cryohistolomorphological workflow that was performed on the femur and vertebral compartments on 220 randomly selected knockouts (KOs) and 36 control bone samples over a 2 1/2 year collection period provided by one of the production/phenotyping centers. The performance of the screening protocol was designed to balance throughput and cost versus sensitivity and informativeness such that the output would be of value to the skeletal biology community. Here we report the reliability of this screening protocol to establish criteria for control skeletal variance at the architectural, dynamic and cellular histomorphometric level. Unexpected properties of the control population include unusually high variance in BV/TV in male femurs and greater bone formation and bone turnover rates in the female femur and vertebral trabeculae bone compartments. However, the manner for maintaining bone formation differed between these two bone sites. The vertebral compartment relies on maintaining a greater number of bone forming surfaces while the femoral compartment utilized more matrix production per cell. The comparison of the architectural properties obtained by µCT and histomorphology revealed significant differences in values for BV/TV, Tb.Th and Tb.N which is attributable to sampling density of the two methods. However, as a screening tool, expressing the ratio of KO to the control line as obtained by either method was remarkably similar. It identified KOs with significant variance which led to a more detailed histological analysis. Our findings are exemplified by the Efna4 KO, in which a high BV/TV was identified by µCT and confirmed by histomorphometry in the femur but not in the vertebral compartment. Dynamic labeling showed a marked increase in BFR which was attributable to increased labeling surfaces. Cellular analysis confirmed partitioning of osteoblast to labeling surfaces and a marked decrease in osteoclastic activity on both labeling and quiescent surfaces. This pattern of increased bone modeling would not be expected based on prior studies of the Ephrin-Ephrin receptor signaling pathways between osteoblasts and osteoclasts. Overall, our findings underscore why unbiased screening is needed because it can reveal unknown or unanticipated genes that impact skeletal variation.

Association of polymorphisms in C1orf106, IL1RN, and IL10 with post-induction infliximab trough level in Crohn's disease patients.

BACKGROUND: Trough levels of the post-induction serum infliximab (IFX) are associated with short-term and long-term responses of Crohn's disease patients to IFX, but the inter-individual differences are large. We aimed to elucidate whether single gene polymorphisms (SNPs) within FCGR3A, ATG16L1, C1orf106, OSM, OSMR, NF-κB1, IL1RN, and IL10 partially account for these differences and employed a multivariate regression model to predict patients' post-induction IFX levels. METHODS: The retrospective study included 189 Crohn's disease patients undergoing IFX therapy. Post-induction IFX levels were measured and 41 tag SNPs within eight genes were genotyped. Associations between SNPs and IFX levels were analysed. Then, a multivariate logistic-regression model was developed to predict whether the patients' IFX levels achieved the threshold of therapy (3 µg/mL). RESULTS: Six SNPs (rs7587051, rs143063741, rs442905, rs59457695, rs3213448, and rs3021094) were significantly associated with the post-induction IFX trough level (P = 0.015, P < 0.001, P = 0.046, P = 0.022, P = 0.011, P = 0.013, respectively). A multivariate prediction model of the IFX level was established by baseline albumin (P = 0.002), rs442905 (P = 0.025), rs59457695 (P = 0.049), rs3213448 (P = 0.056), and rs3021094 (P = 0.047). The area under the receiver operating characteristic curve (AUROC) of this prediction model in a representative training dataset was 0.758. This result was verified in a representative testing dataset, with an AUROC of 0.733. CONCLUSIONS: Polymorphisms in C1orf106, IL1RN, and IL10 play an important role in the variability of IFX post-induction levels, as indicated in this multivariate prediction model of IFX levels with fair performance.

Determination of apatinib and its three active metabolites by UPLC-MS/MS in a Phase IV clinical trial in NSCLC patients.

Aim: To develop and validate a simple method using UPLC-MS/MS for determination of apatinib and its three active metabolites in a Phase IV clinical trial. Materials & methods: All compounds were separated on a Hypersil GOLD™ aQ C18 Polar Endcapped LC column (50 × 2.1 mm, 1.9 µm, Thermo) using 5 mmol/l ammonium acetate with 0.1% formic acid:acetonitrile (20:80, v/v) as the mobile phase after a rapid liquid-liquid extraction. This method was validated over the linear concentration range of 1.00-1000 ng/ml for each compound. Results: The interassay precision and accuracy were less than ±15%. The validated method was successfully applied to determine concentrations of clinical samples in non-small-cell lung cancer patients.

miR-429 suppresses cell proliferation, migration and invasion in nasopharyngeal carcinoma by downregulation of TLN1.

BACKGROUND: miR-429 and TLN1 have been shown to affect the biological behaviours of many carcinomas. However, their effects in nasopharyngeal carcinoma (NPC) are not yet clear. Here, we investigated their regulatory relationships and effects on NPC cells. METHODS: TargetScan was used to predict the regulatory relationships of miR-429 and TLN1 in NPC cells. Then, Western blotting and quantitative real-time PCR (qPCR) were performed to examine TLN1 levels, and qPCR was used to determine miR-429 levels in NPC cell lines with different metastatic characteristics (5-8F, CNE-2, CNE-1, 6-10B and NP69), to investigate whether TLN1 and miR-429 are correlated with the metastatic characteristics of these cells. Next, we upregulated or downregulated miR-429 in 5-8F and 6-10B cells, which have different tumourigenicity and transferability, and examined TLN1 expression by western blotting and qPCR after transfection. QPCR was also performed to confirm successful transfection of miR-429 mimic into 5-8F and 6-10B cells. Dual luciferase reporter gene assay was performed to investigate whether miR-429 regulates TLN1 by binding to its 3'UTR. After transfection, Cell Counting Kit-8 (CCK8) and IncuCyte were used to examine the proliferation of these cells, and wound-healing assay, Transwell migration assay, and invasion assays were performed to investigate the changes in migration and invasion after transfection. RESULTS: Western blotting and qPCR analyses showed that the protein level of TLN1 was negatively correlated with miR-429 in NPC cell lines (P < 0.05), while the mRNA level showed no relation with miR429 expression (P > 0.05). In addition, cells with high transferability showed high TLN1 expression at the protein level, while miR429 expression showed the opposite trend (P < 0.05), but there were no differences at the mRNA level between the different cell lines. Overexpression of miR429 in 5-8F and 6-10B cells was accompanied by downregulation of TLN1 at the protein level (P < 0.05), while there were no significant differences at the mRNA level (P > 0.05). In addition, transferability, proliferation, and invasion were downregulated by miR429 overexpression (P < 0.05). However, dual-luciferase reporter gene assay indicated that TLN1 was not a direct target of miR-429. CONCLUSION: This study showed that miR-429 functions as a tumour suppressor in NPC by downregulation of TLN1, although the relationship is not direct.

Screening Gene Knockout Mice for Variation in Bone Mass: Analysis by µCT and Histomorphometry.

PURPOSE OF REVIEW: The international mouse phenotyping consortium (IMPC) is producing defined gene knockout mouse lines. Here, a phenotyping program is presented that is based on micro-computed tomography (µCT) assessment of distal femur and vertebra. Lines with significant variation undergo a computer-based bone histomorphometric analysis. RECENT FINDINGS: Of the 220 lines examined to date, approximately 15% have a significant variation (high or low) by µCT, most of which are not identified by the IMPC screen. Significant dimorphism between the sexes and bone compartments adds to the complexity of the skeletal findings. The µCT information that is posted at www.bonebase.org can group KOMP lines with similar morphological features. The histological data is presented in a graphic form that associates the cellular features with a specific anatomic group. The web portal presents a bone-centric view appropriate for the skeletal biologist/clinician to organize and understand the large number of genes that can influence skeletal health. Cataloging the relative severity of each variant is the first step towards compiling the dataset necessary to appreciate the full polygenic basis of degenerative bone disease.