Rational Regulation of Reaction Specificity of Nitrilase for Efficient Biosynthesis of 2-Chloronicotinic Acid through a Single Site Mutation.

Nitrilase-catalyzed hydrolysis of 2-chloronicotinonitrile (2-CN) is a promising approach for the efficient synthesis of 2-chloronicotinic acid (2-CA). The development of nitrilase with ideal catalytic properties is crucial for the biosynthetic route with industrial potential. Herein, a nitrilase from Rhodococcus zopfii (RzNIT), which showed much higher hydration activity than hydrolysis activity, was designed for efficient hydrolysis of 2-CN. Two residues (N165 and W167) significantly affecting the reaction specificity were precisely identified. By tuning these two residues, a single mutation of W167G with abolished hydration activity and 20-fold improved hydrolysis activity was obtained. Molecular dynamics simulation and molecular docking revealed that the mutation generated a larger binding pocket, causing the substrate 2-CN to bind more deeply in the pocket and form a delocalized π bond between the residues W190 and Y196, which reduced the negative influence of steric hindrance and electron effect caused by chlorine substituent. With mutant W167G as biocatalyst, 100 mM 2-CN was exclusively converted into 2-CA within 16 h. The study provides useful guidance in nitrilase engineering for simultaneous improvement of reaction specificity and catalytic activity, which are highly desirable in value-added carboxylic acids production from nitriles hydrolysis. IMPORTANCE 2-CA is an important building block for agrochemicals and pharmaceuticals with a rapid increase in demand in recent years. It is currently manufactured from 3-cyanopyridine by chemical methods. However, during the final step of 2-CN hydrolysis under high temperature and strong alkaline conditions, the byproduct 2-CM was generated except for the target product, leading to low yield and tedious separation steps. Nitrilase-mediated hydrolysis is regarded as a promising alternative for 2-CA production, which proceeded under mild conditions. Nevertheless, nitrilase capable of efficient hydrolysis of 2-CN has not been reported because the enzymes showed either extremely low activity or surprisingly high hydration activity toward 2-CN. Herein, the reaction specificity of RzNIT was precisely tuned through a single site mutation. The mutant exhibited remarkably enhanced hydrolysis activity without the formation of byproducts, providing a robust biocatalyst for 2-CA biosynthesis with industrial potential.

Assessment of serum Tim-3 and Gal-9 levels in predicting the risk of infection after kidney transplantation.

Infection remains a major cause of morbidity and mortality after kidney transplantation (KT). Reliable biomarkers to predict post-transplant infection are lacking. We investigated the predictive performance of pre- and post-transplant levels of T-cell immunoglobulin and mucin domain-3 (Tim-3) and Galectin-9 (Gal-9), two pleiotropic immunomodulatory molecules, in early identification of infection. Serum Tim-3 and Gal-9 were paired measured before and 30 days after transplantation (PTD 30) in 95 KT recipients (KTRs). The decline rates of Tim-3 and Gal-9 were calculated relative to pre-transplant levels. KTRs with infection history had significantly higher levels of PTD 30 Tim-3 and Gal-9, and slower decrease rates of Gal-9 compared to non-infected recipients, while no difference was observed between two groups regarding pre-transplant levels. The AUCs for predicting 1-year post-transplant infection were 0.653 and 0.711 for post-transplant Tim-3 and Gal-9, 0.664 and 0.670 for relative Tim-3 and Gal-9, respectively. After adjusting for potential confounders, PTD 30 Tim-3, Gal-9 and relative Gal-9 remained as independent risk factors for post-transplant infection. Our results suggested that PTD 30 Tim-3 and Gal-9 and relative decrease of Gal-9 were promising predictors for identifying KTRs with high risk of infection, while pre-transplant Tim-3 and Gal-9 showed no predictive power to infection.

Comparison of urine and blood NGAL for early prediction of delayed graft function in adult kidney transplant recipients: a meta-analysis of observational studies.

BACKGROUND: Neutrophil gelatinase-assoicated lipocalin (NGAL) appears to be a promising proximal tubular injury biomarker for early prediction of delayed graft function (DGF) in kidney transplant recipients. However, its predictive values in urine and blood were varied among different studies. Here, we performed the meta-analysis to compare the predictive values of urine NGAL (uNGAL) and blood NGAL (bNGAL) for DGF in adult kidney transplant recipients. METHODS: We systematically searched Medline, Cochrane library and Embase for relevant studies from inception to May 2018. The summary receiver operating characteristic (SROC) curves, the pooled sensitivity, specificity and diagnostic odds ratio (DOR) were used to evaluate the prognostic performance of uNGAL and bNGAL for the identification of DGF. RESULTS: A total of 1036 patients from 14 eligible studies were included in the analysis. 8 studies focused on NGAL in urine and 6 reported NGAL in serum or plasma. The composite area under the ROC (AUC) for 24 h uNGAL was 0.91 (95% CI, 0.89-0.94) and the overall DOR for 24 h uNGAL was 24.17(95% CI, 9.94-58.75) with a sensitivity of 0.88 (95% CI, 0.75-0.94) and a specificity of 0.81 (95% CI, 0.68-0.89). The composite AUC for 24 h bNGAL was 0.95 (95% CI, 0.93-0.97) and the overall DOR for 24 h bNGAL was 43.11 (95% CI, 16.43-113.12) with a sensitivity of 0.91 (95% CI, 0.81-0.96) and a specificity of 0.86 (95% CI, 0.78-0.92). CONCLUSIONS: Urine and serum/plasma NGAL were valuable biomarkers for early identification of DGF in kidney transplantation. In addition, the bNGAL was superior to uNGAL in early prediction of DGF.

Correlation of CYP2C19 genotype with plasma voriconazole exposure in South-western Chinese Han patients with invasive fungal infections.

The aim of this study was to investigate the correlation between CYP2C19 genotype and dose-adjusted voriconazole (VCZ) trough concentrations (C0/dose).We analyzed the correlation between CYP2C192(681G>A), CYP2C193(636G>A), and CYP2C1917(-806C>T) genetic polymorphisms and the dose-corrected pre-dose concentration (C0/dose) in 106 South-western Chinese Han patients.The frequencies of variant alleles of CYP2C192, 3, and 17 were 29.7%, 4.25%, and 0.92%. For 49.3% of the VCZ samples, the therapeutic window between 1.5 and 5.5 µg/ml was reached. Following the first dose VCZ measurement, in subsequent samples the proportion of VCZ C0 within the therapeutic window increased, suggesting effective therapeutic drug monitoring (TDM) (P = .001). The VCZ C0 was significantly different (P = .010) between patients with normal metabolism (NMs), intermediate metabolism (IMs), and poor metabolism (PMs). The VZC C0/dose was 12.2 (interquartile range (IQR), 8.33-18.2 µg·ml/kg·day), and 7.68 (IQR, 4.07-16.3 µg·ml/kg·day) in PMs and IMs patients, respectively, which was significantly higher than in NMs phenotype patients (4.68; IQR, 2.51-8.87 µg·ml/kg·day, P = .008 and P = .014).This study demonstrated that the VCZ C0/dose was significantly influenced by the CYP2C19 genotype in South-western Chinese Han patients. In this patient population, more over-exposure was observed in patients with a CYP2C19 genotype associated with poor or intermediate metabolism. CYP2C19 genotype-based dosing combined with TDM will support individualization of VCZ dosing, and potentially will minimize toxicity and maximize therapeutic efficacy.

A Low Fixed Tacrolimus Starting Dose Is Effective and Safe in Chinese Renal Transplantation Recipients.

BACKGROUND We investigated whether a low fixed Tac starting dose regimen could lead to a better achievement of Tac target concentrations, as well as an effective immunosuppressive treatment, in Chinese kidney transplant recipients (KTRs). MATERIAL AND METHODS We collected whole-blood and serum samples from 189 KTRs and the Tac starting dose was 2, 2.5, or 3 mg/day. Information on Tac C0, dose, body weight, body mass index (BMI), Scr, eGFR, and CYP3A5 genotypes were collected from a routine therapeutic drug monitoring database. The correlation between Tac C0 and body weight (or BMI) was investigated by calculating the goodness of fit. Multivariable logistic regression was performed to estimate the independent associated factors. RESULTS The patients with 3 mg per day of Tac had higher C0 at day 7 compared to those with 2 or 2.5 mg. For patients receiving the same Tac starting dose, no significant difference was found in Tac C0 at day 7 among different body weight or BMI groups. There was no significant difference in Scr or eGFR at 1 year after transplant, nor was there a significant difference in the rates of DGF or AR at post-transplant day 30 among different Tac starting dose groups or among the 3 Tac C0 range groups. CYP3A5 genotype and Tac initial dose were independently associated with Tac C0. CONCLUSIONS CYP3A5 genotype and Tac initial dose were independently associated with Tac C0 in renal transplant recipients. Our results suggest that a low Tac target C0 range (5-8 ng/ml) with a low fixed starting dose (3 mg/day) would be safe and effective among Chinese KTRs.

Soluble Tim-3 and Gal-9 are associated with renal allograft dysfunction in kidney transplant recipients: A cross-sectional study.

BACKGROUND: T cell immunoglobulin mucin-3 (Tim-3) has been reported to participate in the regulation of immune response and the induction of allograft tolerance. However, the association between Tim-3 and renal allograft dysfunction is unclear. We studied the expression of cellular and soluble Tim-3 (sTim-3), soluble galectin-9 (sGal-9) and carcinoembryonic antigen-related cell adhesion molecule-1 (sCEACAM-1) in kidney transplantation recipients (KTRs) to explore their roles in allograft dysfunction. METHODS: 96 KTRs (53 with stable graft and 43 with graft dysfunction) and 30 healthy controls (HC) were enrolled. Among the KTRs, 55 used Tacrolimus (TAC) and 41 used Sirolimus (SRL). In the dysfunction group, 29 recipients have undergone graft biopsy and 14 were classified as biopsy-proven rejection (BPR). Cellular Tim-3 was determined by flow cytometry. sTim-3 was determined by ELISA. sGal-9 and sCEACAM-1 were determined by Bio-Plex® suspension array system. RESULTS: KTRs with renal dysfunction showed significantly higher levels of sTim-3 and sGal-9 but similar levels of cellular Tim-3 and sCEACAM-1 compared with stable recipients. Correlation analysis revealed that estimated glomerular filtration rate (eGFR) was negatively associated with sTim-3 and sGal-9. Both BPR and non-BPR groups showed comparable levels of Tim-3, Gal-9 and CEACAM-1. Moreover, SRL group showed significantly higher levels of sCEACAM-1 than TAC and HC groups. CONCLUSIONS: sTim-3 and sGal-9 were promising biomarkers for allograft dysfunction, but unable to differentiate allograft rejection from other causes of renal dysfunction in KTRs. Moreover, long-term administration of sirolimus would up-regulate sCEACAM-1 level, while exert similar regulatory effects on Tim-3 and Gal-9 compared to tacrolimus.

Impact of immunosuppressive drugs on circulating Tfh cells in kidney transplant recipients: A pilot study.

BACKGROUND: T follicular helper cells (Tfh) are recently revealed to be vital in antibody-mediated rejection (AMR) in kidney transplant recipients (KTRs). However, the impact of immunosuppressive drugs on Tfh cells is not fully understood. The purpose of this study was to investigate the variation of Tfh cells phenotypically and functionally in KTRs treated with different immunosuppression regimens. METHODS: We recruited 26 KTRs treated with tacrolimus (TAC) -based regimen, 13 with sirolimus (SRL) -based regimen and 10 healthy controls (HC) in this study. The percentage and absolute number of circulating Tfh cells and the co-expression of Tfh related molecules including inducible costimulatory molecule (ICOS), programmed cell death protein 1 (PD-1), interleukin-21 (IL-21) and signal transducer and activator of transcription 3 (STAT3) were analyzed by flow cytometry, while serum IL-6 was detected by electrochemiluminescence immunoassay. RESULTS: The percentage and absolute number of Tfh cells and the co-expression of PD-1, STAT3 in Tfh cells were significantly higher in TAC group than that in SRL group. While no difference was found in regard to IL-21 and ICOS co-expressed with Tfh cells among three groups. Multiple linear regression analysis results showed that pre-transplant PRA level was the significant confounder affecting the absolute numbers of Tfh and CD4+CXCR5+PD-1+ T cells. In addition, correlation analysis showed that CD4+CXCR5+STAT3+ T cells were positively correlated to Tfh cells. CONCLUSIONS: Our study indicates that sirolimus can suppress the quantity of Tfh cells more significantly than tacrolimus. The higher level of circulating Tfh cells in tacrolimus group might be related to STAT3 signaling.

Correlation of HLA-DP/DQ polymorphisms with transplant etiologies and prognosis in liver transplant recipients.

Previous study has identified that the genetic variants in the human leukocyte antigen (HLA)-DP/DQ region were strongly associated with hepatitis B virus (HBV) infection. But their roles in liver function recovery after hepatic transplantation were still obscure. This study aimed to investigate whether HLA-DP/DQ polymorphisms were associated with post-transplant etiologies and prognosis in Chinese liver transplant recipients.A total of 144 liver transplant recipients were enrolled, which were divided into 2 groups according to the transplant etiology: HBV-related disease and non-HBV-related disease. HBV-related disease includes 3 subgroups: liver cirrhosis, hepatocellular carcinoma, and progressive HBV hepatitis. Three single-nucleotide polymorphisms HLA-DP (rs3077 and rs9277535) and HLA-DQ (rs7453920) were studied in all recipients by high-resolution melting curve analysis. Liver function indices (albumin, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, gamma-glutamyltranspeptidase, direct bilirubin, total bilirubin) and coagulation indices (prothrombin time, platelet, international normalized ratio, fibrinogen) were routinely tested. After transplant, 10 recipients who were positive for HBsAg or with elevation in HBV virus load were regarded as HBV recurrence.No significant association of HLA-DP/DQ polymorphisms with HBV recurrence or transplant etiology was observed (P < .05). Recipients with HLA-DQ (rs7453920) AG and AA genotype had lower direct bilirubin levels than GG genotype individuals, especially on the 14th day after surgery (17.80 vs. 5.35, P  =  .038). Patients with A alleles displayed earlier liver function recovery than patients with G alleles (7 vs. 6 months). No significant correlation was shown in HLA-DP rs3077 and rs9277535 with HBV infection or liver function recovery (P < .05).Our study concluded that HLA-DP (rs3077 and rs9277535) and HLA-DQ (rs7453920) were not significantly associated with HBV recurrence or HBV susceptibility, but HLA-DQ rs7453920 was related to prognosis of liver transplant recipients. HLA-DQ rs7453920 A might be used as an indicator of earlier recovery and better prognosis after transplantation.

Overweight Kidney Transplant Recipients Are at Risk of Being Overdosed Following Standard Bodyweight-Based Tacrolimus Starting Dose.

BACKGROUND: Bodyweight-based dosing of tacrolimus (Tac) is considered standard care, even though the available evidence is thin. An increasing proportion of transplant recipients is overweight, prompting the question if the starting dose should always be based on bodyweight. METHODS: For this analysis, data were used from a randomized-controlled trial in which patients received either a standard Tac starting dose or a dose that was based on CYP3A5 genotype. The hypothesis was that overweight patients would have Tac overexposure following standard bodyweight-based dosing. RESULTS: Data were available for 203 kidney transplant recipients, with a median body mass index (BMI) of 25.6 (range, 17.2-42.2). More than 50% of the overweight or obese patients had a Tac predose concentration above the target range. The CYP3A5 nonexpressers tended to be above target when they weighed more than 67.5 kg or had a BMI of 24.5 or higher. Dosing guidelines were proposed with a decrease up to 40% in Tac starting doses for different BMI groups. The dosing guideline for patients with an unknown genotype was validated using the fixed-dose versus concentration controlled data set. CONCLUSIONS: This study demonstrates that dosing Tac solely on bodyweight results in overexposure in more than half of overweight or obese patients.

Different characteristics of cryptococcal meningitis between HIV-infected and HIV-uninfected patients in the Southwest of China.

Cryptococcus is the major pathogen that causes fungal meningitis. In the People's Republic of China, especially in the Southwest area, cryptococcal meningitis (CM) in HIV-uninfected patients is more common than in HIV-infected patients. We compared clinical features and laboratory data pertaining to CM in patients with different immunological statuses. Demographic data, clinical manifestations, and laboratory data from inpatients in West China Hospital Sichuan University were collected from June 2009 to June 2014. Patients were grouped according to HIV status. Continuous variables were evaluated by Student t-test or Wilcoxon rank sum tests. Categorical variables were analyzed by χ2 test. Among 85 patients with CM were identified, 53 (62.4%) were HIV-uninfected patients. CM occurred more frequently in males in the HIV-infected group. Compared with HIV-infected patients, HIV-uninfected patients had more leukocytes in their blood and more leukocytes and protein in cerebrospinal fluid. More HIV-uninfected patients had increased cerebrospinal fluid (CSF)/serum albumin ratios, while intrathecal immunoglobulin G (IgG) synthesis was significantly increased. The rate of in-hospital mortality of HIV-infected CM patients was higher. Clinical signs are similar between HIV-uninfected and HIV-infected CM patients. Fewer leukocytes and protein was detected in the CSF and lower local synthesis of IgG in the central nervous system in HIV-infected patients, which reflects their diminished immune response. These characteristics should be noted in order to avoid misdiagnosis. Meningeal enhancement and intrathecal IgG synthesis in the HIV-uninfected group was significantly higher, that may be performance of aggressive inflammatory response and might contribute to a better outcome.

The influence of living donor SHROOM3 and ABCB1 genetic variants on renal function after kidney transplantation.

OBJECTIVE: A genome-wide association study has identified several gene polymorphisms associated with loss of renal function. The effect of these variants on renal function in kidney transplant recipients receiving immunosuppressive treatment is unknown. MATERIALS AND METHODS: A cohort of 189 kidney transplant recipients and their living donors were recruited from West China Hospital of Sichuan University, on whom we assessed the association of five single nucleotide polymorphisms with renal function after kidney transplantation. RESULTS: Glomerular filtration rate estimated by serum creatinine was significantly higher in recipients carrying allograft with the A allele at rs17319721 in SHROOM3 (shroom family member 3) than those in the group with the GG genotype from month 1 to month 6 after transplantation (P=0.020). Covariate adjustment analysis showed that the variant at rs17319721 in SHROOM3 was an independent risk factor for renal dysfunction after the first month after transplantation (P=0.022). The estimated glomerular filtration rate was the lowest in recipients with allograft carrying both the A allele at rs17319721 in SHROOM3 and the CC genotype at rs1045642 in ABCB1 (P<0.05). CONCLUSION: The genetic variants in SHROOM3 and ABCB1 in donors were associated closely with renal function after kidney transplantation.

The pharmacokinetics and pharmacodynamics of mycophenolate mofetil in younger and elderly renal transplant recipients.

AIMS: Elderly transplant recipients have a lower incidence of acute rejection, and a higher risk to die from infectious complications. A potential cause may be differences in the pharmacokinetics (PK) or pharmacodynamics (PD) of the immunosuppressive drugs they are taking. This study was designed to comprehensively evaluate the influence of age on the PK and PD of mycophenolic acid (MPA). METHODS: In this study the PK and PD of MPA was studied in 26 elderly and 54 younger renal transplant recipients treated with mycophenolate mofetil and tacrolimus. Patients were sampled repetitively, both before and during the first 6 months after kidney transplantation. Age-related variability in MPA PK, baseline IMPDH activity, as well as MPA-induced IMPDH inhibition were studied. RESULTS: The IMPDH activity pre-transplantation did not differ between elderly and younger patients. Neither IMPDH activity pre-transplantation nor maximum IMPDH inhibition was significantly correlated with the patients' age. The area under the MPA plasma concentration-time curve (AUC0-12h ) and the area under the effect (IMPDH activity)-time curve (AEC0-12h ) from 0 to 12 h were also not significantly different between the two groups. We found no significant differences in EC50 and Emax between elderly and younger patients. CONCLUSIONS: Age did not significantly affect the PK or PD of MPA. It is unlikely that the lower incidence of acute rejection in elderly patients, or the higher risk to die from a severe infection in elderly patients is due to different handling of MPA in the elderly.

Donor ABCB1 3435 C>T genetic polymorphisms influence early renal function in kidney transplant recipients treated with tacrolimus.

AIM: Determine the effect of genetic variants of donors and recipients on early renal function and tacrolimus pharmacokinetics in kidney transplant recipients. PATIENTS & METHODS: We measured CYP3A5 (6986A>G), ABCB1 (3435C>T, 2677G>T/A, 1236C>T) genetic polymorphisms in 120 renal transplant recipients and donors by high-resolution melting curve analysis. The renal function and tacrolimus trough concentrations were analyzed. RESULTS: The genotype of CYP3A5 (6986A>G) in recipients showed strong influence on tacrolimus pharmacokinetics. The ABCB1 3435 CC genotype in donor was significantly associated with lower estimated glomerular filtration in recipients within 1 month (p < 0.05) and correlated with delayed creatinine recovery (p = 0.007). CONCLUSIONS: ABCB1 3435 CC genotype in donor influences early renal function and creatinine recovery in tacrolimus-treated kidney transplant recipients.

[Diagnostic Value of Immunofixation Electrophoresis and KAP/LAM Ratio in Multiple Myeloma Patients with Renal Injury].

OBJECTIVES: To explore the diagnostic value of immunofixation electrophoresis and Kappa/Lambda (KAP/LAM) ratio in multiple myeloma patients with renal injury. METHODS: The serum of 822 patients of renal disease were collected for the examnation of immunofixation electrophoresis, KAP/LAM ratio, serum immunoglobulin levels and renal function, including serum urea nitrogen (BUN), serum creatinine (Crea), cystatin C (Cys-C) and estimated glomerular filtration rate (eGFR). To analyze the diagnostic value of immunofixation and KAP/LAM ratio in the differentiation of renal injury of multiple myeloma from primary renal injury diseases. RESULTS: M protein was observed in 75 patients (9.1%). The ratio of each type was IgG 49.3%(37/75), IgA 34.7%(26/75), IgM 5.3%(4/75) and LAM 10.7%(8/75). There was significant difference of KAP/LAM ratio between M protein group and non-M protein group. The KAP/LAM ratio was significant higher in KAP group, compared to non-M protein group. Reverse result was obtained in LAM group. There were higher Crea level and lower eGFR value in pure LAM light chain group, compared with IgG, IgA and IgM groups. CONCLUSIONS: Immunofixation electrophoresis and KAP/LAM ratio may play an important role in the diagnosis of multiple myeloma patients with renal injury, so could be early screening markers.

Procalcitonin levels predict acute kidney injury and prognosis in acute pancreatitis: a prospective study.

BACKGROUND: Acute kidney injury (AKI) has been proposed as a leading cause of mortality for acute pancreatitis (AP) patients admitted to the intensive care unit (ICU). This study investigated the predictive value of procalcitonin (PCT) for AKI development and relevant prognosis in patients with AP, and compared PCT's predictive power with that of other inflammation-related variables. METHODS: Between January 2011 and March 2013, we enrolled 305 cases with acute pancreatitis admitted to ICU. Serum levels of PCT, serum amyloid A (SAA), interleukin-6 (IL-6), and C reactive protein (CRP) were determined on admission. Serum PCT was tested in patients who developed AKI on the day of AKI occurrence and on either day 28 after occurrence (for survivors) or on the day of death (for those who died within 28 days). RESULTS: Serum PCT levels were 100-fold higher in the AKI group than in the non-AKI group on the day of ICU admission (p<0.05). The area under the receiver-operating characteristic (ROC) curve of PCT for predicting AKI was 0.986, which was superior to SAA, CRP, and IL-6 (p<0.05). ROC analysis revealed all variables tested had lower predictive performance for AKI prognosis. The average serum PCT level on day 28 (2.67 (0.89, 7.99) ng/ml) was significantly (p<0.0001) lower than on the day of AKI occurrence (43.71 (19.24,65.69) ng/ml) in survivors, but the serum PCT level on death (63.73 (34.22,94.30) ng/ml) was higher than on the day of AKI occurrence (37.55 (18.70,74.12) ng/ml) in non-survivors, although there was no significant difference between the two days in the latter group (p = 0.1365). CONCLUSION: Serum PCT is superior to CRP, IL-6, and SAA for predicting the development of AKI in patients with AP, and also can be used for dynamic evaluation of AKI prognosis.