Phase 1-2 Trial of AAVS3 Gene Therapy in Patients with Hemophilia B.

BACKGROUND: FLT180a (verbrinacogene setparvovec) is a liver-directed adeno-associated virus (AAV) gene therapy that uses a synthetic capsid and a gain-of-function protein to normalize factor IX levels in patients with hemophilia B. METHODS: In this multicenter, open-label, phase 1-2 trial, we assessed the safety and efficacy of varying doses of FLT180a in patients with severe or moderately severe hemophilia B (factor IX level, ≤2% of normal value). All the patients received glucocorticoids with or without tacrolimus for immunosuppression to decrease the risk of vector-related immune responses. After 26 weeks, patients were enrolled in a long-term follow-up study. The primary end points were safety and efficacy, as assessed by factor IX levels at week 26. RESULTS: Ten patients received one of four FLT180a doses of vector genomes (vg) per kilogram of body weight: 3.84×1011 vg, 6.40×1011 vg, 8.32×1011 vg, or 1.28×1012 vg. After receiving the infusion, all the patients had dose-dependent increases in factor IX levels. At a median follow-up of 27.2 months (range, 19.1 to 42.4), sustained factor IX activity was observed in all the patients except one, who resumed factor IX prophylaxis. As of the data-cutoff date (September 20, 2021), five patients had normal factor IX levels (range, 51 to 78%), three patients had levels from 23 to 43%, and one had a level of 260%. Of the reported adverse events, approximately 10% were related to FLT180a and 24% to immunosuppression. Increases in liver aminotransferase levels were the most common FLT180a-related adverse events. Late increases in aminotransferase levels occurred in patients who had received prolonged tacrolimus beyond the glucocorticoid taper. A serious adverse event of arteriovenous fistula thrombosis occurred in the patient with high factor IX levels. CONCLUSIONS: Sustained factor IX levels in the normal range were observed with low doses of FLT180a but necessitated immunosuppression with glucocorticoids with or without tacrolimus. (Funded by Freeline Therapeutics; ClinicalTrials.gov numbers, NCT03369444 and NCT03641703; EudraCT numbers, 2017-000852-24 and 2017-005080-40.).

Protease inhibitor-based direct-acting antivirals are associated with increased risk of aminotransferase elevations but not hepatic dysfunction or decompensation.

BACKGROUND & AIMS: Cases of acute liver injury (ALI) have been reported among chronic HCV-infected patients receiving protease inhibitor (PI)-based direct-acting antiviral (DAA) regimens, but no analyses have compared the risk of ALI in patients receiving PI- vs. non-PI-based DAAs. Thus, we compared the risk of 3 ALI outcomes between patients (by baseline Fibrosis-4 [FIB-4] group) receiving PI-based or non-PI-based DAAs. METHODS: We conducted a cohort study of 18,498 patients receiving PI-based DAA therapy (paritaprevir/ritonavir/ombitasvir±dasabuvir, elbasvir/grazoprevir, glecaprevir/pibrentasvir) matched 1:1 on propensity score to those receiving non-PI-based DAAs (sofosbuvir/ledipasvir, sofosbuvir/velpatasvir) in the 1945-1965 Veterans Birth Cohort (2014-2019). During exposure to DAA therapy, we determined development of: i) alanine aminotransferase (ALT) >200 U/L, ii) severe hepatic dysfunction (coagulopathy with hyperbilirubinemia), and iii) hepatic decompensation. We used Cox regression to determine hazard ratios (HRs) with 95% CIs for each ALI outcome within groups defined by baseline FIB-4 (≤3.25; >3.25). RESULTS: Among patients with baseline FIB-4 ≤3.25, those receiving PIs had a higher risk of ALT >200 U/L (HR 3.98; 95% CI 2.37-6.68), but not severe hepatic dysfunction (HR 0.67; 95% CI 0.19-2.39) or hepatic decompensation (HR 1.01; 95% CI 0.29-3.49), compared to those receiving non-PI-based regimens. For those with baseline FIB-4 >3.25, those receiving PIs had a higher risk of ALT >200 U/L (HR, 2.15; 95% CI 1.09-4.26), but not severe hepatic dysfunction (HR, 1.23 [0.64-2.38]) or hepatic decompensation (HR, 0.87; 95% CI 0.41-1.87), compared to those receiving non-PI-based regimens CONCLUSION: While risk of incident ALT elevations was increased in those receiving PI-based DAAs in both FIB-4 groups, the risk of severe hepatic dysfunction and hepatic decompensation did not differ between patients receiving PI- or non-PI-based DAAs in either FIB-4 group. LAY SUMMARY: Cases of liver injury have been reported among patients treated with protease inhibitor-based direct-acting antivirals for hepatitis C infection, but it is not clear if the risk of liver injury among people starting these drugs is increased compared to those starting non-protease inhibitor-based therapy. In this study, patients receiving protease inhibitor-based treatment had a higher risk of liver inflammation than those receiving a non-protease inhibitor-based treatment, regardless of the presence of pre-treatment advanced liver fibrosis/cirrhosis. However, the risk of severe liver dysfunction and decompensation were not higher for patients treated with protease inhibitor-based regimens.

Prognostic significance of branched-chain amino acid transferase 1 and CD133 in triple-negative breast cancer.

BACKGROUND: Previous studies have shown that branched-chain amino acid transferase 1 (BCAT1) is associated with tumour progression in triple-negative breast cancer (TNBC). Furthermore, CD133 has emerged as a novel cancer stem cell marker for indicating tumour progression. However, the prognostic significance of these two markers remains to be verified. This study was conducted to investigate the correlation between BCAT1 and CD133 expression and clinicopathological features, as well as the prognosis of patients with TNBC. METHODS: The study cohort included 291 patients with TNBC. Tissue microarrays were constructed for both cancer and normal tissues. The expression of BCAT1 and CD133 was detected by immunohistochemical staining, and the levels were evaluated using an H-scoring system. Cut-off points for BCAT1 and CD133 expression were determined using receiver operating characteristic curves. RESULTS: The median follow-up time for the study participants was 68.73 months (range: 1.37-103.6 months). The 5-year disease-free survival (DFS) and overall survival (OS) rates of the 291 patients with TNBC were 72.51 and 82.47%, respectively. Higher levels of BCAT1 and CD133 expression independently indicated shorter DFS and OS. High levels of both BCAT1 and CD133 expression were detected in 36 (12.37%) patients, who had significantly shorter DFS and OS (both P < 0.001) compared to other patients. CONCLUSION: BCAT1 and CD133 can be considered as biomarkers with prognostic significance for TNBC.

Kynurenine Aminotransferases I, II and III Are Present in Saliva.

BACKGROUND/AIMS: Fluids of the human body such as serum, cerebrospinal fluid and saliva contain a wide variety of proteins. Because kynurenic acid (KYNA) has been detected in human saliva, we wondered if KYNA could be produced in saliva by KYNA-synthesising enzymes, namely the kynurenine aminotransferases KAT I, KAT II and KAT III. METHODS: Thirty samples of human saliva from control volunteers were investigated. KAT activity was measured in the presence of 1 mM pyruvate and 2 µM or 100 µM L-kynurenine and KYNA production was assessed by high-performance liquid chromatography. RESULTS: Saliva dose- and time-dependently produced KYNA. KAT activity ranged between 900 and 1050 pmol/mg protein/h: 900 for KAT I, 950 for KAT III and 1050 for KAT II. KYNA was synthesised in saliva at a physiological concentration of 2 µM L-kynurenine and at a higher concentration of 100 µM. Investigation of the distributions of the enzymes in saliva revealed that KAT I, KAT II and KAT III activity in a centrifuge-obtained pellet ranged from ~100% to 120%; in the supernatant, the percentage was between 0% and 20%. We observed a nonsignificant tendency for lower KAT activity in women's saliva than in men's. KATs present in saliva were sensitive to the GABA-transaminase inhibitor γ-acetylenic GABA, with a concentration of 100 µM γ-acetylenic GABA significantly blocking the formation of KYNA (50% of control, p < 0.05). Furthermore, KATs in saliva were sensitive to anti-dementia drugs, such as D-cycloserine and cerebrolysin, in an in vitro study. CONCLUSION: Our data revealed for the first time the presence of KAT I, KAT II and KAT III proteins in human saliva. KAT activity was found mostly in pelleted cells, suggesting their presence in salivary gland cells. KAT proteins in saliva are sensitive to drugs blocking KYNA formation. Our data indicate the presence of cells in saliva involved in the biochemical machinery of the kynurenine pathway. Their role in the digestive process remains to be clarified. We speculate that modulation of KYNA formation in the mouth by food and/or drugs might affect glutamate neurotransmission and cholinergic activity in the CNS and/or periphery and play a role under physiological as well as pathological conditions.

High-Throughput Nanoelectrospray Ionization-Mass Spectrometry Analysis of Microfluidic Droplet Samples.

Droplet microfluidics enables high-throughput manipulation of fL-µL volume samples. Methods implemented for the chemical analysis of microfluidic droplets have been limited in scope, leaving some applications of droplet microfluidics difficult to perform or out of reach entirely. Nanoelectrospray ionization-mass spectrometry (nESI-MS) is an attractive approach for droplet analysis, because it allows rapid, label-free, information-rich analysis with high mass sensitivity and resistance to matrix effects. Previous proof-of-concept systems for the nESI-MS analysis of droplets have been limited by the microfluidics used so that stable, long-term operation needed for high-throughput applications has not been demonstrated. We describe a platform for the stable analysis of microfluidic droplet samples by nESI-MS. Continuous infusion of droplets to an nESI emitter was demonstrated for as long as 2.5 h, corresponding to analysis of over 20 000 samples. Stable signal was observed for droplets as small as 65 pL and for throughputs as high as 10 droplets/s. A linear-concentration-based response and sample-to-sample carryover of <3% were also shown. The system is demonstrated for measuring products of in-droplet enzymatic reactions.

Liver fibrosis during clinical ascertainment of glycogen storage disease type III: a need for improved and systematic monitoring.

PURPOSE: In glycogen storage disease type III (GSD III), liver aminotransferases tend to normalize with age giving an impression that hepatic manifestations improve with age. However, despite dietary treatment, long-term liver complications emerge. We present a GSD III liver natural history study in children to better understand changes in hepatic parameters with age. METHODS: We reviewed clinical, biochemical, histological, and radiological data in pediatric patients with GSD III, and performed a literature review of GSD III hepatic findings. RESULTS: Twenty-six patients (median age 12.5 years, range 2-22) with GSD IIIa (n = 23) and IIIb (n = 3) were enrolled in the study. Six of seven pediatric patients showed severe fibrosis on liver biopsy (median [range] age: 1.25 [0.75-7] years). Markers of liver injury (aminotransferases), dysfunction (cholesterol, triglycerides), and glycogen storage (glucose tetrasaccharide, Glc4) were elevated at an early age, and decreased significantly thereafter (p < 0.001). Creatine phosphokinase was also elevated with no significant correlation with age (p = 0.4). CONCLUSION: Liver fibrosis can occur at an early age, and may explain the decrease in aminotransferases and Glc4 with age. Our data outlines the need for systematic follow-up and specific biochemical and radiological tools to monitor the silent course of the liver disease process.

Evolution Under Normothermic Machine Perfusion of Type 2 Donation After Cardiac Death Livers Discarded as Nontransplantable.

BACKGROUND: Type 2 donation after cardiac death (DCD) represents an underused source of grafts for liver transplantation. In our center, normothermic regional perfusion and strict selection criteria have led to acceptable postoperative results after transplanting type 2 DCD livers. However, many of these grafts are still discarded before transplantation. We believe that the suitability of these organs may be improved by adding normothermic machine perfusion (NMP) to our current procedure. MATERIALS AND METHODS: A total of 5 type 2 DCD livers discarded for transplantation were submitted to normothermic regional perfusion and 12 h of NMP. The macroscopic aspect of the liver, vascular and bile flows, and pH were continuously monitored. Serial perfusate analyses and liver biopsies were performed. After NMP, the microscopic appearance of the liver parenchyma and the bile ducts was analyzed. RESULTS: All the grafts showed hemodynamic stability during the NMP. The alanine aminotransferase peak during NMP correlated with the warm ischemia time (Pearson correlation of 0.933, p 0.021). After an initial period of acidosis, the grafts were generally able to spontaneously correct pH and lactate levels without the need for additional bicarbonate. Livers with favorable bile duct histology generally started bile production earlier and registered higher bile flows. CONCLUSIONS: NMP represents a feasible procedure for use with type 2 DCD livers. The pH and lactate correction and the bile flows appear to be significant factors associated with graft viability. However, these favorable results should be confirmed in a clinical transplant setting.

Fast and Cost-Effective Biochemical Spectrophotometric Analysis of Solution of Insect "Blood" and Body Surface Elution.

Using insect hemolymph ("blood") and insect body surface elutions, researchers can perform rapid and cheap biochemical analyses to determine the insect's immunology status. The authors of this publication describe a detailed methodology for a quick marking of the concentration of total proteins and evaluation of the proteolytic system activity (acid, neutral, and alkaline proteases and protease inhibitors), as well as a methodology for quick "liver" tests in insects: alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and urea and glucose concentration analyses. The meaning and examples of an interpretation of the results of the presented methodology for biochemical parameter determination are described for the example of honey bees.

Clinical Characteristics and Outcome of Hepatic Sarcoidosis: A Population-Based Study 1976-2013.

OBJECTIVES: Data on clinical manifestations and outcome of hepatic sarcoidosis are scarce. This study aimed to use a population-based cohort of patients with incident sarcoidosis to better describe the characteristics of hepatic sarcoidosis. METHODS: A cohort of incident cases of sarcoidosis in Olmsted County, MN, USA, from 1976 to 2013 was identified from the database. Diagnosis was verified by individual medical record review. Confirmed cases of sarcoidosis were then reviewed for liver involvement. Data on clinical manifestations, imaging study, liver biochemical tests, treatment, and outcome were collected. Cumulative incidence of cirrhosis adjusted for the competing risk of death was estimated. RESULTS: A total of 345 cases of incident sarcoidosis were identified. Of these, 19 cases (6%) had liver involvement (mean age 46.1 years, 53% female and 79% Caucasian). Most patients had asymptomatic liver disease and were discovered in pursuit of abnormal biochemical tests and imaging studies. Alkaline phosphatase (ALP) and gamma-glutamyl transferase (GGT) were elevated in the majority of patients (88 and 90%, respectively). Elevated transaminases were less common and less severe. About half of patients had abnormal imaging study with hypodense nodular lesions being the most common abnormality (six patients) followed by hepatomegaly (three patients). Liver biopsy revealed non-caseating granuloma in 88% (14 of 16 patients). A total of four patients developed cirrhosis. CONCLUSIONS: Involvement of the liver by sarcoidosis was seen in 6% of patients with sarcoidosis. The majority of patients were asymptomatic. Elevated ALP and GGT were the most common abnormal biochemical tests. Liver biopsy revealed non-caseating granuloma in almost all cases. Cirrhosis was seen in a significant number of patients. Generalizability of the observations to other populations may be limited, as the studied population was predominantly Caucasian. The prevalence of liver disease may be higher in more diverse populations.

BCAT1 overexpression is an indicator of poor prognosis in patients with urothelial carcinomas of the upper urinary tract and urinary bladder.

AIMS: Amino acid biosynthesis is one of the cardinal events of carcinogenesis that has not been investigated in urothelial carcinoma (UC). By data-mining a published transcriptomic database of UCs of urinary bladder (UBUCs) (GSE31684), we identified branched-chain amino acid transaminase 1 (BCAT1) as the most significantly stepwise up-regulated gene during tumour progression among those associated with the amino acid biosynthetic process (GO:0008652). Accordingly, we analysed BCAT1 transcript and protein expression with their clinicopathological significance. METHODS AND RESULTS: We used real-time reverse transcription-polymerase chain reaction (RT-PCR) to detect BCAT1 transcript levels in 20 UCs of upper tract (UTUCs) and 20 UBUCs, respectively. Immunohistochemical study was performed to determine BCAT1 protein expression in 340 UTUCs and 295 UBUCs. Higher BCAT1 transcript levels were associated with higher pT status in both groups (P < 0.05). BCAT1 protein overexpression was also associated significantly with adverse clinicopathological features, e.g. advanced pT stage, nodal metastasis, high pathological grade, etc. (P < 0.05). BCAT1 overexpression predicted worse disease-specific survival and metastasis-free survival in both univariate and multivariate analyses (P ≤ 0.001). CONCLUSION: BCAT1 overexpression is associated with advanced tumour status, and implies adverse clinical outcomes of UCs, suggesting that its role in tumour progression could serve as a prognostic biomarker and a novel therapeutic target in UC.

Screening primary sclerosing cholangitis and biliary dysplasia with endoscopic retrograde cholangiography and brush cytology: risk factors for biliary neoplasia.

BACKGROUND AND STUDY AIMS: Primary sclerosing cholangitis (PSC) is associated with increased risk of biliary dysplasia and cholangiocarcinoma (CCA). The aim of this study was to evaluate the role of early endoscopic retrograde cholangiography (ERC) with systematic brush cytology to identify risk factors associated with biliary neoplasia. PATIENTS AND METHODS: Patients who were referred for their first ERC for suspicion of PSC between January 2006 and October 2011 were included in the study. Brush cytology specimens were scored as benign, suspicious, or malignant. End points were CCA, biliary dysplasia, benign histology, or benign disease course for ≥ 2 years. RESULTS: PSC was diagnosed in 261 patients (125 men, 136 women), most of whom were asymptomatic (n = 211). Cholangiographic changes were mild in 57.1 %. Men presented with advanced disease more often than women. Brush cytology was benign in 243, suspicious in 16, and malignant in 2 patients. Follow-up completed in 249 patients indicated a benign disease course in 232 patients. Seven patients were diagnosed with CCA and eight had biliary dysplasia in the explanted liver. Thus, 15 patients had biliary neoplasia, and suspicious or malignant brush cytology had been detected in 8 of them at initial brushing. Advanced extrahepatic cholangiographic changes with elevated aminotransferases at diagnosis seemed to be associated with increased risk of biliary neoplasia. CONCLUSIONS: Even in mostly asymptomatic patients with PSC, 42.9 % had advanced disease and 6.9 % presented with suspicious or malignant brush cytology at first ERC. Advanced extrahepatic ERC changes with elevated aminotransferases at diagnosis might be risk factors for biliary neoplasia.

Continuous colorimetric screening assays for the detection of specific L- or D-α-amino acid transaminases in enzyme libraries.

In the course of a project devoted to the stereoselective synthesis of non-proteinogenic α-amino acids using α-transaminases (α-TA), we report the design and optimization of generic high-throughput continuous assays for the screening of α-TA libraries. These assays are based on the use of L- or D-cysteine sulfinic acid (CSA) as irreversible amino donor and subsequent sulfite titration by colorimetry. The assays' quality was assessed under screening conditions. Hit selection thresholds were accurately determined for every couple of substrates and a library of 232 putative transaminases expressed in Escherichia coli host cells was screened. The reported high throughput screening assays proved very sensitive allowing the detection with high confidence of activities as low as 10 µU (i.e., 0.01 nmol substrate converted per min). The assays were also evidenced to be stereochemically discriminant since L-CSA and D-CSA allowed the exclusive detection of L-TA and D-TA, respectively. These generic assays thus allow testing the stereoselective conversion of a wide range of α-keto acids into α-amino acids of interest. As a proof of principle, the use of 2-oxo-4-phenylbutyric acid as acceptor substrate led to the identification of 54 new α-TA offering an access to valuable L- or D-homophenylalanine.

A Four-Plex Real-Time PCR Assay, Based on rfbE, stx1, stx2, and eae Genes, for the Detection and Quantification of Shiga Toxin-Producing Escherichia coli O157 in Cattle Feces.

Several real-time polymerase chain reaction (PCR) assays have been developed to detect and quantify Shiga toxin-producing Escherichia coli (STEC) O157:H7, but none have targeted the O-antigen specific gene (rfbEO157) in combination with the three major virulence genes, stx1, stx2, and eae. Our objectives were to develop and validate a four-plex, quantitative PCR (mqPCR) assay targeting rfbE(O157), stx1, stx2, and eae for the detection and quantification of STEC O157 in cattle feces, and compare the applicability of the assay to detect STEC O157 to a culture method and conventional PCR (cPCR) targeting the same four genes. Specificity of the mqPCR assay to differentially detect the four genes was confirmed with strains of O157 and non-O157 STEC with different profiles of target genes. In cattle feces spiked with pure cultures, detection limits were 2.8×10(4) and 2.8×10(0) colony-forming units/g before and after enrichment, respectively. Detection of STEC O157 in feedlot cattle fecal samples (n=278) was compared between mqPCR, cPCR, and a culture method. The mqPCR detected 48.9% (136/278) of samples as positive for E. coli O157. Of the 100 samples that were randomly picked from 136 mqPCR-positive samples, 35 and 48 tested positive by cPCR and culture method, respectively. Of the 100 samples randomly chosen from 142 mqPCR-negative samples, all were negative by cPCR, but 21 samples tested positive by the culture method. McNemar's chi-square tests indicated significant disagreement between the proportions of positive samples detected by the three methods. In conclusion, the mqPCR assay that targets four genes is a novel and more sensitive method than the cPCR or culture method to detect STEC O157 in cattle feces. However, the use of real-time PCR as a screening method to identify positive samples and then subjecting only positive samples to a culture method may underestimate the presence of STEC O157 in fecal samples.

MiRNA regulation of TRAIL expression exerts selective cytotoxicity to prostate carcinoma cells.

Prostate carcinoma is the most common cancer for men and among the leading cancer-related causes. Many evidences have shown that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) potently induces apoptosis in cancer cells, and thus, is a promising biologic agent for prostate carcinoma therapy. However, TRAIL expression mediated by the current vectors lacks tumor specificity, thereby exerting cytotoxicity to normal cells. To solve this problem, we inserted miRNA response elements (MREs), miR-143 and miR-145, expression levels of which were reduced in prostate carcinoma, as well as that of miR-122, which is specifically expressed in hepatic cells, into adenoviral vectors to control TRAIL expression (Ad-TRAIL-M3). qPCR data confirmed that miR-143, miR-145, and miR-122 levels were all decreased in prostate carcinoma cell lines and prostate cancer samples from patients. Luciferase assays showed that MREs-regulated luciferase expression was potently suppressed in normal cells, but not in prostate cancer cells. Ad-TRAIL-M3, which expresses TRAIL in a MREs-regulated manner, produced high level of TRAIL and suppressed the survival of prostate cancer cells by inducing apoptosis, while Ad-TRAIL-M3 had no TRAIL expression in normal cells and thus exerted no cytotoxicity to them. The studies on PC-3 tumor xenograft in mice further confirmed that Ad-TRAIL-M3 was able to inhibit the growth of tumors and possessed high biosafety. In conclusion, we successfully generated an adenoviral vector that expresses TRAIL in miRNA-regulated mechanism. This miRNA-based gene therapy may be promising for prostate carcinoma treatment.

Physiological alterations in Bradybaena similaris (Stylommatophora: Bradybaenidae) induced by the entomopathogenic nematode Heterorhabditis indica (Rhabditida: Heterorhabditidae) strain LPP1.

Heterorhabditis is a nematode found in the soil that is used as an important biological control agent against various organisms. However, few studies have been performed of its use against snails and the present study is the first to investigate the effect of experimental exposure of Bradybaena similaris to Heterorhabditis indica LPP1. Two groups of 16 snails were formed: the control group (not exposed) and the treatment, which was exposed for three weeks to infective juveniles (J3) of H. indica LPP1. The entire experiment was conducted in duplicate, using a total of 64 snails. After this period, the snails were dissected to collect the hemolymph to evaluate the possible physiological alterations, namely total proteins, uric acid and hemolymph urea, as well as the activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) as a result of the infection. The terrariums were analyzed on alternate days throughout the experiment to count the dead snails. Intense proteolysis was observed in the infected snails. An increase in the level of uric acid and reduction of the hemolymph urea content indicated that the infection by H. indica results in the inversion of the excretion pattern of the host snail. Variations in the aminotransferase activities were also observed, with the infected group presenting significantly higher values (p<0.05) than the control group for both ALT and AST. The exposure to H. indica LPP1 caused 55% mortality, with the highest rate observed in the first week after exposure (30%). These results suggest that the use of H. indica LPP1 is a feasible alternative for the biological control of B. similaris.

Evaluation of cephalometric, hormonal and enzymatic parameters in young obese subjects.

AIM: The aim of the present investigation was to analyse cephalometric skeletal structures and hormonal and enzymatic parameters in young obese subjects in comparison with those of normal weight subjects. MATERIALS AND METHODS: The whole sample consisted of 50 Caucasian patients (28 males and 22 females) whose lateral radiographs, laboratory hormonal and enzymatic analyses were already available. The test group included 25 obese patients (11 females and 14 males, average age: 9.8 +/- 2.11 years old), while the control group included 25 normal weight subjects matched for age and sex (11 females and 14 males, 9.9 +/- 2.5 years old). Data were statistically analysed: Student's t-test for independent samples was adopted and the level of significance was set at: p < 0.05. RESULTS: As regards cephalometric records, the anterior cranial base length was significantly greater in the test group (S-N: 69.9 +/- 4 mm) compared to the controls (S-N: 68.1 +/-2.7 mm). Moreover, the maxillary lenght was higher in the test group (Pm-A: 48.5 +/- 2.5 mm) in comparison to the control group (Pm-A: 46.1 +/- 1.9 mm). As regards skeletal class and vertical dimension, no significant differences were found between the two groups, with the exception of the intermaxillary plane angle, which was significantly lower in the obese subjects in comparison to the controls. Laboratory analysis showed significant (p < 0.05) higher levels of leptin and insulin in the test group in comparison with control subjects. Furthermore, LH, FSH, IGF-1 values were significantly (p < 0.05) lower in the test group in comparison with the control group. CONCLUSION: Obese subjects exhibited an increase of some craniofacial parameters and alteration of some laboratory parameters that may be involved in the process of skeletal maturation, in comparison to normal weight subjects. These findings may be of interest in orthodontics, as young obese subjects may need a different orthodontic treatment plan in comparison to normal weight subjects of the same age.

Hepatic transaminase and alkaline phosphatase enzyme levels in HIV/HBV co-infected and HIV mono-infected patients in Maiduguri, Nigeria.

BACKGROUND: Studies have shown that HIV-HBV co-infected patients have an increased risk of liver-related morbidity and mortality compared to their HIV-mono-infected counterparts. Furthermore, it has been reported that HIV-HBV co-infected patients have a significantly high incidence of drug-induced hepatotoxicity following commencement of HAART than HIV-mono-infected patients. OBJECTIVES: To compare the levels of aspartate amino transferase (AST), alanine amino transferase (ALT) and alkaline phosphatase (ALKPO 4 ) enzyme levels between HAART naïve HIV-HBV co-infected patients and their HIV-mono-infected counterparts. MATERIALS AND METHODS: A cross-sectional descriptive study in which 142 newly diagnosed HIV/HBV co-infected and HIV mono-infected adults were investigated for alkaline aminotransferase, aspartate aminotransferase and alkaline phosphatase enzyme levels. RESULTS: The study subjects comprised of 80 (56.3%) females and 62 (46.7%) males. The age range of the study population was 15-65 years. The mean ages of male and female subjects were 45.5 ± 10.5 years and 39.1 ± 7.5 years respectively ( P < 0.05). Sixty-three (44.4%) study subjects were HIV/HBV co-infected while 79 (55.6%) were HIV mono-infected. The mean ALT enzyme level of HIV/HBV co-infected subjects was significantly higher than that of HIV mono-infected ones i.e., 42.12 IU/l vs. 27.86 IU/l, ( P = 0.038). However, there was no statistically significant difference in the mean AST (30.14 IU/l vs. 29.09 IU/l, P = 0.893) and ALKPO 4 (55.86 IU/l vs. 60.97 IU/l, P = 0.205) enzyme levels between HIV-HBV co-infected and HIV mono-infected subjects albeit the two enzymes were moderately elevated in both categories of subjects. CONCLUSION: The significantly elevated ALT enzyme levels amongst HIV-HBV co-infected subjects suggest that HIV-HBV co-infected patients may have an increased risk of liver-related morbidity and mortality than their HIV mono-infected counterparts. Screening for serological markers of chronic HBV infection, as well as hepatic transaminase enzyme levels in all newly diagnosed HIV-positive patients is therefore recommended before commencement of HAART.

Rational assignment of key motifs for function guides in silico enzyme identification.

Biocatalysis has emerged as a powerful alternative to traditional chemistry, especially for asymmetric synthesis. One key requirement during process development is the discovery of a biocatalyst with an appropriate enantiopreference and enantioselectivity, which can be achieved, for instance, by protein engineering or screening of metagenome libraries. We have developed an in silico strategy for a sequence-based prediction of substrate specificity and enantiopreference. First, we used rational protein design to predict key amino acid substitutions that indicate the desired activity. Then, we searched protein databases for proteins already carrying these mutations instead of constructing the corresponding mutants in the laboratory. This methodology exploits the fact that naturally evolved proteins have undergone selection over millions of years, which has resulted in highly optimized catalysts. Using this in silico approach, we have discovered 17 (R)-selective amine transaminases, which catalyzed the synthesis of several (R)-amines with excellent optical purity up to >99% enantiomeric excess.

Phytochemical characterization of Morus nigra fruit ultrasound-assisted ethanolic extract for its cardioprotective potential.

The current work investigated the phytochemical profile of ultrasound-assisted ethanolic extract of Morus nigra (M. nigra) fruit. FTIR analysis of M. nigra fruit extract revealed the presence of alcohols (O-H), alkanes (C-H stretch), alkenes (C=C), and alkynes (C≡C). The HPLC analysis quantified the quercetin, gallic acid, vanillic acid, chlorogenic acid, syringic acid, cinnamic acid, sinapic acid, and kaempferol. Furthermore, the cardioprotective activity of ethanolic extract of M. nigra fruit was investigated. Cholesterol supplementation (2%) in the daily diet and exposure to cigarette smoke (2 cigarettes twice a day) were to induce hypertension in rats. The experimental animals were categorized into four groups: G0 (negative control), G1 (positive control), G2 (standard drug), and G3 (M. nigra fruit). The fruit extract administration at 300 mg/kg BW/day orally for 2 months significantly (p < .001) enhanced the activities of serum and cardiac tissue antioxidants in hypertensive rats. Meanwhile, the fruit extract reduced the elevated serum lipid profile while significantly increasing the high-density lipoproteins in G3 than G1 and G2. The increase in blood pressure, liver transaminases, and serum lactate dehydrogenase also reduced significantly in M. nigra fruit extract-treated rats. Histopathological findings revealed mild normalization of cardiac myocytes with central nuclei, branching, and cross-striations. Consequently, the M. nigra fruit extract exerted the cardioprotective potential via increasing the antioxidant enzymes and reducing the lipids, lactate dehydrogenase, liver transaminases, and blood pressure. The therapeutic potential of M. nigra fruit can be due to flavonols and phenolic acids. PRACTICAL APPLICATIONS: The present work quantified the Morus nigra fruit phytochemicals and its significant role in reducing lipid markers and blood pressure and improving antioxidant status in rats fed a hypercholesterolemic diet and exposed to cigarette smoke. Conclusively, the inclusion of M. nigra fruit in daily diet could improve the cardiac health of the individuals. Furthermore, the therapeutic potential of M. nigra fruit and its isolated constituents in modulating the gene expression against cardiac problems can explore after clinical trials and standardization in higher animals.

Development of a microplate fluorescence assay for kynurenine aminotransferase.

Inhibition of kynurenine aminotransferases (KATs) is a strategy to therapeutically reduce levels of kynurenic acid (KYNA), an endogenous antagonist of glutamatergic N-methyl-D-aspartate (NMDA) and cholinergic α7 nicotinic receptors. Several methods of measuring KAT activity in vitro have been developed, but none is well-suited to high throughput and automation. In this article, we describe a modification of existing high-performance liquid chromatography (HPLC)-based methods that enables the development of a 96-well microplate assay in both enzyme- and cell-based formats using human KAT I as an example. KYNA enzymatically produced from L-kynurenine is measured directly in a reaction mixture fluorimetrically.