The In Vitro Evaluation of Rooster Semen Pellets Frozen with Dimethylacetamide.

Sperm cryopreservation is an effective technique for conserving animal genetic diversity and transmitting superior genetic backgrounds, maintained via a non-invasive sampling and collection of huge quantities of sperm. Nevertheless, cryopreservation in avian species is not commercially viable because of the rooster sperm's susceptibility to damage. This study aims to estimate the impact of dimethylacetamide (DMA) as a cryoprotectant at different levels (3%, 6%, or 9%) on the post-thawed sperm quality, motility, antioxidant-biomarkers, and the expression of anti-freeze related genes. Semen samples were collected twice a week from twelve roosters aged 40 wk, weighing 3400 ± 70 g, and belonging to the Cairo-B2 chicken strain. Fresh semen samples were rapidly appraised, pooled, diluted with two volumes of a basic extender, and divided equally into three groups. The diluted groups were chilled at -20 °C for 7 min, then gently supplemented with 3, 6, or 9% pre-cooled DMA and equilibrated at 5 °C for a further 10 min. Semen pellets were formed by pipetting drops 7 cm above liquid nitrogen (LN2), which were then kept inside cryovials in the LN2. Thawing was performed 2 months later by taking 3-4 pellets of the frozen semen into a glass tube and warming it in a water bath for 8 s at 60 °C. The results showed that 3% DMA increased the proportion of total motile sperm, progressivity, viability, and plasma membrane integrity (%) compared to the 6% and 9% DMA groups. The lipid peroxidation and antioxidant enzyme activity were improved in the 3% group. At the same time, some anti-freeze-related genes' (including ras homolog family member A (RHOA), heat shock protein 70 (HSP70), and small nuclear ribonucleoprotein polypeptide A (SNRPA1)) expressions were upregulated within the 3% DMA group relative to other groups. In conclusion, the 3% DMA group maintained higher post-thawed sperm quality than the other tested groups.

HCC-Mark: a simple non-invasive model based on routine parameters for predicting hepatitis C virus related hepatocellular carcinoma.

BACKGROUND: Early detection of hepatocellular carcinoma (HCC) is crucial in providing more effective therapies. As routine laboratory variables are readily accessible, this study aimed to develop a simple non-invasive model for predicting hepatocellular cancer. METHODS: Two groups of patients were recruited: an estimation group (n = 300) and a validation group (n = 625). Each comprised two categories: hepatocellular cancer and liver cirrhosis. Logistic regression analyses and receiver operating characteristic (ROC) curves were used to develop and validate the HCC-Mark model comprising AFP, high-sensitivity C-reactive protein, albumin and platelet count. This model was tested in cancer patients classified by the Barcelona Clinic Liver Cancer (BCLC), Cancer of Liver Italian Program (CLIP) and Okuda systems, and was compared with other non-invasive models for predicting hepatocellular cancer. RESULTS: HCC-Mark produced a ROC AUC of 0.89 (95% CI 0.85-0.90) for discriminating hepatocellular carcinoma from liver cirrhosis in the estimation group and 0.90 (0.86-0.90) in the validation group (both p < 0.0001). This AUC exceeded all other models, that had AUCs from 0.41 to 0.81. AUCs of HCC-Mark for discriminating patients with a single focal lesion, absent macrovascular invasion, tumour size <2 cm, BCLC (0-A), CLIP (0-1) and Okuda (stage Ι) from cirrhotic patients were 0.88 (0.85-0.90), 0.87 (0.85-0.89), 0.89 (0.85-0.93), 0.87 (0.84-0.89), 0.85 (0.82-0.87) and 0.86 (0.83-0.89), respectively (all p < 0.0001). CONCLUSION: HCC-Mark is an accurate and validated model for the detection of hepatocellular cancer and certain of its clinical features.

Diagnostic role of collagen-III and matrix metalloproteinase-1 for early detection of hepatocellular carcinoma.

Background: As the poor prognosis of hepatocellular carcinoma (HCC) is mostly due to late detection at an advanced stage there is a strong need for establishing more effective strategies for early identification. We hypothesized that collagen-III and matrix metalloproteinase-1 (MMP-1) and their ratio (CMR) are effective markers for identifying early-HCC when used alongside serum AFP, alkaline phosphatase and bilirubin.Methods: We recruited 148 patients with HCC, 133 with cirrhosis and 121 with fibrosis. Liver fibrosis was staged according to METAVIR, HCC was diagnosed by on histological findings or typical imaging characteristics by ultrasound and computed tomography. Collagen-III and MMP-1 were identified based on Western blotting and quantified in sera using ELISA, liver function tests (LFTs) by routine methods.Results: Patients with HCC showed a significantly (P < 0.05) higher collagen-III and collagen-III/MMP-1 ratio (CMR) than fibrotic and cirrhotic patients. Patients with HCC showed significantly (P < 0.05) lower concentration of MMP-1 than those without. As expected, numerous LFTs were also abnormal. A score of AFP, alkaline phosphatase and bilirubin together with CMR (the HCC-ABC test) was then constructed, This yielded ROC area under curves of 0.85 (95% CI 0.79-0.98) for identifying small tumour size (<3 cm), 0.87 (0.79-0.98) for identifying CLIP (0-1) [Cancer of the Liver Italian Program] disease severity, and 0.87 (0.74-0.93) for identifying BCLC disease severity (all p < 0.001), which is each case exceeded the predictive value of AFP.Conclusion: HCC-ABC diagnostic Test is a promising index for HCC early detection with a high degree of accuracy that may facilitate therapy.

Monocyte/granulocyte to lymphocyte ratio and the MELD score as predictors for early recurrence of hepatocellular carcinoma after trans-arterial chemoembolization.

BACKGROUND: The first-line treatment option for intermediate-stage hepatocellular carcinoma is trans-arterial chemoembolization (TACE). Blood indices, such as lymphocyte/monocyte ratio (LMR), lymphocyte count, neutrophil/lymphocyte ratio (NLR), platelet/lymphocyte ratio (PLR), monocyte-granulocyte/lymphocyte ratio (MGLR) and red blood cell distribution width (RDW), are prognostic biomarkers in certain diseases. The model for end-stage liver disease (MELD) and Child-Turcotte-Pugh (CTP) scores have been designed for patients with cirrhosis waiting for liver transplantation and in patients with hepatocellular carcinoma. We hypothesized possible roles for these blood indices, and the MELD and CTP scores as predictors for early recurrence of hepatocellular carcinoma after TACE. METHODS: Routine laboratory indices determined the NLR, LMR, MGLR, RDW, PLR, as well as MELD and CTP scores in 147 patients. Sensitivity and specificity of the indices for hepatocellular carcinoma recurrence 36 months after TACE were estimated by receiver operator characteristic curve. RESULTS: In multivariate regression analysis, only male sex, the lymphocyte count, CTP, the MGLR and the MELD score significantly (P < 0.01) predicted recurrence. The area under curve (AUC) for detection of recurrence for MGLR at a cut-off value 2.75 was 0.63 (95% CI 0.54-0.72) with sensitivity 70.7%, specificity 59.2% and accuracy 63%. The MELD score at cut-off value 9.5 had diagnostic performance with AUC 0.71 (0.63-0.79), sensitivity 80% and specificity 55.8% and accuracy 71.3%. CONCLUSIONS: High MGLR and MELD scores are linked to increasing frequency of hepatocellular carcinoma recurrence after TACE and could be used as novel, simple, non-invasive prognostic tests.

Combined use of nuclear phosphoprotein c-Myc and cellular phosphoprotein p53 for hepatocellular carcinoma detection in high-risk chronic hepatitis C patients.

BACKGROUND: Hepatocellular carcinoma (HCC) is a multistage process resulting from various genetic changes. We aimed to determine nuclear phosphoprotein c-Myc and cellular phosphoprotein p53 expression and to evaluate their importance in HCC diagnosis. METHODS: One hundred and twenty chronic hepatitis C (CHC) patients (60 non-HCC CHC patients and 60 HCC patients who had a single small (<5 cm) tumour) were recruited. The gene products of c-Myc and p53 were identified in liver tissues and serum samples using immunostaining, western blot and ELISA. RESULTS: Immunohistochemical detection of c-Myc and p53 with monospecific antibodies revealed intense and diffuse cytoplasmic staining patterns. Accumulated mutant proteins, released from tumour cells into the extracellular serum, were detected at 62 KDa, for c-Myc, and 53 KDa, for p53, using western blotting. In contrast to alpha feto-protein, there was a significant increase (p < 0.0001) in the positivity rate of c-Myc (86.7% vs. 6.7%) and p53 (78.3% vs. 8.3%) in the malignant vs. non-malignant patients. The parallel combination of c-Myc and p53 reach the absolute sensitivity (100%), for more accurate and reliable HCC detection (specificity was 87%). CONCLUSION: c-Myc and p53 are potential HCC diagnostic biomarkers, and convenient combinations of them could improve diagnostic accuracy of HCC.

HCC-ART score, a simple, highly sensitive and specific test for early diagnosis of hepatocellular carcinoma: a large-scale, multicentre study.

BACKGROUND: A simple scoring system is needed to discriminate HCC from patients with chronic liver diseases (CLD). The simplest score would be one that requires only variables that can be documented simply from routine laboratory tests without the need for sophisticated tests. METHODS: Data from the estimation group (1351 patients) and the validation group (2208 patients) were retrospectively analysed. Liver fibrosis-negative control and liver cirrhosis were compared with HCC. Area under ROC curve (AUC) were used to develop HCC-α-fetoprotein-routine test (HCC-ART). RESULTS: Hepatocellular carcinoma-AFP-routine test showed diagnostic accuracy for liver cirrhosis vs HCC with ROC curves of 0.99%, sensitivity of 97%, and specificity of 96% in the estimation, and 0.95%, 90%, and 83%, respectively, in the validation. Sensitivity (97%) and specificity (100%) were obtained to discriminate HCC from liver fibrosis. Area under curve for AFP at 400 U l(-1) was 0.70, sensitivity was 41%, and specificity was 99% in the estimation, and 0.77%, 54%, and 99%, respectively, in the validation. The AUC for HCC-ART in HCC with single tumour, absent vascular invasion, size <2 cm and CLIP score (0-1) were 0.95, 0.93, 0.86, 0.87, respectively, compared with 0.72, 0.71, 0.71, 0.50, respectively, for AFP. CONCLUSION: Hepatocellular carcinoma-AFP-routine test could increase the accuracy of HCC screening and surveillances and could be used worldwide without extra efforts.

Trp53 deficiency protects against acute intestinal inflammation.

The p53 protein has not only important tumor suppressor activity but also additional immunological and other functions, whose nature and extent are just beginning to be recognized. In this article, we show that p53 has a novel inflammation-promoting action in the intestinal tract, because loss of p53 or the upstream activating kinase, ATM, protects against acute intestinal inflammation in murine models. Mechanistically, deficiency in p53 leads to increased survival of epithelial cells and lamina propria macrophages, higher IL-6 expression owing to enhanced glucose-dependent NF-κB activation, and increased mucosal STAT3 activation. Blockade or loss of IL-6 signaling reverses the protective effects of p53 deficiency. Conversely, IL-6 treatment protects against acute colitis in a manner dependent on STAT3 signaling and induction of cytoprotective factors in epithelial cells. Together, these results indicate that p53 promotes inflammation in the intestinal tract through suppression of epithelium-protective factors, thus significantly expanding the spectrum of physiological and immunological p53 activities unrelated to cancer formation.

Determination of fluoxetine and norfluoxetine in human plasma by capillary gas chromatography with electron-capture detection.

A capillary gas chromatographic method with 63Ni electron-capture detection is reported for the determination of fluoxetine (Prozac) and its metabolite norfluoxetine in human plasma. A liquid-liquid extraction is used, followed by derivatization with heptafluorobutyric anhydride to increase the sensitivity of detection. A 30 m x 0.25 mm I.D. DB-17 capillary column resolves the compounds from endogenous matrix interferences. The limit of quantitation by this method is 5 ng/ml for each compound. Stability studies show that fluoxetine and norfluoxetine are stable in human plasma for up to 96 h at room temperature and up to one year at -20 degrees C.

Kinetics of the aspartyl transpeptidation of daptomycin, a novel lipopeptide antibiotic.

Two degradation products of the lipopeptide antibiotic, daptomycin, were identified and the reaction pathway and kinetics were delineated in aqueous solution at 60 degrees C, pH range 3 to 8 and ionic strength 0.01. The degradation products were 1) a succinimido intermediate (anhydro-daptomycin) formed by attack of side-chain carbonyl on the peptide-bond nitrogen in the asp-gly sequence and 2) a beta-asp daptomycin isomer formed by rehydration of the anhydrodaptomycin succinimide. This aspartyl transpeptidation pathway was found to be reversible. Formation of the anhydrodaptomycin from either daptomycin or beta-asp daptomycin was pH dependent but the pH-rate profiles for anhydrodaptomycin formation were not mechanistically interpretable. The pH-rate profiles for the formation of daptomycin or beta-asp daptomycin from the anhydrodaptomycin were linear with slopes = 1, which is consistent with nucleophilic hydroxide ion attack of the succinimido intermediate at either the alpha-carbonyl, giving rise to the beta-asp daptomycin, or the beta-carbonyl, giving rise to daptomycin.

Determination of fluoxetine and norfluoxetine in plasma by gas chromatography with electron-capture detection.

This gas-chromatographic method for assay of fluoxetine and norfluoxetine in human plasma involves extraction of the drugs and use of a 63Ni electron-capture detector. The linear range of detection is 25 to 800 micrograms/L for each drug. Overall precision (CV) in the concentration range of 10 to 100 micrograms/L for both drugs was approximately 10%. Accuracy (relative error) in the same concentration range was approximately +10%. None of the commonly prescribed antidepressants or tranquilizers that we tested interfere with the assay.