The introduction of metagenomics into an undergraduate biochemistry laboratory course yielded a predicted reductase that decreases triclosan susceptibility in Escherichia coli

Traditional undergraduate science classes often include a laboratory component aimed at enabling the students to experience the classroom topics firsthand. Typically, these experiments are chosen because they have known outcomes that will clearly demonstrate particular aspects of scientific theory. While this approach has its benefits in skill development and concept reinforcement, the lack of novelty inherent in repeating experiments that have been repeated for many years does not accurately convey the feeling of true scientific discovery to the students. In this work, we have designed and implemented a series of experiments into an undergraduate biochemistry curriculum that incorporates the opportunity for scientific discovery, while simultaneously creating an environment for learning routine laboratory techniques. Through this set of experiments, students enrolled in the course were successful in identifying and beginning to characterize an unknown bacterial gene that confers increased tolerance to triclosan on its host.     

High-content imaging technology for the evaluation of drug-induced steatosis using a multiparametric cell-based assay

In the present study, we developed a cell-based protocol for the identification of drugs able to induce steatosis. The assay measures multiple markers of toxicity in a 96-well plate format using high-content screening (HCS) technology. After treating HepG2 cells with increasing concentrations of the tested compounds, toxicity parameters were analyzed using fluorescent probes: BODIPY493/503 (lipid content), 2',7'-dihydrodichlorofluorescein diacetate (reactive oxygen species [ROS] generation), tetramethyl rhodamine methyl ester (mitochondrial membrane potential), propidium iodide (cell viability), and Hoechst 33342 (nuclei staining). A total of 16 drugs previously reported to induce liver steatosis through different mechanisms (positive controls) and six nonsteatotic compounds (negative controls) were included in the study. All the steatosis-positive compounds significantly increased BODIPY493/503 fluorescence in HepG2 cells, whereas none of the negative controls induced lipid accumulation. In addition to effects on fat levels, increased ROS generation was produced by certain compounds, which could be indicative of increased risk of liver damage. Our results suggest that this in vitro approach is a simple, rapid, and sensitive screening tool for steatosis-inducing drugs. This conclusion should be confirmed by testing a larger number of steatosis-positive and -negative inducers.     

Population stratification may bias analysis of PGC-1α as a modifier of age at Huntington disease motor onset

Huntington’s disease (HD) is an inherited neurodegenerative disorder characterized by motor, cognitive and behavioral disturbances, caused by the expansion of a CAG trinucleotide repeat in the HD gene. The CAG allele size is the major determinant of age at onset (AO) of motor symptoms, although the remaining variance in AO is highly heritable. The rs7665116 SNP in PPARGC1A, encoding the mitochondrial regulator PGC-1α, has been reported to be a significant modifier of AO in three European HD cohorts, perhaps due to affected cases from Italy. We attempted to replicate these findings in a large collection of (1,727) HD patient DNA samples of European origin. In the entire cohort, rs7665116 showed a significant effect in the dominant model (p value?=?0.008) and the additive model (p value?=?0.009). However, when examined by origin, cases of Southern European origin had an increased rs7665116 minor allele frequency (MAF), consistent with this being an ancestry-tagging SNP. The Southern European cases, despite similar mean CAG allele size, had a significantly older mean AO (p?<?0.001), suggesting population-dependent phenotype stratification. When the generalized estimating equations models were adjusted for ancestry, the effect of the rs7665116 genotype on AO decreased dramatically. Our results do not support rs7665116 as a modifier of AO of motor symptoms, as we found evidence for a dramatic effect of phenotypic (AO) and genotypic (MAF) stratification among European cohorts that was not considered in previously reported association studies. A significantly older AO in Southern Europe may reflect population differences in genetic or environmental factors that warrant further investigation.     

Immunohistochemical analysis of the ubiquitin-conjugating enzyme UbcH10 in lung cancer: a useful tool for diagnosis and therapy

The ubiquitin-conjugating enzyme (UbcH10) plays important roles in the regulation of cell cycle progression. Recently, UbcH10 expression has been demonstrated in several human and experimental tumors, and proteasome inhibitors have been tested in trials for pulmonary neoplasms; however, the underlying mechanisms as well as the clinicopathological relevance of UbcH10 in the genesis and progression of lung cancer remain largely unknown. Therefore, the authors evaluated the expression of UbcH10 in human lung cancer and evaluated its possible diagnostic and prognostic use. They found that most cases of lung adenocarcinoma, squamous cell carcinoma, and large cell and small cell carcinoma were positive for UbcH10. The expression levels of UbcH10 progressively increased with decreasing degree of tumor differentiation. There was a statistically significant difference of UbcH10 positivity between grade I/III of lung adenocarcinoma (p=0.013) and squamous cell carcinoma (p=0.002). No significant differences were found between histological types (p=0.072). In the case of cell blocks prepared from pleural effusions, inflammatory and reactive mesothelial elements did not show appreciable UbcH10 expression, whereas neoplastic cells exhibited clear UbcH10 positivity. The results suggest that UbcH10 might represent a new and promising diagnostic and prognostic marker in both histologic and cytologic specimens of lung cancer.     

Effects of Single Amino Acid Substitutions on Aggregation and Cytotoxicity Properties of Amyloid β Peptide

International Journal of Peptide Research and Therapeutics - Alzheimer’s disease is the main cause of dementia and the deposition of amyloid beta peptide (Aβ) in the brain is the key...     

Emerging and re-emerging viral infections in Europe

Emerging viral infections are becoming a serious problem in Europe in the recent years. This is particularly true for severe acute respiratory syndrome (SARS), West Nile virus (WNV) disease, Toscana virus (TOSV) disease, and potentially for avian influenza virus (H5N1). In contrast, emergence or re-emergence of severe viral infections, including tick borne encephalitis virus, and viral haemorrhagic fever caused by Hantavirus and dengue virus have been frequently reported in several European countries. Laboratory diagnosis of these viral infections based on viral isolation or detection by immune electron microscopy, immunoassay and polymerase chain reaction (PCR) has dramatically improved in the recent years, and SARS represents a good example of a diagnostic approach to emerging viral infections. Finally, old and new promising agents are in the pipeline of pharmaceutical companies to treat emerging viral infections. However only prevention based on large epidemiological studies, and research and development of new vaccines may be able to control and eventually eradicate these deadly viral infections.     

Modulation of the proteolytic activity of matrix metalloproteinase-2 (gelatinase A) on fibrinogen

The proteolytic processing of bovine fibrinogen by MMP-2 (gelatinase A), which brings about the formation of a product unable to form fibrin clots, has been studied at 37 degrees C. Catalytic parameters, although showing a somewhat lower catalytic efficiency with respect to thrombin and plasmin, indeed display values indicating a pathophysiological significance of this process. A parallel molecular modelling study predicts preferential binding of MMP-2 to the beta-chain of fibrinogen through its haemopexin-like domain, which has been directly demonstrated by the inhibitory effect in the presence of the exogenous haemopexin-like domain. However, the removal of this domain does not impair the interaction between MMP-2 and fibrinogen, but it dramatically alters the proteolytic mechanism, producing different fragmentation intermediates. The investigation at various pH values between 6.0 and 9.3 indicates a proton-linked behaviour, which is relevant for interpreting the influence on the process by environmental conditions occurring at the site of an injury. Furthermore, the action of MMP-2 on peroxynitrite-treated fibrinogen has been investigated, a situation possibly occurring under oxidative stress. The chemical alteration of fibrinogen, which has been shown to abolish its clotting activity, brings about only limited modifications of the catalytic parameters without altering the main enzymatic mechanism.     

Effect of chronic Sildenafil treatment on the prostate of C57Bl/6 mice

Sildenafil is a potent and selective inhibitor of phosphodiesterase-5 (PDE5) and is considered first-line therapy for erectile dysfunction. Nowadays, Sildenafil is used extensively throughout the world on patients with pulmonary hypertension. However, few studies have evaluated the possible side effects of chronic Sildenafil treatment on the male reproductive system, specifically in the prostate. In the present study, it was demonstrated via morphological and ultrastructural analysis that chronic treatment with Sildenafil induced an enhancement of the glandular activity of the prostate. In addition, mice treated with Sildenafil showed a significant increase in testosterone serum levels. However, no statistically significant differences were observed in nitric oxide serum levels, or in sGC, eNOS, PSA and TGF-β prostatic expression. In conclusion, the present study suggests that chronic use of Sildenafil does not cause evident prostatic damage, and therefore, can be used pharmacologically to treat a variety of disorders.     

Phosphocaveolin-1 Enforces Tumor Growth and Chemoresistance in Rhabdomyosarcoma

Caveolin-1 (Cav-1) can ambiguously behave as either tumor suppressor or oncogene depending on its phosphorylation state and the type of cancer. In this study we show that Cav-1 was phosphorylated on tyrosine 14 (pCav-1) by Src-kinase family members in various human cell lines and primary mouse cultures of rhabdomyosarcoma (RMS), the most frequent soft-tissue sarcoma affecting childhood. Cav-1 overexpression in the human embryonal RD or alveolar RH30 cells yielded increased pCav-1 levels and reinforced the phosphorylation state of either ERK or AKT kinase, respectively, in turn enhancing in vitro cell proliferation, migration, invasiveness and chemoresistance. In contrast, reducing the pCav-1 levels by administration of a Src-kinase inhibitor or through targeted Cav-1 silencing counteracted the malignant in vitro phenotype of RMS cells. Consistent with these results, xenotransplantation of Cav-1 overexpressing RD cells into nude mice resulted in substantial tumor growth in comparison to control cells. Taken together, these data point to pCav-1 as an important and therapeutically valuable target for overcoming the progression and multidrug resistance of RMS.