Post-Operative Plasma Osteopontin Predicts Distant Metastasis in Human Colorectal Cancer

BackgroundThe overall prognosis of colorectal cancer (CRC) patients is unsatisfactory due to cancer metastasis after operation. This study aims to investigate the clinical significance of plasma osteopontin (OPN) levels as minimally invasive, predictive, and surrogate biomarkers for prognosis of CRC patients.MethodsThis randomized study design consists of pre-operative and post-operative plasma samples from a total of 79 patients. We determined plasma levels of OPN by ELISA and examined their correlation with the clinicopathological parameters of CRC patients. The effects of endogenous and exogenous OPN on CRC metastasis were investigated by examination of the effect on regulators of epithelial to messenchymal transition and migration assay.ResultsOur findings demonstrated for the first time the clinical correlation of plasma OPN with metastasis of CRC patients. High post-operative plasma OPN level (>153.02 ng/ml) associated with development of metastasis after curative resection (p<0.001). Moreover, post-operative plasma OPN level correlated with disease-free survival of CRC patients (p=0.009) and was an independent factor for predicting development of metastasis in CRC patients after curative resection (p=0.036). Our in vitro model showed that OPN ectopic expression induced DLD1 cell migration through Snail and Twist1 overexpression and E-cadherin repression, and secretory OPN level enhanced cell migration.ConclusionsThe results of the current study suggest that post-operative plasma OPN correlated with post-operative metastasis, suggesting that it is a potential non-invasive biomarker for the development of future metastasis in CRC patients. In addition, OPN was shown to be involved in the metastatic process and thus inhibition of OPN is a potential therapeutic approach to treat CRC patients.     

Loss from Treatment for Drug Resistant Tuberculosis: Risk Factors and Patient Outcomes in a Community-Based Program in Khayelitsha,South Africa

Background

A community based drug resistant tuberculosis (DR-TB) program has been incrementally implemented in Khayelitsha, a high HIV and TB burden community in South Africa. We investigated loss from treatment (LFT), and post treatment outcomes of DR-TB patients in this setting.

Methodology

LFT, defined as interruption of treatment for ≥2 consecutive months was assessed among patients initiating DR-TB treatment for the first time between January 2009 and July 2011. Patients were traced through routine data sources to identify those who subsequently restarted treatment and those who died. Additional information on patient status and survival after LTF was obtained from community DR-TB counselors and from the national death registry. Post treatment outcomes were observed until July 2013.

Results

Among 452 patients initiating treatment for the first time within the given period, 30% (136) were LFT, with 67% retention at 18 months. Treatment was restarted in 27 (20%) patients, with additional resistance recorded in 2/25 (8%), excluding two with presumed DR-TB. Overall, 34 (25%) patients died, including 11 who restarted treatment. Males and those in the age category 15-25 years had a greater hazard of LFT; HR 1.93 (95% CI 1.35-2.75), and 2.43 (95% CI 1.52-3.88) respectively. Older age (>35 years) was associated with a greater hazard of death; HR 3.74 (1.13- 12.37) post treatment. Overall two-year survival was 62%. It was lower (45%) in older patients, and was 92% among those who received >12 months treatment.

Conclusion

LFT was high, occurred throughout the treatment period and was particularly high among males and those aged 15-25 years. Overall long term survival was poor. High rates of LFT should however not preclude scale up of community based care given its impact in increasing access to treatment. Further research is needed to support retention of DR-TB patients on treatment, even within community based treatment programs.     

Exosomes Are Unlikely Involved in Intercellular Nef Transfer

HIV-1 Nef is an important pathogenic factor for HIV/AIDS pathogenesis. Several recent studies including ours have demonstrated that Nef can be transferred to neighboring cells and alters the function of these cells. However, how the intercellular Nef transfer occurs is in dispute. In the current study, we attempted to address this important issue using several complementary strategies, a panel of exosomal markers, and human CD4+ T lymphocyte cell line Jurkat and a commonly used cell line 293T. First, we showed that Nef was transferred from Nef-expressing or HIV-infected Jurkat to naïve Jurkat and other non-Jurkat cells and that the transfer required the membrane targeting function of Nef and was cell density-dependent. Then, we showed that Nef transfer was cell-cell contact-dependent, as exposure to culture supernatants or exosomes from HIV-infected Jurkat or Nef-expressing Jurkat and 293T led to little Nef detection in the target cells Jurkat. Thirdly, we demonstrated that Nef was only detected to be associated with HIV virions but not with acetylcholinesterase (AChE+) exosomes from HIV-infected Jurkat and not in the exosomes from Nef-expressing Jurkat. In comparison, when it was over-expressed in 293T, Nef was detected in detergent-insoluble AChE+/CD81^low/TSG101^low exosomes, but not in detergent-soluble AChE-/CD81^high/TSG101^high exosomes. Lastly, microscopic imaging showed no significant Nef detection in exosomal vesicle-like structures in and out 293T. Taken together, these results show that exosomes are unlikely involved in intercellular Nef transfer. In addition, this study reveals existence of two types of exosomes: AChE+/CD81^low/TSG101^low exosomes and AChE-/CD81^high/TSG101^high exosomes.     

The MMS22L-TONSL complex mediates recovery from replication stress and homologous recombination

Genome integrity is jeopardized each time DNA replication forks stall or collapse. Here we report the identification of a complex composed of MMS22L (C6ORF167) and TONSL (NFKBIL2) that participates in the recovery from replication stress. MMS22L and TONSL are homologous to yeast Mms22 and plant Tonsoku/Brushy1, respectively. MMS22L-TONSL accumulates at regions of ssDNA associated with distressed replication forks or at processed DNA breaks, and its depletion results in high levels of endogenous DNA double-strand breaks caused by an inability to complete DNA synthesis after replication fork collapse. Moreover, cells depleted of MMS22L are highly sensitive to camptothecin,?a topoisomerase I poison that impairs DNA replication progression. Finally, MMS22L and TONSL are necessary for the efficient formation of RAD51 foci after DNA damage, and their depletion impairs homologous recombination. These results indicate that MMS22L and TONSL are genome caretakers that stimulate the recombination-dependent repair of stalled or collapsed replication forks.     

Assessment of environmental stress by the micronucleus and comet assays on Limnoperna fortunei exposed to Guaíba hydrographic region samples (Brazil) under laboratory conditions

The Guaíba Basin is a source of drinking water for Porto Alegre (RS, Brazil). The water from this basin receives industrial, urban, and rural waste from many sources. The mussel species Limnoperna fortunei was chosen based on population data, distribution, and sensitivity. Previous tests with comet assay and micronuclei frequency in this freshwater mussel have shown to be successful in biomonitoring studies. The aim of this study was to evaluate the genotoxic contamination of the Guaíba Lake Hydrographic Region, through the determination of damage by the micronuclei and comet assays in L. fortunei (golden mussel). Nine sampling sites were evaluated in three different seasons: five sites in the mouths of the main rivers that flow into Guaíba lake; one site at the mouth of a stream; one major site of sewage discharge; two sites at Guaíba lake, near a sewage discharge; and the control site in a preservation area. DNA damage was detected by the single cell gel assay, as well as the frequency of micronuclei in hemocytes of mussels exposed under laboratory conditions for 7 days to water and sediment samples. Significant results were found in different seasons in almost all sampling sites (P<0.05, ANOVA Dunnet's test). Most of the positive results were found in samples affected mainly by urban effluents. It was possible to observe that there was a weak relation between mutagenic and genotoxic responses and mussels inorganic elements contents. Seasonal variation was observed at different sampling sites, but always indicating a huge contamination near urban sewage discharge. These results are consistent with previous studies, allowing us to infer that urban contamination is the biggest problem in this region. It is also possible to infer that L. fortunei is a good sentinel organism for the Guaíba Basin.     

Prospective identification of myogenic endothelial cells in human skeletal muscle

We document anatomic, molecular and developmental relationships between endothelial and myogenic cells within human skeletal muscle. Cells coexpressing myogenic and endothelial cell markers (CD56, CD34, CD144) were identified by immunohistochemistry and flow cytometry. These myoendothelial cells regenerate myofibers in the injured skeletal muscle of severe combined immunodeficiency mice more effectively than CD56+ myogenic progenitors. They proliferate long term, retain a normal karyotype, are not tumorigenic and survive better under oxidative stress than CD56+ myogenic cells. Clonally derived myoendothelial cells differentiate into myogenic, osteogenic and chondrogenic cells in culture. Myoendothelial cells are amenable to biotechnological handling, including purification by flow cytometry and long-term expansion in vitro, and may have potential for the treatment of human muscle disease.     

Virtual screening using molecular simulations

Effective virtual screening relies on our ability to make accurate prediction of protein-ligand binding, which remains a great challenge. In this work, utilizing the molecular-mechanics Poisson-Boltzmann (or Generalized Born) surface area approach, we have evaluated the binding affinity of a set of 156 ligands to seven families of proteins, trypsin β, thrombin α, cyclin-dependent kinase (CDK), cAMP-dependent kinase (PKA), urokinase-type plasminogen activator, β-glucosidase A, and coagulation factor Xa. The effect of protein dielectric constant in the implicit-solvent model on the binding free energy calculation is shown to be important. The statistical correlations between the binding energy calculated from the implicit-solvent approach and experimental free energy are in the range of 0.56-0.79 across all the families. This performance is better than that of typical docking programs especially given that the latter is directly trained using known binding data whereas the molecular mechanics is based on general physical parameters. Estimation of entropic contribution remains the barrier to accurate free energy calculation. We show that the traditional rigid rotor harmonic oscillator approximation is unable to improve the binding free energy prediction. Inclusion of conformational restriction seems to be promising but requires further investigation. On the other hand, our preliminary study suggests that implicit-solvent based alchemical perturbation, which offers explicit sampling of configuration entropy, can be a viable approach to significantly improve the prediction of binding free energy. Overall, the molecular mechanics approach has the potential for medium to high-throughput computational drug discovery.     

"My parents decide if I can. I decide if I want to." Children's views on participation in medical research

The participation of children in medical research raises many ethical issues, in particular regarding assent. However, little is known about children's own views on participation. This study presents results from interviews with children 10-12 years old with and without experience in a large-scale longitudinal screening study. We identified five themes: (1) knowledge about research, (2) a sense of altruism, (3) shared decision-making and right to dissent, (4) notions of integrity, privacy, and access, and (5) understanding of disease risk and personal responsibilities. We conclude that the children feel positive towards medical research, and want to take an active part in decisions and have their integrity respected. However, the study also indicates that children who had participated in longitudinal screening had a limited understanding, suggesting the vital importance of providing information appropriate to their age and maturity. This information should be provided out of respect for the children as persons, but also to promote their willingness to continue participating in longitudinal studies.