Passaging capability of human corneal endothelial cells derived from old donors with and without accelerating cell attachment

In a recent report, we showed that it is possible to establish the culture of Human Corneal Endothelial Cells (HCEnCs) from older donor corneas (usually over 65 year olds) when left to attach in the presence of a viscoelastic solution, potentially increasing the donor pool for culturing HCEnCs. Therefore, we set out to evaluate the outcome of using a viscoelastic solution (Viscoat) to accelerate the attachment of passaged cultured human corneal endothelial cells (HCEnCs). The cells from 28 donor tissues were isolated using peel-and-digest method and evenly seeded into two wells of an 8-well chamber slide. The cells were left to attach after topical application of Viscoat. At confluence, one well was subjected to end-stage characterization, whereas the other well was passaged into another two wells. The cells at P1 were attached with and without the use of Viscoat. The growth rate was monitored; and at confluence, morphometric analysis, corneal endothelial specific (CD166-Tag1A3 & PRDX6-Tag2A12), mitochondrial and respiration assessment (Tom-20 and Seahorse); function-associated (Na+/K+ATPase & ZO-1); proliferative (Ki-67) marker analysis, and viability (Hoechst, Ethidium Homodimer and Calcein AM-HEC) studies were performed. Cells at P0 (with Viscoat) showed 100% confluence at day 9. Cells at P1 with and without Viscoat showed significant difference of confluence 67.0% v 18.8% respectively (p < 0.05). Confluence rate, cell density, hexagonality, Ki-67 positivity and mitochondrial intensity was significantly higher (p < 0.05), whereas cell-area and polymorphism was significantly lower (p < 0.05) in the cells attached with Viscoat compared with the cells attached without Viscoat. There was no significant difference in oxygen consumption rate between the groups. In conclusion, we observed that acceleration in the attachment of passaged HCEnCs with the assistance of Viscoat, could be beneficial for the propagation of HCEnCs isolated from older donors, to increase their propensity to proliferate, without loss of the expression of vital proteins and heterogeneity in cellular morphology

Automotive leaf spring design and manufacturing process improvement using failure mode and effects analysis (FMEA)

Nowadays human safety and comfort are the most considerable parameters in designing and manufacturing of a vehicle, that is why every organization ensures the quality and reliability of components used in the vehicle. Leaf spring is also a component of vehicle which plays an important role in human safety and comfort. It acts as a structural member and an integral part of suspension system. It is important to eliminate the failures in designing and manufacturing process of leaf springs because of its importance in functionality and safety of vehicle. In this research, failure mode and effects analysis has been used to analyze and reduce the risks of 42 possible failures that can occur in automotive leaf spring. It starts from determining, classifying, and analyzing all potential failures and then rating them with the help numeric scores. The four numeric scores namely severity, occurrence, detection, and Risk Priority Number (RPN) are used to find the high potential failures of semi-elliptical leaf springs. In the end, actions are recommended for RPN greater than 250, to increase quality and reliably of product. </jats:p

From Nonspecific DNA–Protein Encounter Complexes to the Prediction of DNA–Protein Interactions

©2009 Gao, Skolnick. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.doi:10.1371/journal.pcbi.1000341DNA–protein interactions are involved in many essential biological activities. Because there is no simple mapping code between DNA base pairs and protein amino acids, the prediction of DNA–protein interactions is a challenging problem. Here, we present a novel computational approach for predicting DNA-binding protein residues and DNA–protein interaction modes without knowing its specific DNA target sequence. Given the structure of a DNA-binding protein, the method first generates an ensemble of complex structures obtained by rigid-body docking with a nonspecific canonical B-DNA. Representative models are subsequently selected through clustering and ranking by their DNA–protein interfacial energy. Analysis of these encounter complex models suggests that the recognition sites for specific DNA binding are usually favorable interaction sites for the nonspecific DNA probe and that nonspecific DNA–protein interaction modes exhibit some similarity to specific DNA–protein binding modes. Although the method requires as input the knowledge that the protein binds DNA, in benchmark tests, it achieves better performance in identifying DNA-binding sites than three previously established methods, which are based on sophisticated machine-learning techniques. We further apply our method to protein structures predicted through modeling and demonstrate that our method performs satisfactorily on protein models whose root-mean-square Ca deviation from native is up to 5 Å from their native structures. This study provides valuable structural insights into how a specific DNA-binding protein interacts with a nonspecific DNA sequence. The similarity between the specific DNA–protein interaction mode and nonspecific interaction modes may reflect an important sampling step in search of its specific DNA targets by a DNA-binding protein

Optimal Surgical Extent for Memory and Seizure Outcome in Temporal Lobe Epilepsy

OBJECTIVE: Postoperative memory decline is an important consequence of anterior temporal lobe resection (ATLR) for temporal lobe epilepsy (TLE), and the extent of resection may be a modifiable factor. This study aimed to define optimal resection margins for cognitive outcome while maintaining a high rate of postoperative seizure freedom. METHODS: This cohort study evaluated the resection extent on postoperative structural MRI using automated voxel-based methods and manual measurements in 142 consecutive patients with unilateral drug refractory TLE (74 left, 68 right TLE) who underwent standard ATLR. RESULTS: Voxel-wise analyses revealed that postsurgical verbal memory decline correlated with resections of the posterior hippocampus and inferior temporal gyrus, whereas larger resections of the fusiform gyrus were associated with worsening of visual memory in left TLE. Limiting the posterior extent of left hippocampal resection to 55% reduced the odds of significant postoperative verbal memory decline by a factor of 8.1 (95% CI 1.5-44.4, p=0.02). Seizure freedom was not related to posterior resection extent, but to the piriform cortex removal after left ATLR. In right TLE, variability of the posterior extent of resection was not associated with verbal and visual memory decline or seizures after surgery. INTERPRETATION: The extent of surgical resection is an independent and modifiable risk factor for cognitive decline and seizures after left ATLR. Adapting the posterior extent of left ATLR might optimize postoperative outcome, with reduced risk of memory impairment while maintaining comparable seizure-freedom rates. The current, more lenient, approach might be appropriate for right ATLR. This article is protected by copyright. All rights reserved

Targeted knock-down of miR21 primary transcripts using snoMEN vectors induces apoptosis in human cancer cell lines

We have previously reported an antisense technology, 'snoMEN vectors', for targeted knock-down of protein coding mRNAs using human snoRNAs manipulated to contain short regions of sequence complementarity with the mRNA target. Here we characterise the use of snoMEN vectors to target the knock-down of micro RNA primary transcripts. We document the specific knock-down of miR21 in HeLa cells using plasmid vectors expressing miR21-targeted snoMEN RNAs and show this induces apoptosis. Knock-down is dependent on the presence of complementary sequences in the snoMEN vector and the induction of apoptosis can be suppressed by over-expression of miR21. Furthermore, we have also developed lentiviral vectors for delivery of snoMEN RNAs and show this increases the efficiency of vector transduction in many human cell lines that are difficult to transfect with plasmid vectors. Transduction of lentiviral vectors expressing snoMEN targeted to pri-miR21 induces apoptosis in human lung adenocarcinoma cells, which express high levels of miR21, but not in human primary cells. We show that snoMEN-mediated suppression of miRNA expression is prevented by siRNA knock-down of Ago2, but not by knock-down of Ago1 or Upf1. snoMEN RNAs colocalise with Ago2 in cell nuclei and nucleoli and can be co-immunoprecipitated from nuclear extracts by antibodies specific for Ago2

Optimizing CIGB-300 intralesional delivery in locally advanced cervical cancer

Background:We conducted a phase 1 trial in patients with locally advanced cervical cancer by injecting 0.5 ml of the CK2-antagonist CIGB-300 in two different sites on tumours to assess tumour uptake, safety, pharmacodynamic activity and identify the recommended dose.Methods:Fourteen patients were treated with intralesional injections containing 35 or 70 mg of CIGB-300 in three alternate cycles of three consecutive days each before standard chemoradiotherapy. Tumour uptake was determined using 99 Tc-radiolabelled peptide. In situ B23/nucleophosmin was determined by immunohistochemistry.Results:Maximum tumour uptake for CIGB-300 70-mg dose was significantly higher than the one observed for 35 mg: 16.1±8.9 vs 31.3±12.9 mg (P=0.01). Both, AUC 24h and biological half-life were also significantly higher using 70 mg of CIGB-300 (P<0.001). Unincorporated CIGB-300 diffused rapidly to blood and was mainly distributed towards kidneys, and marginally in liver, lungs, heart and spleen. There was no DLT and moderate allergic-like reactions were the most common systemic side effect with strong correlation between unincorporated CIGB-300 and histamine levels in blood. CIGB-300, 70 mg, downregulated B23/nucleophosmin (P=0.03) in tumour specimens.Conclusion:Intralesional injections of 70 mg CIGB-300 in two sites (0.5 ml per injection) and this treatment plan are recommended to be evaluated in phase 2 studies.Fil: Sarduy, M. R.. Medical-surgical Research Center; CubaFil: García, I.. Centro de Ingeniería Genética y Biotecnología; CubaFil: Coca, M. A.. Clinical Investigation Center; CubaFil: Perera, A.. Clinical Investigation Center; CubaFil: Torres, L. A.. Clinical Investigation Center; CubaFil: Valenzuela, C. M.. Centro de Ingeniería Genética y Biotecnología; CubaFil: Baladrón, I.. Centro de Ingeniería Genética y Biotecnología; CubaFil: Solares, M.. Hospital Materno Ramón González Coro; CubaFil: Reyes, V.. Center For Genetic Engineering And Biotechnology Havana; CubaFil: Hernández, I.. Isotope Center; CubaFil: Perera, Y.. Centro de Ingeniería Genética y Biotecnología; CubaFil: Martínez, Y. M.. Medical-surgical Research Center; CubaFil: Molina, L.. Medical-surgical Research Center; CubaFil: González, Y. M.. Medical-surgical Research Center; CubaFil: Ancízar, J. A.. Centro de Ingeniería Genética y Biotecnología; CubaFil: Prats, A.. Clinical Investigation Center; CubaFil: González, L.. Centro de Ingeniería Genética y Biotecnología; CubaFil: Casacó, C. A.. Clinical Investigation Center; CubaFil: Acevedo, B. E.. Centro de Ingeniería Genética y Biotecnología; CubaFil: López Saura, P. A.. Centro de Ingeniería Genética y Biotecnología; CubaFil: Alonso, Daniel Fernando. Universidad Nacional de Quilmes; ArgentinaFil: Gómez, R.. Elea Laboratories; ArgentinaFil: Perea Rodríguez, S. E.. Center For Genetic Engineering And Biotechnology Havana; Cuba. Centro de Ingeniería Genética y Biotecnología; Cub

MicroRNAs targeting oncogenes are down-regulated in pancreatic malignant transformation from benign tumors

BACKGROUND MicroRNA (miRNA) expression profiles have been described in pancreatic ductal adenocarcinoma (PDAC), but these have not been compared with pre-malignant pancreatic tumors. We wished to compare the miRNA expression signatures in pancreatic benign cystic tumors (BCT) of low and high malignant potential with PDAC, in order to identify miRNAs deregulated during PDAC development. The mechanistic consequences of miRNA dysregulation were further evaluated. METHODS Tissue samples were obtained at a tertiary pancreatic unit from individuals with BCT and PDAC. MiRNA profiling was performed using a custom microarray and results were validated using RT-qPCR prior to evaluation of miRNA targets. RESULTS Widespread miRNA down-regulation was observed in PDAC compared to low malignant potential BCT. We show that amongst those miRNAs down-regulated, miR-16, miR-126 and let-7d regulate known PDAC oncogenes (targeting BCL2, CRK and KRAS respectively). Notably, miR-126 also directly targets the KRAS transcript at a "seedless" binding site within its 3'UTR. In clinical specimens, miR-126 was strongly down-regulated in PDAC tissues, with an associated elevation in KRAS and CRK proteins. Furthermore, miR-21, a known oncogenic miRNA in pancreatic and other cancers, was not elevated in PDAC compared to serous microcystic adenoma (SMCA), but in both groups it was up-regulated compared to normal pancreas, implicating early up-regulation during malignant change. CONCLUSIONS Expression profiling revealed 21 miRNAs down-regulated in PDAC compared to SMCA, the most benign lesion that rarely progresses to invasive carcinoma. It appears that miR-21 up-regulation is an early event in the transformation from normal pancreatic tissue. MiRNA expression has the potential to distinguish PDAC from normal pancreas and BCT. Mechanistically the down-regulation of miR-16, miR-126 and let-7d promotes PDAC transformation by post-transcriptional up-regulation of crucial PDAC oncogenes. We show that miR-126 is able to directly target KRAS; re-expression has the potential as a therapeutic strategy against PDAC and other KRAS-driven cancers