Validation and reproducibility of computerised cell-viability analysis of tissue slices

BACKGROUND: The identification of live cells using membrane integrity dyes has become a frequently used technique, especially with articular cartilage and chondrocytes in situ where tissue slices are used to assess cell recovery as a function of location. The development of a reproducible computerised method of cell evaluation would eliminate many variables associated with manual counting and significantly reduce the amount of time required to evaluate experimental results. METHODS: To validate a custom computerised counting program, intra-person and inter-person cell counts of nine human evaluators (three groups – unskilled, novice, and experienced) were compared with repeated pixel counts of the custom program on 15 digitised images (in triplicate) of chondrocytes in situ stained with fluorescent dyes. RESULTS: Results indicated increased reproducibility with increased experience within evaluators [Intraclass Correlation Coefficient (ICC) range = 0.67 (unskilled) to 0.99 (experienced)] and between evaluators [ICC = 0.47 (unskilled), 0.85 (novice), 0.93 (experienced)]. The computer program had perfect reproducibility (ICC = 1.0). There was a significant relationship between the average of the experienced evaluators results and the custom program results (ICC = 0.77). CONCLUSIONS: This study demonstrated that increased experience in cell counting resulted in increased reproducibility both within and between human evaluators but confirmed that the computer program was the most reproducible. There was a good correlation between the intact cell recovery determined by the computer program and the experienced human evaluators. The results of this study showed that the computer counting program was a reproducible tool to evaluate intact cell recovery after use of membrane integrity dyes on chondrocytes in situ. This and the significant decrease in the time used to count the cells by the computer program advocate its use in future studies because it has significant advantages

KamLAND Sensitivity to Neutrinos from Pre-Supernova Stars

In the late stages of nuclear burning for massive stars (M>8~M_{\sun}), the production of neutrino-antineutrino pairs through various processes becomes the dominant stellar cooling mechanism. As the star evolves, the energy of these neutrinos increases and in the days preceding the supernova a significant fraction of emitted electron anti-neutrinos exceeds the energy threshold for inverse beta decay on free hydrogen. This is the golden channel for liquid scintillator detectors because the coincidence signature allows for significant reductions in background signals. We find that the kiloton-scale liquid scintillator detector KamLAND can detect these pre-supernova neutrinos from a star with a mass of 25~M_{\sun} at a distance less than 690~pc with 3$\sigma$ significance before the supernova. This limit is dependent on the neutrino mass ordering and background levels. KamLAND takes data continuously and can provide a supernova alert to the community.Comment: 19 pages, 6 figures, 1 tabl

Oral carcinoma after hematopoietic stem cell transplantation – a new classification based on a literature review over 30 years

BACKGROUND: Patients undergoing hematopoietic stem cell transplantation (HSCT) have a higher risk of developing secondary solid tumors, in particular squamous cell carcinoma, because of several risk factors, including full-body irradiation (TBI), chemotherapy, and chronic graft versus host disease (GVHD). Based on the review presented here, a classification of oral changes is suggested in order to provide a tool to detect high-risk patients. Methods and Results The literature over the last 30 years was reviewed for development of malignoma of the oral cavity after HSCT. Overall, 64 cases were found. In 16 out of 30 cases, the tongue was the primary location, followed by the salivary gland (10 out of 30); 56.4% appeared in a latency time of 5 to 9 years after HSCT. In 76.6%, GVHD was noticed before the occurrence of oral malignancy. Premalignant changes of the oral mucosa were mucositis, xerostomia, and lichenoid changes, developing into erosive form. CONCLUSION: All physicians involved in the treatment of post-HSCT patients should be aware of the increased risk, even after 5 years from the development of oral malignancy, in particular when oral graft versus host changes are visible. In order to develop evidence management and to detect and offer adequate therapy as early as possible in this patient group, multicenter studies, involving oncologists and head and neck surgeons, should be established

Anaerobic Carbon Monoxide Dehydrogenase Diversity in the Homoacetogenic Hindgut Microbial Communities of Lower Termites and the Wood Roach

Anaerobic carbon monoxide dehydrogenase (CODH) is a key enzyme in the Wood-Ljungdahl (acetyl-CoA) pathway for acetogenesis performed by homoacetogenic bacteria. Acetate generated by gut bacteria via the acetyl-CoA pathway provides considerable nutrition to wood-feeding dictyopteran insects making CODH important to the obligate mutualism occurring between termites and their hindgut microbiota. To investigate CODH diversity in insect gut communities, we developed the first degenerate primers designed to amplify cooS genes, which encode the catalytic (β) subunit of anaerobic CODH enzyme complexes. These primers target over 68 million combinations of potential forward and reverse cooS primer-binding sequences. We used the primers to identify cooS genes in bacterial isolates from the hindgut of a phylogenetically lower termite and to sample cooS diversity present in a variety of insect hindgut microbial communities including those of three phylogenetically-lower termites, Zootermopsis nevadensis, Reticulitermes hesperus, and Incisitermes minor, a wood-feeding cockroach, Cryptocercus punctulatus, and an omnivorous cockroach, Periplaneta americana. In total, we sequenced and analyzed 151 different cooS genes. These genes encode proteins that group within one of three highly divergent CODH phylogenetic clades. Each insect gut community contained CODH variants from all three of these clades. The patterns of CODH diversity in these communities likely reflect differences in enzyme or physiological function, and suggest that a diversity of microbial species participate in homoacetogenesis in these communities

Increased susceptibility of Huh7 cells to HCV replication does not require mutations in RIG-I

<p>Abstract</p> <p>Background</p> <p>The cytosolic retinoic acid-inducible gene I (RIG-I) is a pattern recognition receptor that senses HCV double-stranded RNA and triggers type I interferon pathways. The clone Huh7.5 of human hepatoma Huh7 cells contains a mutation in RIG-I that is believed to be responsible for the improved replication of HCV in these cells relative to the parental strain. We hypothesized that, in addition to RIG-I, other determinant(s) outside the RIG-I coding sequence are involved in limiting HCV replication in cell culture. To test our hypothesis, we analyzed Huh7 cell clones that support the efficient replication of HCV and analyzed the RIG-I gene.</p> <p>Results</p> <p>One clone, termed Huh7D, was more permissive for HCV replication and more efficient for HCV-neomycin and HCV-hygromycin based replicon colony formation than parental Huh7 cells. Nucleotide sequence analysis of the RIG-I mRNA coding region from Huh7D cells showed no mutations relative to Huh7 parental cells.</p> <p>Conclusions</p> <p>We derived a new Huh7 cell line, Huh7D, which is more permissive for HCV replication than parental Huh7 cells. The higher permissiveness of Huh7D cells is not due to mutations in the RIG-I protein, indicating that cellular determinants other than the RIG-I amino-acid sequence are responsible for controlling HCV replication. In addition, we have selected Huh7 cells resistant to hygromycin via newly generated HCV-replicons carrying the hygromycin resistant gene. Further studies on Huh7D cells will allow the identification of cellular factors that increased the susceptibility to HCV infection, which could be targeted for anti-HCV therapies.</p

Image-guided versus blind corticosteroid injections in adults with shoulder pain: A systematic review

<p>Abstract</p> <p>Background</p> <p>Corticosteroid injections can be performed blind (landmark-guided) or with image guidance, and this may account for variable clinical outcomes. The objective of this study was to assess the effectiveness and safety of image-guided versus blind corticosteroid injections in improving pain and function among adults with shoulder pain.</p> <p>Methods</p> <p>MEDLINE, the Cochrane Controlled Trials Register and EMBASE were searched to May 2010. Additional studies were identified by searching bibliographies of shortlisted articles. Search items included blind, landmark, anatomical, clinical exam, image-guided, ultrasound, fluoroscopy, steroid injection, frozen shoulder, random allocation, randomized controlled trial (RCT) and clinical trial.</p> <p>Randomized controlled studies comparing image-guided versus blind (landmark-guided) corticosteroid shoulder injections that examined pain, function and/or adverse events were included. Independent extraction was done by two authors using a form with pre-specified data fields, including risk of bias appraisal. Conflicts were resolved by discussion. The decision to pool data was based on assessment of clinical design homogeneity. When warranted, studies were pooled under a random-effects model.</p> <p>Results</p> <p>Two RCTs for pain, function and adverse events (n = 101) met eligibility criteria. No serious threats to validity were found. Both trials compared ultrasound-guided versus landmark-guided injections and were judged similar in clinical design. Low to moderate heterogeneity was observed: shoulder pain I²= 60%, function I²= 22%. A meta-analysis demonstrated greater improvement with ultrasound-guided injections at 6 weeks after injection in both pain (mean difference = 2.23 [95% CI: 1.27, 3.18]), as assessed with a 0 to 10 visual analogue scale, and shoulder function (standardised mean difference = 1.09 [95% CI: 0.61, 1.57]) as assessed with shoulder function scores. Although more adverse events (all mild) were reported with landmark-guided injections, the difference was not statistically significant (risk ratio = 0.20 [95% CI: 0.04, 1.13]).</p> <p>This review was only based on two moderate-sized trials. Blinding of patients was not performed in both trials, causing some risk of bias in outcome assessment since primary endpoints were wholly or partially patient-reported.</p> <p>Conclusion</p> <p>There is a paucity of RCTs on image-guided versus landmark-guided corticosteroid shoulder injections examining pain, function and adverse events. In this review, patients who underwent image-guided (ultrasound) injections had statistically significant greater improvement in shoulder pain and function at 6 weeks after injection. Image-guided (ultrasound) corticosteroid injections potentially offer a significantly greater clinical improvement over blind (landmark-guided) injections in adults with shoulder pain. However, this apparent benefit requires confirmation from further studies (adequately-powered and well-executed RCTs).</p

Platinum Assisted Vapor–Liquid–Solid Growth of Er–Si Nanowires and Their Optical Properties

We report the optical activation of erbium coated silicon nanowires (Er–SiNWs) grown with the assist of platinum (Pt) and gold (Au), respectively. The NWs were grown on Si substrates by using a chemical vapor transport process using SiCl4 and ErCl4 as precursors. Pt as well as Au worked successfully as vapor–liquid–solid (VLS) catalysts for growing SiNWs with diameters of ~100 nm and length of several micrometers, respectively. The SiNWs have core–shell structures where the Er-crystalline layer is sandwiched between silica layers. Photoluminescence spectra analyses showed the optical activity of SiNWs from both Pt and Au. A stronger Er3+ luminescence of 1,534 nm was observed from the SiNWs with Pt at room- and low-temperature (25 K) using the 488- and/or 477-nm line of an Ar laser that may be due to the uniform incorporation of more Er ions into NWs with the exclusion of the formation of catalyst-induced deep levels in the band-gap. Pt would be used as a VLS catalyst for high performance optically active Er–SiNWs