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The central importance of osteocytes in regulating bone homeostasis is becoming increasingly apparent. However, the study of these cells has been restricted by the relative paucity of cell line models, especially those of human origin. Therefore, we investigated the extent to which SaOS2 human osteosarcoma cells can differentiate into osteocyte-like cells. During culture under the appropriate mineralising conditions, SaOS2 cells reproducibly synthesised a bone-like mineralised matrix and temporally expressed the mature osteocyte marker genes SOST , DMP1 , PHEX and MEPE and down-regulated expression of RUNX2 and COL1A1 . SaOS2 cells cultured in 3D collagen gels acquired a dendritic morphology, characteristic of osteocytes, with multiple interconnecting cell processes. These findings suggest that SaOS2 cells have the capacity to differentiate into mature osteocyte-like cells under mineralising conditions. PTH treatment of SaOS2 cells resulted in strong down-regulation of SOST mRNA expression at all time points tested. Interestingly, PTH treatment resulted in the up-regulation of RANKL mRNA expression only at earlier stages of differentiation. These findings suggest that the response to PTH is dependent on the differentiation stage of the osteoblast/osteocyte. Together, our results demonstrate that SaOS2 cells can be used as a human model to investigate responses to osteotropic stimuli throughout differentiation to a mature osteocyte-like stage.

We sequenced four mitochondrial subgenomes from the potato cyst nematode Globodera pallida, previously characterized as one of the few animals to have a multipartite mitochondrial genome. The sequence data indicate that three of these subgenomic mitochondrial circles are mosaics, comprising long, multigenic fragments derived from fragments of the other circles. This pattern is consistent with the operation of intermitochondrial recombination, a process generally considered absent in animal mitochondria. We also report that many of the duplicated genes contain deleterious mutations, ones likely to render the gene nonfunctional; gene conversion does not appear to be homogenizing the different gene copies. The proposed nonfunctional copies are clustered on particular circles, whereas copies that are likely to code functional gene products are clustered on others.

Objective Positive human epidermal growth factor 2 (HER2) expression and its predictive clinicopathological features remain unclear in Sri Lankan gastric cancer (GC) patients. Here, we aimed to determine GC HER2 status predictors by analyzing associations between clinicopathological features and HER2 expression using immunohistochemistry (IHC) and silver in situ hybridization (SISH). Methods During this 4-year prospective study, clinicopathological data were collected from participants in the National Hospital of Sri Lanka. HER2 IHC and SISH were performed using commercial reagents. Using chi-square tests, associations of HER2-IHC/SISH with clinicopathological features were analyzed. Results Overall, 145 GC patients were included, 69 had gastrectomies and 76 had biopsies. Positive HER2 expression by IHC was associated with age <60 years, high T stage (assessed pathologically in resections and radiologically in biopsies), high nuclear grade, tumor necrosis, mitosis >5/high-power field, with additional perineural invasion and lymphovascular invasion in resections. These features, excluding lymphovascular invasion but including male sex, were associated with HER2 expression by SISH. Conclusions Age <60 years, high nuclear grade, tumor necrosis, and perineural invasion are associated factors of HER2 status. These could be used to triage GC patients for HER2 status testing in limited resource settings where IHC/SISH analysis is costly.

IntroductionGastric adenocarcinoma(GC) patient selection for antiHER2 therapy is dependent on accurate HER2 status. It is assessed immunohistochemically(IHC) for protein expression and by silver in-situ hybridization(SISH) for gene copy number. This study aimed to evaluate the concordance of HER2 status by IHC/SISH analyses and HER2-SISH based survival.MethodsThis prospective study includes 145 GC’s(excluding gastro-oesophageal-junction tumours) from the National Hospital of Sri Lanka. HER2-IHC was assessed by DAKO A0485, RealTM Envision system and interpreted using Ruschoff criteria. HER2-SISH was assessed with INFORM HER2 dual ISH DNA Probe Cocktail. Concordance between HER2 IHC/SISH results was determined by Cohens kappa statistics. SISH based survival of GC patients who did not receive antiHER2 therapy, was analyzed by Kaplan-Meier method and log-rank test.ResultsOf the 69 gastrectomies and 76 biopsies, 8.3% (n=12) were HER2-IHC positive (n=7, +2 and n=5, +3). HER2-SISH positivity was 4.8%( n=7). All IHC+3 were SISH positive, while two, +2 were SISH positive. Concordance for IHC 0, +1, +3 were 100%. There was a significant overall correlation (kappa=0.72, p<0.001) between HER2-IHC and HER2-SISH indicating substantial concordance. The mean overall survival of HER2-SISH negative and positive patients were 41.7(0–210) and 14.6(3–51) weeks respectively. The mean duration of follow up was 40.4 weeks (range 0–210). Survival was significantly poor(p=0.018) with HER2-SISH positivity.ConclusionsHER2-IHC was well concordant with HER2-SISH for 0, +1, +3 scores and could be used for treatment and prognostication in low resource settings. HER2-IHC+2 without gene amplification may be due to transcriptional activation by other genes or post-transcriptional events, mandating further evaluation by SISH. Survival of GC patients is significantly affected by HER2-SISH positive status.

Background HER2 protein expression indicates adverse prognosis in gastric adenocarcinoma (GCa). GCa HER2 positivity ranges from 10 to 22.8%. Similar data are scarce in South Asia and unavailable in Sri Lanka. Aim To evaluate HER2 protein expression, its clinicopathological relationship and survival in a Sri Lankan GCa cohort. Methods One hundred consecutive GCa patients were recruited prospectively for 2 years. Histological diagnosis was confirmed on endoscopic biopsies/gastrectomy specimens. Clinicopathological and overall survival data were collected. HER2 expression was assessed using immunohistochemistry. 2+ and 3+ scores were considered positive. HER2 expression and clinicopathological parameters were analyzed by Chi-squared test and multivariate analysis with logistic regression using SPSS-21. Kaplan–Meier method and log-rank test were used for survival analysis. Results Study includes 56 biopsies and 44 resections. Male/female ratio was 1.9:1. Mean age of diagnosis was 61.1 years (range 32–82). Majority tumors were proximally located (58%). HER2 positivity was 9%. Even though intestinal subtype predominated HER2 positivity was mostly among diffuse variant (14.8%). In multivariate analysis, mitotic count >5/hpf, high nuclear grade and tumor necrosis were significantly associated with HER2 positivity, while poor differentiation, signet cells, extracellular mucin, perineural invasion and pathological nodal metastasis (all p  < 0.05) showed a correlation in univariate analysis. Mean follow-up duration was 37.4 weeks (range 0–104). HER2 positivity was associated with a significantly lower median overall survival ( p  = 0.046). Conclusion GCa HER2 positivity was 9%, associated with a lower median overall survival. Adverse histological features had a positive correlation with HER2 positivity. These histological features could direct patients for confirmatory HER2 testing in limited resource settings.