Hierarchical clustering of flow cytometry data for the study of conventional central chondrosarcoma

We have investigated the use of hierarchical clustering of flow cytometry data to classify samples of conventional central chondrosarcoma, a malignant cartilage forming tumor of uncertain cellular origin, according to similarities with surface marker profiles of several known cell types. Human primary chondrosarcoma cells, articular chondrocytes, mesenchymal stem cells, fibroblasts, and a panel of tumor cell lines from chondrocytic or epithelial origin were clustered based on the expression profile of eleven surface markers. For clustering, eight hierarchical clustering algorithms, three distance metrics, as well as several approaches for data preprocessing, including multivariate outlier detection, logarithmic transformation, and z-score normalization, were systematically evaluated. By selecting clustering approaches shown to give reproducible results for cluster recovery of known cell types, primary conventional central chondrosacoma cells could be grouped in two main clusters with distinctive marker expression signatures: one group clustering together with mesenchymal stem cells (CD49b-high/CD10-low/CD221-high) and a second group clustering close to fibroblasts (CD49b-low/CD10-high/CD221-low). Hierarchical clustering also revealed substantial differences between primary conventional central chondrosarcoma cells and established chondrosarcoma cell lines, with the latter not only segregating apart from primary tumor cells and normal tissue cells, but clustering together with cell lines from epithelial lineage. Our study provides a foundation for the use of hierarchical clustering applied to flow cytometry data as a powerful tool to classify samples according to marker expression patterns, which could lead to uncover new cancer subtypes.

Mortality associated with discordant responses to antiretroviral therapy in resource-constrained settings

Objectives: We assessed mortality associated with immunologic and virologic patterns of response at 6 months of highly active antiretroviral therapy (HAART) in HIV-infected individuals from resource-limited countries in Africa and South America. Methods: Patients who initiated HAART between 1996 and 2007, aged 16 years or older, and had at least 1 measurement (HIV-1 RNA plasma viral load or CD4 cell count) at 6 months of therapy (3-9 month window) were included. Therapy response was categorized as complete, discordant (virologic only or immunologic only), and absent. Associations between 6-month response to therapy and all-cause mortality were assessed by Cox proportional hazards regression. Robust standard errors were calculated to account for intrasite correlation. Results: A total of 7160 patients, corresponding to 15,107 person-years, were analyzed. In multivariable analysis adjusted for age at HAART initiation, baseline clinical stage and CD4 cell count, year of HAART initiation, clinic, occurrence of an AIDS-defining condition within the first 6 months of treatment, and discordant and absent responses were associated with increased risk of death. Conclusions: Similar to reports from high-income countries, discordant immunologic and virologic responses were associated with intermediate risk of death compared with complete and no response in this large cohort of HIV-1 patients from resource-limited countries. Our results support a recommendation for wider availability of plasma viral load testing to monitor antiretroviral therapy in these settings.

Premature Osteoblast Clustering by Enamel Matrix Proteins Induces Osteoblast Differentiation through Up-Regulation of Connexin 43 and N-Cadherin

In recent years, enamel matrix derivative (EMD) has garnered much interest in the dental field for its apparent bioactivity that stimulates regeneration of periodontal tissues including periodontal ligament, cementum and alveolar bone. Despite its widespread use, the underlying cellular mechanisms remain unclear and an understanding of its biological interactions could identify new strategies for tissue engineering. Previous in vitro research has demonstrated that EMD promotes premature osteoblast clustering at early time points. The aim of the present study was to evaluate the influence of cell clustering on vital osteoblast cell-cell communication and adhesion molecules, connexin 43 (cx43) and N-cadherin (N-cad) as assessed by immunofluorescence imaging, real-time PCR and Western blot analysis. In addition, differentiation markers of osteoblasts were quantified using alkaline phosphatase, osteocalcin and von Kossa staining. EMD significantly increased the expression of connexin 43 and N-cadherin at early time points ranging from 2 to 5 days. Protein expression was localized to cell membranes when compared to control groups. Alkaline phosphatase activity was also significantly increased on EMD-coated samples at 3, 5 and 7 days post seeding. Interestingly, higher activity was localized to cell cluster regions. There was a 3 fold increase in osteocalcin and bone sialoprotein mRNA levels for osteoblasts cultured on EMD-coated culture dishes. Moreover, EMD significantly increased extracellular mineral deposition in cell clusters as assessed through von Kossa staining at 5, 7, 10 and 14 days post seeding. We conclude that EMD up-regulates the expression of vital osteoblast cell-cell communication and adhesion molecules, which enhances the differentiation and mineralization activity of osteoblasts. These findings provide further support for the clinical evidence that EMD increases the speed and quality of new bone formation in vivo.

Constrained pattern of viral evolution in acute and early HCV infection limits viral plasticity

Cellular immune responses during acute Hepatitis C virus (HCV) and HIV infection are a known correlate of infection outcome. Viral adaptation to these responses via mutation(s) within CD8+ T-cell epitopes allows these viruses to subvert host immune control. This study examined HCV evolution in 21 HCV genotype 1-infected subjects to characterise the level of viral adaptation during acute and early HCV infection. Of the total mutations observed 25% were within described CD8+ T-cell epitopes or at viral adaptation sites. Most mutations were maintained into the chronic phase of HCV infection (75%). The lack of reversion of adaptations and high proportion of silent substitutions suggests that HCV has structural and functional limitations that constrain evolution. These results were compared to the pattern of viral evolution observed in 98 subjects during a similar phase in HIV infection from a previous study. In contrast to HCV, evolution during acute HIV infection is marked by high levels of amino acid change relative to silent substitutions, including a higher proportion of adaptations, likely reflecting strong and continued CD8+ T-cell pressure combined with greater plasticity of the virus. Understanding viral escape dynamics for these two viruses is important for effective T cell vaccine design.

Does published orthodontic research account for clustering effects during statistical data analysis?

In orthodontics, multiple site observations within patients or multiple observations collected at consecutive time points are often encountered. Clustered designs require larger sample sizes compared to individual randomized trials and special statistical analyses that account for the fact that observations within clusters are correlated. It is the purpose of this study to assess to what degree clustering effects are considered during design and data analysis in the three major orthodontic journals. The contents of the most recent 24 issues of the American Journal of Orthodontics and Dentofacial Orthopedics (AJODO), Angle Orthodontist (AO), and European Journal of Orthodontics (EJO) from December 2010 backwards were hand searched. Articles with clustering effects and whether the authors accounted for clustering effects were identified. Additionally, information was collected on: involvement of a statistician, single or multicenter study, number of authors in the publication, geographical area, and statistical significance. From the 1584 articles, after exclusions, 1062 were assessed for clustering effects from which 250 (23.5 per cent) were considered to have clustering effects in the design (kappa = 0.92, 95 per cent CI: 0.67-0.99 for inter rater agreement). From the studies with clustering effects only, 63 (25.20 per cent) had indicated accounting for clustering effects. There was evidence that the studies published in the AO have higher odds of accounting for clustering effects [AO versus AJODO: odds ratio (OR) = 2.17, 95 per cent confidence interval (CI): 1.06-4.43, P = 0.03; EJO versus AJODO: OR = 1.90, 95 per cent CI: 0.84-4.24, non-significant; and EJO versus AO: OR = 1.15, 95 per cent CI: 0.57-2.33, non-significant). The results of this study indicate that only about a quarter of the studies with clustering effects account for this in statistical data analysis.

Clustering of health behaviours in adult survivors of childhood cancer and the general population

Little is known about engagement in multiple health behaviours in childhood cancer survivors.

Enumerating constrained elementary flux vectors of metabolic networks

The concept of elementary vector is generalised to the case where the steady-state space of the metabolic network is not a flux cone but is a general polyhedron due to further inhomogeneous constraints on the flows through some of the reactions. On one hand, this allows to selectively enumerate elementary modes which satisfy certain optimality criteria and this can yield a large computational gain compared with full enumeration. On the other hand, in contrast to the single optimum found by executing a linear program, this enables a comprehensive description of the set of alternate optima often encountered in flux balance analysis. The concepts are illustrated on a metabolic network model of human cardiac mitochondria.

Discordant responses to potent antiretroviral treatment in previously naive HIV-1-infected adults initiating treatment in resource-constrained countries: the antiretroviral therapy in low-income countries (ART-LINC) collaboration

OBJECTIVES: To assess the frequency of and risk factors for discordant responses at 6 months on highly active antiretroviral therapy (HAART) in previously treatment-naive HIV patients from resource-limited countries. METHODS: The Antiretroviral Therapy in Low-Income Countries Collaboration is a network of clinics providing care and treatment to HIV-infected patients in Africa, Latin America, and Asia. Patients who initiated therapy between 1996 and 2004, were aged 16 years or older, and had a baseline CD4 cell count were included in this analysis. Responses were defined based on plasma viral load (PVL) and CD4 cell count at 6 months as complete virologic and immunologic (VR(+)IR(+)), virologic only (VR(+)IR(-)), immunologic only (VR(-)IR(+)), and nonresponse (VR(-)IR(-)). Multinomial logistic regression was used to assess the association between therapy responses and clinical and demographic variables. RESULTS: Of the 3111 patients eligible for analysis, 1914 had available information at 6 months of therapy: 1074 (56.1%) were VR(+)IR(+), 364 (19.0%) were VR(+)IR(-), 283 (14.8%) were (VR(-)IR(+)), and 193 (10.1%) were VR(-)IR(-). OF THE 3111 patients eligible for analysis, 1914 had available information at 6 months of therapy: 1074 (56.1%) were VRIR, 364 (19.0%) were VRIR, 283 (14.8%) were (VRIR), and 193 (10.1%) were VRIR. Compared with complete responders, virologic-only responders were older, had a higher baseline CD4 cell count, had a lower baseline PVL, and were more likely to have received a nonstandard HAART regimen; immunologic-only responders were younger, had a lower baseline CD4 cell count, had a higher baseline PVL, and were more likely to have received a protease inhibitor-based regimen. CONCLUSIONS: The frequency of and risk factors for discordant responses were comparable to those observed in developed countries. Longer follow-up is needed to assess the long-term impact of discordant responses on mortality in these resource-limited settings.