In vitro nuclear interactome of the HIV-1 Tat protein

<p>Abstract</p> <p>Background</p> <p>One facet of the complexity underlying the biology of HIV-1 resides not only in its limited number of viral proteins, but in the extensive repertoire of cellular proteins they interact with and their higher-order assembly. HIV-1 encodes the regulatory protein Tat (86–101aa), which is essential for HIV-1 replication and primarily orchestrates HIV-1 provirus transcriptional regulation. Previous studies have demonstrated that Tat function is highly dependent on specific interactions with a range of cellular proteins. However they can only partially account for the intricate molecular mechanisms underlying the dynamics of proviral gene expression. To obtain a comprehensive nuclear interaction map of Tat in T-cells, we have designed a proteomic strategy based on affinity chromatography coupled with mass spectrometry.</p> <p>Results</p> <p>Our approach resulted in the identification of a total of 183 candidates as Tat nuclear partners, 90% of which have not been previously characterised. Subsequently we applied <it>in silico </it>analysis, to validate and characterise our dataset which revealed that the Tat nuclear interactome exhibits unique signature(s). First, motif composition analysis highlighted that our dataset is enriched for domains mediating protein, RNA and DNA interactions, and helicase and ATPase activities. Secondly, functional classification and network reconstruction clearly depicted Tat as a polyvalent protein adaptor and positioned Tat at the nexus of a densely interconnected interaction network involved in a range of biological processes which included gene expression regulation, RNA biogenesis, chromatin structure, chromosome organisation, DNA replication and nuclear architecture.</p> <p>Conclusion</p> <p>We have completed the <it>in vitro </it>Tat nuclear interactome and have highlighted its modular network properties and particularly those involved in the coordination of gene expression by Tat. Ultimately, the highly specialised set of molecular interactions identified will provide a framework to further advance our understanding of the mechanisms of HIV-1 proviral gene silencing and activation.</p

Theorising links between context and structure to introduce powerful statistical ideas in the early years

Recent literature in the early years has emphasised the benefits of introducing children to powerful disciplinary ideas. Powerful ideas in statistics such as variability, aggregate, population, the need for data, data representation and statistical inquiry are generally introduced in the later years of schooling or university and therefore may be considered too difficult for young children. However, at an informal level, these ideas arise in contexts that are accessible to young children. The aim of this chapter is to theorise important relations between children’s contextual experiences and key structures in statistics. It introduces the notion of statistical context–structures, which characterise aspects of contexts that can expose children to important statistical ideas. A classroom case study involving statistical inquiry by children in their first year of schooling (ages 4–5) is included to illustrate characteristics of age-appropriate links between contexts and structures in statistics. Over time, engaging children in significant activities that rely on statistical context–structures can provide children with multiple opportunities to experience statistics as a coherent and purposeful discipline and develop rich networks of informal statistical concepts well before ideas are formalised. For teachers and curriculum writers, statistical context–structures provide a framework to design statistical inquiries that directly address learning intentions and curricular goals

Clinical utility of chitotriosidase enzyme in nephropathic cystinosis

BackgroundNephropathic cystinosis is an inherited autosomal recessive lysosomal storage disorder characterized by the pathological accumulation and crystallization of cystine inside different cell types. WBC cystine determination forms the basis for the diagnosis and therapeutic monitoring with the cystine depleting drug (cysteamine). The chitotriosidase enzyme is a human chitinase, produced by activated macrophages. Its elevation is documented in several lysosomal storage disorders. Although, about 6% of Caucasians have enzyme deficiency due to homozygosity of 24-bp duplication mutation in the chitotriosidase gene, it is currently established as a screening marker and therapeutic monitor for Gaucher¿s disease.MethodsPlasma chitotriosidase activity was measured in 45 cystinotic patients, and compared with 87 healthy controls and 54 renal disease patients with different degrees of renal failure (CKD1-5). Chitotriosidase levels were also correlated with WBC cystine in 32 treated patients. Furthermore, we incubated control human macrophages in-vitro with different concentrations of cystine crystals and monitored the response of tumor necrosis factor-alpha (TNF-¿) and chitotriosidase activity. We also compared plasma chitotriosidase activity in cystinotic knocked-out (n¿=¿10) versus wild-type mice (n¿=¿10).ResultsPlasma chitotriosidase activity in cystinotic patients (0¿3880, median 163 nmol/ml/h) was significantly elevated compared to healthy controls (0¿90, median 18 nmol/ml/h) and to CKD patients (0¿321, median 52 nmol/ml/h), P¿<¿0.001 for both groups. Controls with decreased renal function had mild to moderate chitotriosidase elevations; however, their levels were significantly lower than in cystinotic patients with comparable degree of renal insufficiency. Chitotriosidase activity positively correlated with WBC cystine content for patients on cysteamine therapy (r¿=¿0.8), P¿<¿0.001. In culture, human control macrophages engulfed cystine crystals and released TNF-¿ into culture supernatant in a crystal concentration dependent manner. Chitotriosidase activity was also significantly increased in macrophage supernatant and cell-lysate. Furthermore, chitotriosidase activity was significantly higher in cystinotic knocked-out than in the wild-type mice, P¿=¿0.003.ConclusionsThis study indicates that cystine crystals are potent activators of human macrophages and that chitotriosidase activity is a useful marker for this activation and a promising clinical biomarker and therapeutic monitor for nephropathic cystinosis.status: publishe