Solution Structure of the HIV-1 Intron Splicing Silencer and Its Interactions with the UP1 Domain of Heterogeneous Nuclear Ribonucleoprotein (hnRNP) A1

Splicing patterns in human immunodeficiency virus type 1 (HIV-1) are maintained through cis regulatory elements that recruit antagonistic host RNA-binding proteins. The activity of the 3′ acceptor site A7 is tightly regulated through a complex network of an intronic splicing silencer (ISS), a bipartite exonic splicing silencer (ESS3a/b), and an exonic splicing enhancer (ESE3). Because HIV-1 splicing depends on protein-RNA interactions, it is important to know the tertiary structures surrounding the splice sites. Herein, we present the NMR solution structure of the phylogenetically conserved ISS stem loop. ISS adopts a stable structure consisting of conserved UG wobble pairs, a folded 2X2 (GU/UA) internal loop, a UU bulge, and a flexible AGUGA apical loop. Calorimetric and biochemical titrations indicate that the UP1 domain of heterogeneous nuclear ribonucleoprotein A1 binds the ISS apical loop site-specifically and with nanomolar affinity. Collectively, this work provides additional insights into how HIV-1 uses a conserved RNA structure to commandeer a host RNA-binding protein.     

Small Intestine Inflammation in Roquin-Mutant and Roquin-Deficient Mice

Roquin, an E3 ubiquitin ligase that localizes to cytosolic RNA granules, is involved in regulating mRNA stability and translation. Mice that have a M199R mutation in the Roquin protein (referred to as sanroque or Roquin^san/san mice) develop autoimmune pathologies, although the extent to which these occur in the intestinal mucosa has not been determined. Here, we demonstrate that Roquin^san/san mice reproducibly develop intestinal inflammation in the small intestine but not the colon. Similarly, mice generated in our laboratory in which the Roquin gene was disrupted by insertion of a gene trap cassette (Roquin^gt/gt mice) had small intestinal inflammation that mimicked that of Roquin^san/san mice. MLN cells in Roquin^san/san mice consisted of activated proliferating T cells, and had increased numbers of CD44^hi CD62L^lo KLRG1⁺ short-lived effector cells. Proportionally more small intestinal intraepithelial lymphocytes in Roquin^san/san mice expressed the ICOS T cell activation marker. Of particular interest, small intestinal lamina propria lymphocytes in Roquin^san/san mice consisted of a high proportion of Gr-1⁺ T cells that included IL-17A⁺ cells and CD8⁺ IFN-γ⁺ cells. Extensive cytokine dysregulation resulting in both over-expression and under-expression of chemotactic cytokines occurred in the ileum of Roquin^san/san mice, the region most prone to the development of inflammation. These findings demonstrate that chronic inflammation ensues in the intestine following Roquin alteration either as a consequence of protein mutation or gene disruption, and they have implications for understanding how small intestinal inflammation is perpetuated in Crohn''s disease (CD). Due to the paucity of animal models of CD-like pathophysiology in the small intestine, and because the primary gene/protein defects of the Roquin animal systems used here are well-defined, it will be possible to further elucidate the underlying genetic and molecular mechanisms that drive the disease process.     

Adenovirus type 5 intrinsic adsorption rates measured by surface plasmon resonance

Intrinsic adsorption rates of whole adenovirus type 5 (Ad5) onto a diethylaminoethyl (DEAE) anion exchange surface are measured for the first time by surface plasmon resonance (SPR). Fitting SPR sensorgrams to a two-compartment mass transport reaction model distinguishes intrinsic adsorption rates from slow diffusive Ad5 mass transport. Ad5 is a widely used viral vector for gene therapy that binds electrostatically to surfaces of cells and synthetics such as membranes, chromatographic resins, and glass. Increasing NaCl concentration from 4.8 to 14.4mM shifts binding of whole Ad5 from diffusion control to a regime where both sorption and diffusion affect binding. Intrinsic adsorption rates for Ad5-DEAE interaction are 16 times faster than intrinsic adsorption rates for Ad5 fiber knob interacting with soluble extracellular domain of coxsackievirus adenovirus receptors (s-CAR).     

Impact of restricted marital practices on genetic variation in an endogamous Gujarati group

Recent studies have examined the influence on patterns of human genetic variation of a variety of cultural practices. In India, centuries‐old marriage customs have introduced extensive social structuring into the contemporary population, potentially with significant consequences for genetic variation. Social stratification in India is evident as social classes that are defined by endogamous groups known as castes. Within a caste, there exist endogamous groups known as gols (marriage circles), each of which comprises a small number of exogamous gotra (lineages). Thus, while consanguinity is strictly avoided and some randomness in mate selection occurs within the gol, gene flow is limited with groups outside the gol. Gujarati Patels practice this form of “exogamic endogamy.” We have analyzed genetic variation in one such group of Gujarati Patels, the Chha Gaam Patels (CGP), who comprise individuals from six villages. Population structure analysis of 1,200 autosomal loci offers support for the existence of distinctive multilocus genotypes in the CGP with respect to both non‐Gujaratis and other Gujaratis, and indicates that CGP individuals are genetically very similar. Analysis of Y‐chromosomal and mitochondrial haplotypes provides support for both patrilocal and patrilineal practices within the gol, and a low‐level of female gene flow into the gol. Our study illustrates how the practice of gol endogamy has introduced fine‐scale genetic structure into the population of India, and contributes more generally to an understanding of the way in which marriage practices affect patterns of genetic variation. Am J PhyAnthropol 2012. © Wiley Periodicals, Inc.     

Characterization of M. tuberculosis SerB2, an Essential HAD-Family Phosphatase,Reveals Novel Properties

M. tuberculosis harbors an essential phosphoserine phosphatase (MtSerB2, Rv3042c) that contains two small- molecule binding ACT-domains (Pfam 01842) at the N-terminus followed by the phosphoserine phosphatase (PSP) domain. We found that exogenously added MtSerB2 elicits microtubule rearrangements in THP-1 cells. Mutational analysis demonstrates that phosphatase activity is co-related to the elicited rearrangements, while addition of the ACT-domains alone elicits no rearrangements. The enzyme is dimeric, exhibits divalent metal- ion dependency, and is more specific for l- phosphoserine unlike other classical PSPases. Binding of a variety of amino acids to the ACT-domains influences MtSerB2 activity by either acting as activators/inhibitors/have no effects. Additionally, reduced activity of the PSP domain can be enhanced by equimolar addition of the ACT domains. Further, we identified that G18 and G108 of the respective ACT-domains are necessary for ligand-binding and their mutations to G18A and G108A abolish the binding of ligands like l- serine. A specific transition to higher order oligomers is observed upon the addition of l- serine at ∼0.8 molar ratio as supported by Isothermal calorimetry and Size exclusion chromatography experiments. Mutational analysis shows that the transition is dependent on binding of l- serine to the ACT-domains. Furthermore, the higher-order oligomeric form of MtSerB2 is inactive, suggesting that its formation is a mechanism for feedback control of enzyme activity. Inhibition studies involving over eight inhibitors, MtSerB2, and the PSP domain respectively, suggests that targeting the ACT-domains can be an effective strategy for the development of inhibitors.     

B cell responses to a peptide epitope. VIII. Immune complex-mediated regulation of memory B cell generation within germinal centers.

Using an in vivo reconstitution assay, we examine here the role of immune complexes in both formation of germinal centers (GC) and processes that occur subsequently within. The presence of Ag, as immune complexes, was found not to constitute a limiting requirement for the initiation of GC formation. No detrimental effect either on numbers or sizes of the resulting GC was observed when Ag-containing immune complexes were omitted during reconstitution. Thus, both recruitment and proliferation of Ag-activated B cells within GC appear not to be limited by Ag concentrations. In contrast, the presence of immune complexes was observed to be obligatory for the generation of Ag-specific memory B cells. This optimally required immune complexes to be constituted by IgG-class Abs with epitope specificities that were homologous to those of the GC B cells. The GC reaction was also found to be characterized by an enhancement of Ab specificity for the homologous epitope. Although some improvement in specificity was noted in recall responses from immune complex-deficient GC, the presence of appropriate immune complexes served to further optimize the outcome. Here again, isotype and epitope-specificity of the Ab constituent in immune complexes proved to be important.     

Impacts of anthropogenic disturbances on forest succession in the mid-montane forests of Central Himalaya

We studied the influence of anthropogenic drivers on the distribution and regeneration of tree species in vegetation at different stages of succession from grasslands to oak forests in mid-montane Central Himalaya. We found fire, grazing, and lopping as the main factors hindering a progressive successional regime towards a late-successional oak community. Succession was studied in five vegetation formations (grasslands, pine, pine–oak, open oak, and dense oak), with similar site conditions, representing a theoretical successional sequence from early- to late-successional stages. A structured survey with uniform distribution of sampling plots in the five selected vegetation formations was conducted to gather information abut the vegetation communities. Early-successional grasslands and pine forests were found to harbour high densities of pine and oak seedling and sapling regeneration. However, recurring fires and chronic unsustainable levels of grazing in these vegetation formations obstructed progressive succession by eliminating regenerating seedling and saplings from the forest understorey. Similarly, in intermediate- and late-successional stages (including pine–oak, open oak, and dense oak), overexploitation of existing oaks trees via lopping and grazing of regenerating oak seedlings and saplings hampered oak regeneration and development. The possibility to convert pine forests into oak as well as the conservation of existing oak forests through controlled grazing and lopping are management options that can contribute to an enhanced functioning of forest ecosystems in the study area. We conclude that with strategic management that restricts the current anthropogenic disturbances, the extent of oak forest in the study area can be increased.