22q11.2 distal deletion: a recurrent genomic disorder distinct from DiGeorge syndrome and velocardiofacial syndrome

Microdeletions within chromosome 22q11.2 cause a variable phenotype, including DiGeorge syndrome (DGS) and velocardiofacial syndrome (VCFS). About 97% of patients with DGS/VCFS have either a common recurrent ~3 Mb deletion or a smaller, less common, ~1.5 Mb nested deletion. Both deletions apparently occur as a result of homologous recombination between nonallelic flanking low-copy repeat (LCR) sequences located in 22q11.2. Interestingly, although eight different LCRs are located in proximal 22q, only a few cases of atypical deletions utilizing alternative LCRs have been described. Using array-based comparative genomic hybridization (CGH) analysis, we have detected six unrelated cases of deletions that are within 22q11.2 and are located distal to the ~3 Mb common deletion region. Further analyses revealed that the rearrangements had clustered breakpoints and either a ~1.4 Mb or ~2.1 Mb recurrent deletion flanked proximally by LCR22-4 and distally by either LCR22-5 or LCR22-6, respectively. Parental fluorescence in situ hybridization (FISH) analyses revealed that none of the available parents (11 out of 12 were available) had the deletion, indicating de novo events. All patients presented with characteristic facial dysmorphic features. A history of prematurity, prenatal and postnatal growth delay, developmental delay, and mild skeletal abnormalities was prevalent among the patients. Two patients were found to have a cardiovascular malformation, one had truncus arteriosus, and another had a bicuspid aortic valve. A single patient had a cleft palate. We conclude that distal deletions of chromosome 22q11.2 between LCR22-4 and LCR22-6, although they share some characteristic features with DGS/VCFS, represent a novel genomic disorder distinct genomically and clinically from the well-known DGS/VCF deletion syndromes.     

NK cells lyse T regulatory cells that expand in response to an intracellular pathogen

We evaluated the capacity of NK cells to influence expansion of CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs) in response to microbial Ags, using Mycobacterium tuberculosis as a model. We previously found that Tregs expand when CD4(+) cells and monocytes are exposed to M. tuberculosis. Addition of NK cells that were activated by monokines (IL-12, IL-15, and IL-18) or by exposure to M. tuberculosis-stimulated monocytes reduced Treg expansion in response to M. tuberculosis. NK cell inhibition of Treg expansion was not mediated through IFN-gamma. Activated NK cells lysed expanded, but not freshly isolated Tregs. Although monokines increased NK cell expression of the activating receptors NKp46, NKG2D, 2B4, CD16, and DNAM-1, only anti-NKG2D and anti-NKp46 inhibited NK cell lysis of expanded Tregs. Of five NKG2D ligands, only UL16-binding protein 1 (ULBP1) was up-regulated on M. tuberculosis-expanded Tregs, and anti-ULBP1 inhibited NK cell lysis of expanded Tregs. M. tuberculosis-stimulated monocytes activated NK cells to lyse expanded Tregs, and this was also inhibited by anti-NKG2D and anti-ULBP1, confirming the physiological relevance of this effect. Our study identifies a potential new role for NK cells in maintaining the delicate balance between the regulatory and effector arms of the immune response.     

Comparative studies of unfolding and binding of ligands to human serum albumin in the presence of fatty acid: spectroscopic approach

This study was undertaken to investigate the influence of fatty acid binding on the unfolding of HSA and how the fatty acid molecules can influence and/or compete with other ligand molecules bound to the protein. The equilibrium unfolding of fatted and fatty acid free HSA was measured by overlapping of unfolding transition curves monitored by different probes for secondary and tertiary structure and determining changes in free energy of unfolding. Proteins stability was studied by fluorescence spectroscopy, whereas conformational changes were detected by circular dichroism techniques. We have suggested a "molten globule" like intermediate state of HSA at a fairly high concentration of GnHCl (3.2 for fatty acid free and 3.6 for fatted). The free energy of stabilization (DeltaG(D)(H2O)) in the presence of fatty acid was found to be 900 cal mol(-1). We also analyze the effects of fatty acid on binding of ligands using spectroscopic technique and reported the equilibrium constants and free energies obtained from the binding and unfolding experiments.     

Effect of single-residue bulges on RNA double-helical structures: crystallographic database analysis and molecular dynamics simulation studies

Asymmetric bulge loop motifs are widely dispersed in all types of functional RNAs. They are frequently occurring structural motifs in folded RNA structures and appear commonly in pre-microRNA and ribosomes, where they are involved in specific RNA–RNA and RNA–protein interactions. It is therefore necessary to understand such motifs from a structural point of view. We analyzed all available RNA structures and identified quite a few fragments of double helices that contain bulges. We found that these discontinuities often introduce kinks into the double helices, which also affects the stacking overlap between the base pairs across the irregularity. In order to understand the influence of these bulges on stability and flexibility, we carried out molecular dynamics simulations of three different single-residue bulge-containing RNA helices using the CHARMM36 force field. The structural variability at the junctions of RNA bulges is expected to differ from that in continuous double-helical stretches. The structural features of the junction region were observed to vary noticeably depending on the orientation of the bulge residue. When the base of the bulge residue is looped out, the RNA stretch behaves like a standard long A-form RNA double helix, whereas the entire RNA behaves differently when the base of the bulge residue is intercalated between base pairs inside the RNA stem. Such single-base intercalation was found to introduce a permanent kink into the composite double helix, which could be a recognition element for Dicer during the maturation of miRNA.     

Differentiation Induces Dramatic Changes in miRNA Profile,Where Loss of Dicer Diverts Differentiating SH-SY5Y Cells Toward Senescence

MicroRNAs (miRNAs) are generated by endonuclease activity of Dicer, which also helps in loading of miRNAs to their target sequences. SH-SY5Y, a human neuroblastoma and a cellular model of neurodevelopment, consistently expresses genes related to neurodegenerative disorders at different biological levels (DNA, RNA, and proteins). Using SH-SY5Y cells, we have studied the role of Dicer and miRNAs in neuronal differentiation and explored involvement of P53, a master regulator of gene expression in differentiation-induced induction of miRNAs. Knocking down Dicer gene induced senescence in differentiating SH-SY5Y cells, which indicate the essential role of Dicer in brain development. Differentiation of SH-SY5Y cells by retinoic acid (RA) or RA + brain-derived neurotrophic factor (BDNF) induced dramatic changes in global miRNA expression. Fully differentiated SH-SY5Y cells (5-day RA followed by 3-day BDNF) significantly (p < 0.05 and atleast >3-fold change) upregulated and downregulated the expression of 77 and 17 miRNAs, respectively. Maximum increase was observed in the expression of miR-193-5p, miR-199a-5p, miR-192, miR-145, miR-28-5p, miR-29b, and miR-222 after RA exposure and miR-193-5p, miR-146a, miR-21, miR-199a-5p, miR-153, miR-29b, and miR-222 after RA + BDNF exposure in SH-SY5Y cells. Exploring the role of P53 in differentiating SH-SY5Y cells, we have observed that induction of miR-222, miR-192, and miR-145 is P53 dependent and expression of miR-193a-5p, miR-199a-5p, miR-146a, miR-21, miR-153, and miR-29b is P53 independent. In conclusion, decreased Dicer level enforces differentiating cells to senescence, and differentiating SH-SY5Y cells needs increased expression of P53 to cope up with changes in protein levels of mature neurons.     

Are ecological communities the seat of endosymbiont horizontal transfer and diversification? A case study with soil arthropod community

Maternally inherited endosymbionts of arthropods are one of the most abundant and diverse group of bacteria. These bacterial endosymbionts also show extensive horizontal transfer to taxonomically unrelated hosts and widespread recombination in their genomes. Such horizontal transfers can be enhanced when different arthropod hosts come in contact like in an ecological community. Higher rates of horizontal transfer can also increase the probability of recombination between endosymbionts, as they now share the same host cytoplasm. However, reports of community‐wide endosymbiont data are rare as most studies choose few host taxa and specific ecological interactions among the hosts. To better understand endosymbiont spread within host populations, we investigated the incidence, diversity, extent of horizontal transfer, and recombination of three endosymbionts (Wolbachia, Cardinium, and Arsenophonus) in a specific soil arthropod community. Wolbachia strains were characterized with MLST genes whereas 16S rRNA gene was used for Cardinium and Arsenophonus. Among 3,509 individual host arthropods, belonging to 390 morphospecies, 12.05% were infected with Wolbachia, 2.82% with Cardinium and 2.05% with Arsenophonus. Phylogenetic incongruence between host and endosymbiont indicated extensive horizontal transfer of endosymbionts within this community. Three cases of recombination between Wolbachia supergroups and eight incidences of within‐supergroup recombination were also found. Statistical tests of similarity indicated supergroup A Wolbachia and Cardinium show a pattern consistent with extensive horizontal transfer within the community but not for supergroup B Wolbachia and Arsenophonus. We highlight the importance of extensive community‐wide studies for a better understanding of the spread of endosymbionts across global arthropod communities.