Mutations in WDR62, encoding a centrosomal and nuclear protein, in Indian primary microcephaly families with cortical malformations

Primary microcephaly is an autosomal recessive disorder characterized by smaller than normal brain size and mental retardation. It is genetically heterogeneous with seven loci: MCPH1-MCPH7. We have previously reported genetic analysis of 35 families, including the identification of the MCPH7 gene STIL. Of the 35 families, three families showed linkage to the MCPH2 locus. Recent whole-exome sequencing studies have shown that the WDR62 gene, located in the MCPH2 candidate region, is mutated in patients with severe brain malformations. We therefore sequenced the WDR62 gene in our MCPH2 families and identified two novel homozygous protein truncating mutations in two families. Affected individuals in the two families had pachygyria, microlissencephaly, band heterotopias, gyral thickening, and dysplastic cortex. Using immunofluorescence study, we showed that, as with other MCPH proteins, WDR62 localizes to centrosomes in A549, HepG2, and HaCaT cells. In addition, WDR62 was also localized to nucleoli. Bioinformatics analysis predicted two overlapping nuclear localization signals and multiple WD-40 repeats in WDR62. Two other groups have also recently identified WDR62 mutations in MCPH2 families. Our results therefore add further evidence that WDR62 is the MCPH2 gene. The present findings will be helpful in genetic diagnosis of patients linked to the MCPH2 locus.

Studies of phosphazenes .11. syntheses and structures of bis(primary amino)hexachlorocyclotetraphosphazenes and their dimethylamino derivatives

The bis(amino)hexachlorocyclotetraphosphazenes, 2-trans-6-N4P4 (NHR)2Cl6, R [dbnd] Me, Pr n Pr i , Bu n , CH2Ph, Ph, are obtained from the reaction of N4P4Cl8 with four mol. equivalents of the appropriate amine. Isomers with 2,4-structures have been isolated for R [dbnd] Bu n , CH2Ph. The 1H and 31P NMR spectra of these bis(amino) compounds and of their dimethylamino derivatives, 2-trans-6-N4P4 (NMe2)6 (NHR)2 are discussed.

Three-Component Chiral Derivatizing Protocols for NMR Spectroscopic Enantiodiscrimination of Hydroxy Acids and Primary Amines

The novel three-component chiral derivatization protocols have been derived for (1)H and (19)F NMR spectroscopic discrimination of a series of chiral hydroxy acids by their coordination and self-assembly with optically active a-methylbenzylamine and 2-formylphenylboronic acid. In addition, the optically pure (S)-mandelic acid in combination with 2-formylphenylboronic acid permits visualization of enantiomers of primary amines. These protocols have been demonstrated on enantiodiscrimination of chiral amines and hydroxy acids.

A Monoclonal Antibody against Human Notch1 Ligand-Binding Domain Depletes Subpopulation of Putative Breast Cancer Stem-like Cells

Overexpression of Notch receptors and ligands has been associated with various cancers and developmental disorders, making Notch a potential therapeutic target. Here, we report characterization of Notch1 monoclonal antibodies (mAb) with therapeutic potential. The mAbs generated against epidermal growth factor (EGF) repeats 11 to 15 inhibited binding of Jagged1 and Delta-like4 and consequently, signaling in a dose-dependent manner, the antibodies against EGF repeats 11 to 12 being more effective than those against repeats 13 to 15. These data emphasize the role of EGF repeats 11 to 12 in ligand binding. One of the mAbs, 602.101, which specifically recognizes Notch1, inhibited ligand-dependent expression of downstream target genes of Notch such as HES-1, HES-5, and HEY-L in the breast cancer cell line MDA-MB-231. The mAb also decreased cell proliferation and induced apoptotic cell death. Furthermore, exposure to this antibody reduced CD44(Hi)/CD24(Low) subpopulation in MDA-MB-231 cells, suggesting a decrease in the cancer stem-like cell subpopulation. This was confirmed by showing that exposure to the antibody decreased the primary, secondary, and tertiary mammosphere formation efficiency of the cells. Interestingly, effect of the antibody on the putative stem-like cells appeared to be irreversible, because the mammosphere-forming efficiency could not be salvaged even after antibody removal during the secondary sphere formation. The antibody also modulated expression of genes associated with stemness and epithelial-mesenchymal transition. Thus, targeting individual Notch receptors by specific mAbs is a potential therapeutic strategy to reduce the potential breast cancer stem-like cell subpopulation. Mol Cancer Ther; 11(1); 77-86. (C) 2011 AACR.

A non-metal catalysed oxidation of primary azides to nitriles at ambient temperature

A novel non-metal catalyzed oxidation of organic azides to nitriles under solvent-free conditions is presented employing catalytic amounts of KI, and DABCO in aq. TBHP at room temperature. This nonmetal catalyzed oxidation of azides provides good selectivity as double and triple bonds were not oxidized under the present reaction conditions.

Characterization of structural and free energy properties of promoters associated with Primary and Operon TSS in Helicobacter pylori genome and their orthologs

Promoter regions in the genomes of all domains of life show similar trends in several structural properties such as stability, bendability, curvature, etc. In current study we analysed the stability and bendability of various classes of promoter regions (based on the recent identification of different classes of transcription start sites) of Helicobacter pylori 26695 strain. It is found that primary TSS and operon-associated TSS promoters show significantly strong features in their promoter regions. DNA free-energy-based promoter prediction tool PromPredict was used to annotate promoters of different classes, and very high recall values (similar to 80%) are obtained for primary TSS. Orthologous genes from other strains of H. pylori show conservation of structural properties in promoter regions as well as coding regions. PromPredict annotates promoters of orthologous genes with very high recall and precision.

Antibodies against the extracellular domain of human Notch1 receptor reveal the critical role of epidermal-growth-factor-like repeats 25-26 in ligand binding and receptor activation

The Notch signalling pathway is implicated in a wide variety of cellular processes throughout metazoan development. Although the downstream mechanism of Notch signalling has been extensively studied, the details of its ligand-mediated receptor activation are not clearly understood. Although the role of Notch ELRs EGF (epidermal growth factor)-like-repeats] 11-12 in ligand binding is known, recent studies have suggested interactions within different ELRs of the Notch receptor whose significance remains to be understood. Here, we report critical inter-domain interactions between human Notch1 ELRs 21-30 and the ELRs 11-15 that are modulated by calcium. Surface plasmon resonance analysis revealed that the interaction between ELRs 21-30 and ELRs 11-15 is similar to 10-fold stronger than that between ELRs 11-15 and the ligands. Although there was no interaction between Notch 1 ELRs 21-30 and the ligands in vitro, addition of pre-clustered Jagged1Fc resulted in the dissociation of the preformed complex between ELRs 21-30 and 11-15, suggesting that inter-domain interactions compete for ligand binding. Furthermore, the antibodies against ELRs 21-30 inhibited ligand binding to the full-length Notch1 and subsequent receptor activation, with the antibodies against ELRs 25-26 being the most effective. These results suggest that the ELRs 25-26 represent a cryptic ligand-binding site which becomes exposed only upon the presence of the ligand. Thus, using specific antibodies against various domains of the Notch1 receptor, we demonstrate that, although ELRs 11-12 are the principal ligand-binding site, the ELRs 25-26 serve as a secondary binding site and play an important role in receptor activation.

Toll-Like Receptors 7, 8, and 9 Expression and Function in Primary Human Cervical Cancer Langerhans Cells Evidence of Anergy

Objective: Human papillomavirus oncoproteins E6 and E7 down modulate Toll-like receptor (TLR) 9 expression in infected keratinocytes. We explored the status of expression and function of TLR7, TLR8, and TLR9 in primary human Langerhans cells (LCs) isolated from cervical tumors. Methodology: Single-cell suspensions were made from fresh tissues of squamous cell carcinoma (International Federation of Gynecology and Obstetrics stage IB2); myeloid dendritic cells were purified using CD1c magnetic activated cell separation kits. Langerhans cells were further flow sorted into CD1a(+)CD207(+) cells. Acute monocytic leukemia cell line THP-1-derived LCs (moLCs) formed the controls. mRNA from flow-sorted LCs was reverse transcribed to cDNA and TLR7, TLR8, and TLR9 amplified. Monocyte-derived Langerhans cells and cervical tumor LCs were stimulated with TLR7, TLR8, and TLR9 ligands. Culture supernatants were assayed for interleukin (IL) 1 beta, IL-6, IL-10, IL-12p70, interferon (IFN) alpha, interferon gamma, and tumor necrosis factor (TNF) alpha by Luminex multiplex bead array. Human papillomavirus was genotyped. Results: We have for the first time demonstrated that the acute monocytic leukemia cell line THP-1 can be differentiated into LCs in vitro. Although these moLCs. expressed all the 3 TLRs, tumor LCs expressed TLR7 and TLR8, but uniformly lacked TLR9. Also, moLCs secreted IL-6, IL-1 beta, and tumor necrosis factor alpha to TLR8 ligand and interferon alpha in response to TLR9 ligand; in contrast, tumor LCs did not express any cytokine to any of the 3 TLR ligands. Human papillomavirus type 16 was one of the common human papillomavirus types in all cases. Conclusions: Cervical tumor LCs lacked TLR9 expression and were functionally anergic to all the 3: TLR7, TLR8, and TLR9 ligands, which may play a crucial role in immune tolerance. The exact location of block(s) in TLR7 and TLR8 signaling needs to be investigated, which would have important immunotherapeutic implications.

Primary Microcephaly Gene MCPH1 Shows Signatures of Tumor Suppressors and Is Regulated by miR-27a in Oral Squamous Cell Carcinoma

Mutations in the MCPH1 (microcephalin 1) gene, located at chromosome 8p23.1, result in two autosomal recessive disorders: primary microcephaly and premature chromosome condensation syndrome. MCPH1 has also been shown to be downregulated in breast, prostate and ovarian cancers, and mutated in 1/10 breast and 5/41 endometrial tumors, suggesting that it could also function as a tumor suppressor (TS) gene. To test the possibility of MCPH1 as a TS gene, we first performed LOH study in a panel of 81 matched normal oral tissues and oral squamous cell carcinoma (OSCC) samples, and observed that 14/71 (19.72%) informative samples showed LOH, a hallmark of TS genes. Three protein truncating mutations were identified in 1/15 OSCC samples and 2/5 cancer cell lines. MCPH1 was downregulated at both the transcript and protein levels in 21/41 (51.22%) and 19/25 (76%) OSCC samples respectively. A low level of MCPH1 promoter methylation was also observed in 4/40 (10%) tumor samples. We further observed that overexpression of MCPH1 decreased cellular proliferation, anchorage-independent growth in soft agar, cell invasion and tumor size in nude mice, indicating its tumor suppressive function. Using bioinformatic approaches and luciferase assay, we showed that the 3'-UTR of MCPH1 harbors two non-overlapping functional seed regions for miR-27a which negatively regulated its level. The expression level of miR-27a negatively correlated with the MCPH1 protein level in OSCC. Our study indicates for the first time that, in addition to its role in brain development, MCPH1 also functions as a tumor suppressor gene and is regulated by miR-27a.

Throughput analysis of primary and secondary networks in a shared IEEE 802.11 system

In this paper, we analyze the coexistence of a primary and a secondary (cognitive) network when both networks use the IEEE 802.11 based distributed coordination function for medium access control. Specifically, we consider the problem of channel capture by a secondary network that uses spectrum sensing to determine the availability of the channel, and its impact on the primary throughput. We integrate the notion of transmission slots in Bianchi's Markov model with the physical time slots, to derive the transmission probability of the secondary network as a function of its scan duration. This is used to obtain analytical expressions for the throughput achievable by the primary and secondary networks. Our analysis considers both saturated and unsaturated networks. By performing a numerical search, the secondary network parameters are selected to maximize its throughput for a given level of protection of the primary network throughput. The theoretical expressions are validated using extensive simulations carried out in the Network Simulator 2. Our results provide critical insights into the performance and robustness of different schemes for medium access by the secondary network. In particular, we find that the channel captures by the secondary network does not significantly impact the primary throughput, and that simply increasing the secondary contention window size is only marginally inferior to silent-period based methods in terms of its throughput performance.

Synthon identification in co-crystals and polymorphs with IR spectroscopy. Primary amides as a case study

IR spectroscopy has been widely employed to distinguish between different crystal forms such as polymorphs, clathrates, hydrates and co-crystals. IR has been used to monitor co-crystal formation and single synthon detection. In this work, we have developed a strategy to identify multiple supramolecular synthons in polymorphs and co-crystals with this technique. The identification of multiple synthons in co-crystals with IR is difficult for several reasons. In this paper, a four step method involving well assigned IR spectral markers that correspond to bonds in a synthon is used. IR spectra of three forms of the co-crystal system, 4-hydroxybenzoic acid: 4,4'-bipyridine (2 : 1), show clear differences that may be attributed to differences in the synthon combinations existing in the forms (synthon polymorphism). These differences were picked out from the three IR spectra and the bands analysed and assigned to synthons. Our method first identifies IR marker bands corresponding to (covalent) bonds in known/model crystals and then the markers are mapped in known co-crystals having single synthons. Thereafter, the IR markers are queried in known co-crystals with multiple synthons. Finally they are queried in unknown co-crystals with multiple synthons. In the last part of the study, the N-H stretching absorptions of primary amides that crystallize with the amide dimers linked in a ladder like chain show two specific absorptions which are used as marker absorptions and all variations of this band structure have been used to provide details on the environment around the dimer. The extended dimer can accordingly be easily distinguished from the isolated dimer.

Microstructure and texture evolution during sub-transus thermo-mechanical processing of Ti-6Al-4V-0.1B alloy: part II. static annealing in (alpha plus beta) regime

The first part of this study describes the evolution of microstructure and texture in Ti-6Al-4V-0.1B alloy during sub-transus rolling vis-A -vis the control alloy Ti-6Al-4V. In the second part, the static annealing response of the two alloys at self-same conditions is compared and the principal micromechanisms are analyzed. Faster globularization kinetics has been observed in the Ti-6Al-4V-0.1B alloy for equivalent annealing conditions. This is primarily attributed to the alpha colonies, which leads to easy boundary splitting via multiple slip activation in this alloy. The other mechanisms facilitating lamellar to equiaxed morphological transformations, e.g., termination migration and cylinderization, also start early in the boron-modified alloy due to small alpha colony size, small aspect ratio of the alpha lamellae, and the presence of TiB particles in the microstructure. Both the alloys exhibit weakening of basal fiber (ND||aOE (c) 0001 >) and strengthening of prism fiber (RD||aOE (c) aOE(a)) upon annealing. A close proximity between the orientations of fully globularized primary alpha and secondary alpha phases during alpha -> beta -> alpha transformation has accounted for such a texture modification.

Cognitive radio implementation for a frequency hopping primary signal

Frequency hopping communications, used in the military present significant opportunities for spectrum reuse via the cognitive radio technology. We propose a MAC which incorporates hop instant identification, and supports network discovery and formation, QOS Scheduling and secondary communications. The spectrum sensing algorithm is optimized to deal with the problem of spectral leakage. The algorithms are implemented in a SDR platform based test bed and measurement results are presented.

Functional analysis of ultra high information rates conveyed by rat vibrissal primary afferents

Sensory receptors determine the type and the quantity of information available for perception. Here, we quantified and characterized the information transferred by primary afferents in the rat whisker system using neural system identification. Quantification of ``how much'' information is conveyed by primary afferents, using the direct method (DM), a classical information theoretic tool, revealed that primary afferents transfer huge amounts of information (up to 529 bits/s). Information theoretic analysis of instantaneous spike-triggered kinematic stimulus features was used to gain functional insight on ``what'' is coded by primary afferents. Amongst the kinematic variables tested-position, velocity, and acceleration-primary afferent spikes encoded velocity best. The other two variables contributed to information transfer, but only if combined with velocity. We further revealed three additional characteristics that play a role in information transfer by primary afferents. Firstly, primary afferent spikes show preference for well separated multiple stimuli (i.e., well separated sets of combinations of the three instantaneous kinematic variables). Secondly, neurons are sensitive to short strips of the stimulus trajectory (up to 10 ms pre-spike time), and thirdly, they show spike patterns (precise doublet and triplet spiking). In order to deal with these complexities, we used a flexible probabilistic neuron model fitting mixtures of Gaussians to the spike triggered stimulus distributions, which quantitatively captured the contribution of the mentioned features and allowed us to achieve a full functional analysis of the total information rate indicated by the DM. We found that instantaneous position, velocity, and acceleration explained about 50% of the total information rate. Adding a 10 ms pre-spike interval of stimulus trajectory achieved 80-90%. The final 10-20% were found to be due to non-linear coding by spike bursts.