Sequence analysis of the long arm of rice chromosome 11 for rice–wheat synteny

The DNA sequence of 106 BAC/PAC clones in the minimum tiling path (MTP) of the long arm of rice chromosome 11, between map positions 57.3 and 116.2 cM, has been assembled to phase 2 or PLN level. This region has been sequenced to 10× redundancy by the Indian Initiative for Rice Genome Sequencing (IIRGS) and is now publicly available in GenBank. The region, excluding overlaps, has been predicted to contain 2,932 genes using different software. A gene-by-gene BLASTN search of the NCBI wheat EST database of over 420,000 cDNA sequences revealed that 1,143 of the predicted rice genes (38.9%) have significant homology to wheat ESTs (bit score 100). Further BLASTN search of these 1,143 rice genes with the GrainGenes database of sequence contigs containing bin-mapped wheat ESTs allowed 113 of the genes to be placed in bins located on wheat chromosomes of different homoeologous groups. The largest number of genes, about one-third, mapped to the homoeologous group 4 chromosomes of wheat, suggesting a common evolutionary origin. The remaining genes were located on wheat chromosomes of different groups with significantly higher numbers for groups 3 and 5. Location of bin-mapped wheat contigs to chromosomes of all the seven homoeologous groups can be ascribed to movement of genes (transpositions) or chromosome segments (translocations) within rice or the hexaploid wheat genomes. Alternatively, it could be due to ancient duplications in the common ancestral genome of wheat and rice followed by selective elimination of genes in the wheat and rice genomes. While there exists definite conservation of gene sequences and the ancestral chromosomal identity between rice and wheat, there is no obvious conservation of the gene order at this level of resolution. Lack of extensive colinearity between rice and wheat genomes suggests that there have been many insertions, deletions, duplications and translocations that make the synteny comparisons much more complicated than earlier thought. However, enhanced resolution of comparative sequence analysis may reveal smaller conserved regions of colinearity, which will facilitate selection of markers for saturation mapping and sequencing of the gene-rich regions of the wheat genome.     

The inhibition of metallo-beta-lactamase by thioxo-cephalosporin derivatives

The 8-thioxocephalosporins are poor substrates for the B. cereus metallo beta-lactamase (k(cat)/K(m)=61.4M(-1) s(-1)) and act as weak competitive inhibitors (K(i) approximately 700 microM). The hydrolysis product of thioxocephalosporin, a thioacid, also inhibits the enzyme competitively with a K(i)=96 microM, whereas the cyclic thioxo-piperazinedione, formed by intramolecular aminolysis of thioxocephalexin has a K(i) of 29 microM.     

Preclinical safety testing of biotechnology-derived pharmaceuticals

The unique and complex nature of biotechnology-derived pharmaceuticals has meant that it is often not possible to follow the conventional safety testing programs used for chemicals, and hence they are evaluated on a case-by-case basis. Nonclinical safety testing programs must be rationally designed with a strong scientific understanding of the product, including its method of manufacture, purity, sequence, structure, species specificity, pharmacological and immunological effects, and intended clinical use. This knowledge, coupled with a firm understanding of the regulatory requirements for particular product types, will ensure that the most sensitive and regulatory-compliant test systems are used to optimize the chances of gaining regulatory approval for clinical testing or marketing authorization in the shortest possible time frame.     

BAC end sequences and a physical map reveal transposable element content and clustering patterns in the genome of Magnaporthe grisea

Transposable elements (TEs) are viewed as major contributors to the evolution of fungal genomes. Genomic resources such as BAC libraries are an underutilized resource for studying genome-wide TE distribution. Using the BAC end sequences and physical map that are available for the rice blast fungus, Magnaporthe grisea, we describe a likelihood ratio test designed to identify clustering of TEs in the genome. A significant variation in the distribution of three TEs, MAGGY, MGL, and Pot2 was observed among the fingerprint contigs of the physical map. We utilized a draft sequence of M. grisea chromosome 7 to validate our results and found a similar pattern of clustering. By examining individual BAC end sequences, we found evidence for 11 unique integrations of MAGGY or MGL into Pot2 but no evidence for the reciprocal integration of Pot2 into another TE. This suggests that: (a) the presence of Pot2 in the genome predates that of the other TEs, (b) Pot2 was less transpositionally active than other TEs, or (c) that MAGGY and MGL have integration site preference for Pot2. High transition/transversion mutation ratios as well as bias in transition site context was observed in MAGGY and MGL elements, but not in Pot2 elements. These features are consistent with the effects of a Repeat-Induced Point (RIP) mutation-like process occurring in MAGGY and MGL elements. This study illustrates the general utility of a physical map and BAC end sequences for the study of genome-wide repetitive DNA content and organization.     

Activation of phospholipase C-epsilon by heterotrimeric G protein betagamma-subunits

PLC-epsilon was identified recently as a phosphoinositide-hydrolyzing phospholipase C (PLC) containing catalytic domains (X, Y, and C2) common to all PLC isozymes as well as unique CDC25- and Ras-associating domains. Novel regulation of this PLC isozyme by the Ras oncoprotein and alpha-subunits (Galpha(12)) of heterotrimeric G proteins was illustrated. Sequence analyses of PLC-epsilon revealed previously unrecognized PH and EF-hand domains in the amino terminus. The known interaction of Gbetagamma subunits with the PH domains of other proteins led us to examine the capacity of Gbetagamma to activate PLC-epsilon. Co-expression of Gbeta(1)gamma(2) with PLC-epsilon in COS-7 cells resulted in marked stimulation of phospholipase C activity. Gbeta(2) and Gbeta(4) in combination with Ggamma(1), Ggamma(2), Ggamma(3), or Ggamma(13) also activated PLC-epsilon to levels similar to those observed with Gbeta(1)-containing dimers of these Ggamma-subunits. Gbeta(3) in combination with the same Ggamma-subunits was less active, and Gbeta(5)-containing dimers were essentially inactive. Gbetagamma-promoted activation of PLC-epsilon was blocked by cotransfection with either of two Gbetagamma-interacting proteins, Galpha(i1) or the carboxyl terminus of G protein receptor kinase 2. Pharmacological inhibition of PI3-kinase-gamma had no effect on Gbeta(1)gamma(2)-promoted activation of PLC-epsilon. Similarly, activation of Ras in the action of Gbetagamma is unlikely, because a mutation in the second RA domain of PLC-epsilon that blocks Ras activation of PLC failed to alter the stimulatory activity of Gbeta(1)gamma(2). Taken together, these results reveal the presence of additional functional domains in PLC-epsilon and add a new level of complexity in the regulation of this novel enzyme by heterotrimeric G proteins.     

Toward integration of comparative genetic, physical, diversity, and cytomolecular maps for grasses and grains, using the sorghum genome as a foundation

The small genome of sorghum (Sorghum bicolor L. Moench.) provides an important template for study of closely related large-genome crops such as maize (Zea mays) and sugarcane (Saccharum spp.), and is a logical complement to distantly related rice (Oryza sativa) as a "grass genome model." Using a high-density RFLP map as a framework, a robust physical map of sorghum is being assembled by integrating hybridization and fingerprint data with comparative data from related taxa such as rice and using new methods to resolve genomic duplications into locus-specific groups. By taking advantage of allelic variation revealed by heterologous probes, the positions of corresponding loci on the wheat (Triticum aestivum), rice, maize, sugarcane, and Arabidopsis genomes are being interpolated on the sorghum physical map. Bacterial artificial chromosomes for the small genome of rice are shown to close several gaps in the sorghum contigs; the emerging rice physical map and assembled sequence will further accelerate progress. An important motivation for developing genomic tools is to relate molecular level variation to phenotypic diversity. "Diversity maps," which depict the levels and patterns of variation in different gene pools, shed light on relationships of allelic diversity with chromosome organization, and suggest possible locations of genomic regions that are under selection due to major gene effects (some of which may be revealed by quantitative trait locus mapping). Both physical maps and diversity maps suggest interesting features that may be integrally related to the chromosomal context of DNA-progress in cytology promises to provide a means to elucidate such relationships. We seek to provide a detailed picture of the structure, function, and evolution of the genome of sorghum and its relatives, together with molecular tools such as locus-specific sequence-tagged site DNA markers and bacterial artificial chromosome contigs that will have enduring value for many aspects of genome analysis.     

Molecular Engineering Using an Anthanthrone Dye for Low‐Cost Hole Transport Materials: A Strategy for Dopant‐Free,High‐Efficiency,and Stable Perovskite Solar Cells

In this report, highly efficient and humidity‐resistant perovskite solar cells (PSCs) using two new small molecule hole transporting materials (HTM) made from a cost‐effective precursor anthanthrone (ANT) dye, namely, 4,10‐bis(1,2‐dihydroacenaphthylen‐5‐yl)‐6,12‐bis(octyloxy)‐6,12‐dihydronaphtho[7,8,1,2,3‐nopqr]tetraphene (ACE‐ANT‐ACE) and 4,4′‐(6,12‐bis(octyloxy)‐6,12‐dihydronaphtho[7,8,1,2,3‐nopqr]tetraphene‐4,10‐diyl)bis(N,N‐bis(4‐methoxyphenyl)aniline) (TPA‐ANT‐TPA) are presented. The newly developed HTMs are systematically compared with the conventional 2,2′,7,7′‐tetrakis(N,N′‐di‐p‐methoxyphenylamino)‐9,9′‐spirbiuorene (Spiro‐OMeTAD). ACE‐ANT‐ACE and TPA‐ANT‐TPA are used as a dopant‐free HTM in mesoscopic TiO₂/CH₃NH₃PbI₃/HTM solid‐state PSCs, and the performance as well as stability are compared with Spiro‐OMeTAD‐based PSCs. After extensive optimization of the metal oxide scaffold and device processing conditions, dopant‐free novel TPA‐ANT‐TPA HTM‐based PSC devices achieve a maximum power conversion efficiency (PCE) of 17.5% with negligible hysteresis. An impressive current of 21 mA cm^?2 is also confirmed from photocurrent density with a higher fill factor of 0.79. The obtained PCE of 17.5% utilizing TPA‐ANT‐TPA is higher performance than the devices prepared using doped Spiro‐OMeTAD (16.8%) as hole transport layer at 1 sun condition. It is found that doping of LiTFSI salt increases hygroscopic characteristics in Spiro‐OMeTAD; this leads to the fast degradation of solar cells. While, solar cells prepared using undoped TPA‐ANT‐TPA show dewetting and improved stability. Additionally, the new HTMs form a fully homogeneous and completely covering thin film on the surface of the active light absorbing perovskite layers that acts as a protective coating for underlying perovskite films. This breakthrough paves the way for development of new inexpensive, more stable, and highly efficient ANT core based lower cost HTMs for cost‐effective, conventional, and printable PSCs.     

Genetic Ablation of miR-33 Increases Food Intake,Enhances Adipose Tissue Expansion,and Promotes Obesity and Insulin Resistance

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Overexpression of CXCR4 synergizes with LL-37 in the metastasis of breast cancer cells

Chemokine receptor CXCR4 was involved in the progression of breast cancer to a metastatic phenotype, leading to the major cause of death in patients. A more in-depth understanding of signaling mechanism underlying CXCR4 is critical to develop effective therapies toward metastasis. Recently, the role of antimicrobial peptide LL-37 in contributing to the metastasis of breast cancer cells was observed. Clinical analysis of data herein demonstrated for the first time that overexpression of LL-37 and CXCR4 co-existed in human primary breast tumors with lymph node metastases. Further study disclosed that forced expression of CXCR4 led to the enhancement of pro-migratory signaling and migration rate induced by LL-37 in breast cancer cells. Moreover, LL-37 affected tumor microenvironment including induction of migration of mesenchymal stem cells and CXCR4-dependent capillary-like tubule formation. Functional analysis showed that LL-37 induced the internalization of CXCR4 through approaching Glu268, the residue of CXCR4, independent of the binding pocket (Asp171, Asp262, and Glu288) for CXCR4 inhibitor AMD3100, signifying that LL-37 is a distinct agonist of CXCR4. These results suggest the reciprocal roles of LL-37 and CXCR4 in promoting breast cancer cell migration and provide new insight into the design of CXCR4 inhibitor for intervention of metastatic breast cancer.