Epiminocyclohepta[b]indole analogs as 5-HT6 antagonists

A new series of epiminocyclohepta[b]indoles with potent 5-HT(6) antagonist activity were discovered and optimized using in vitro protocols. One compound from this series was progressed to advanced pharmacokinetic (PK) studies followed by 5-HT(6) receptor occupancy studies. The compound was found to have excellent oral absorption, a highly favorable PK profile and demonstrated pharmacodynamic interaction with the 5-HT(6) receptor as shown by ex vivo autoradiography.     

Large scale variation in Enterococcus faecalis illustrated by the genome analysis of strain OG1RF

Background

Enterococcus faecalis has emerged as a major hospital pathogen. To explore its diversity, we sequenced E. faecalis strain OG1RF, which is commonly used for molecular manipulation and virulence studies.

Results

The 2,739,625 base pair chromosome of OG1RF was found to contain approximately 232 kilobases unique to this strain compared to V583, the only publicly available sequenced strain. Almost no mobile genetic elements were found in OG1RF. The 64 areas of divergence were classified into three categories. First, OG1RF carries 39 unique regions, including 2 CRISPR loci and a new WxL locus. Second, we found nine replacements where a sequence specific to V583 was substituted by a sequence specific to OG1RF. For example, the iol operon of OG1RF replaces a possible prophage and the vanB transposon in V583. Finally, we found 16 regions that were present in V583 but missing from OG1RF, including the proposed pathogenicity island, several probable prophages, and the cpsCDEFGHIJK capsular polysaccharide operon. OG1RF was more rapidly but less frequently lethal than V583 in the mouse peritonitis model and considerably outcompeted V583 in a murine model of urinary tract infections.

Conclusion

E. faecalis OG1RF carries a number of unique loci compared to V583, but the almost complete lack of mobile genetic elements demonstrates that this is not a defining feature of the species. Additionally, OG1RF's effects in experimental models suggest that mediators of virulence may be diverse between different E. faecalis strains and that virulence is not dependent on the presence of mobile genetic elements.     

The p23 Protein of Citrus Tristeza Virus Controls Asymmetrical RNA Accumulation

Citrus tristeza virus (CTV), a member of the Closteroviridae, has a 19.3-kb positive-stranded RNA genome that is organized into 12 open reading frames (ORFs) with the 10 3' genes expressed via a nested set of nine or ten 3'-coterminal subgenomic mRNAs (sgRNAs). Relatively large amounts of negative-stranded RNAs complementary to both genomic and sgRNAs accumulate in infected cells. As is characteristic of RNA viruses, wild-type CTV produced more positive than negative strands, with the plus-to-minus ratios of genomic and sgRNAs estimated at 10 to 20:1 and 40 to 50:1, respectively. However, a mutant with all of the 3' genes deleted replicated efficiently, but produced plus to minus strands at a markedly decreased ratio of 1 to 2:1. Deletion analysis of 3'-end genes revealed that the p23 ORF was involved in asymmetric RNA accumulation. A mutation which caused a frameshift after the fifth codon resulted in nearly symmetrical RNA accumulation, suggesting that the p23 protein, not a cis-acting element within the p23 ORF, controls asymmetric accumulation of CTV RNAs. Further in-frame deletion mutations in the p23 ORF suggested that amino acid residues 46 to 180, which contained RNA-binding and zinc finger domains, were indispensable for asymmetrical RNA accumulation, while the N-terminal 5 to 45 and C-terminal 181 to 209 amino acid residues were not absolutely required. Mutation of conserved cysteine residues to alanines in the zinc finger domain resulted in loss of activity of the p23 protein, suggesting involvement of the zinc finger in asymmetric RNA accumulation. The absence of p23 gene function was manifested by substantial increases in accumulation of negative-stranded RNAs and only modest decreases in positive-stranded RNAs. Moreover, the substantial decrease in the accumulation of negative-stranded coat protein (CP) sgRNA in the presence of the functional p23 gene resulted in a 12- to 15-fold increase in the expression of the CP gene. Apparently the excess negative-stranded sgRNA reduces the availability of the corresponding positive-stranded sgRNA as a messenger. Thus, the p23 protein controls asymmetric accumulation of CTV RNAs by downregulating negative-stranded RNA accumulation and indirectly increases expression of 3' genes.     

Discovery and structure-activity relationship of 2-phenyl-oxazole-4-carboxamide derivatives as potent apoptosis inducers

As a continuation of our efforts to discover novel apoptosis inducers as anticancer agents using a cell-based caspase HTS assay, 2-phenyl-oxazole-4-carboxamide derivatives were identified. The structure-activity relationships of this class of molecules were explored. Compound 1k, with EC(50) of 270 nM and GI(50) of 229 nM in human colorectal DLD-1 cells, was selected and demonstrated the ability to cleave PARP and displayed DNA laddering, the hallmarks of apoptosis. Compound 1k showed 63% tumor growth inhibition in human colorectal DLD-1 xenograft mouse model at 50 mpk, bid.     

p75NTR-mediated signaling promotes the survival of myoblasts and influences muscle strength

During muscle development, the p75(NTR) is expressed transiently on myoblasts. The temporal expression pattern of the receptor raises the possibility that the receptor is influencing muscle development. To test this hypothesis, p75(NTR)-deficient mutant mice were tested for muscle strength by using a standard wire gripe strength test and were found to have significantly decreased strength relative to that of normal mice. When normal mybolasts were examined in vivo for expression of NGF receptors, p75(NTR) was detected on myoblasts but the high affinity NGF receptor, trk A, was not co-expressed with p75(NTR). In vitro, proliferating C2C12 and primary myoblasts co-expressed the p75(NTR) and MyoD, but immunofluorescent analysis of primary myoblasts and RT-PCR analysis of C2C12 mRNA revealed that myoblasts were devoid of trk A. In contrast to the cell death functions that characterize the p75(NTR) in neurons, p75(NTR)-positive primary and C2C12 myoblasts did not differentiate or undergo apoptosis in response to neurotrophins. Rather, myoblasts survived and even proliferated when grown at subconfluent densities in the presence of the neurotrophins. Furthermore, when myoblasts treated with NGF were lysed and immunoprecipitated with antibodies against phosphorylated I-kappaB and AKT, the cells contained increased levels of both phospho-proteins, both of which promote cell survival. By contrast, neurotrophin-treated myoblasts did not induce phosphorylation of Map Kinase p42/44 or p38, indicating the survival was not mediated by the trk A receptor. Taken together, the data indicate that the p75(NTR) mediates survival of myoblasts prior to differentiation and that the activity of this receptor during myogenesis is important for developing muscle.     

Cardiomyocytes derived from human embryonic stem cells in pro-survival factors enhance function of infarcted rat hearts

Cardiomyocytes derived from human embryonic stem (hES) cells potentially offer large numbers of cells to facilitate repair of the infarcted heart. However, this approach has been limited by inefficient differentiation of hES cells into cardiomyocytes, insufficient purity of cardiomyocyte preparations and poor survival of hES cell-derived myocytes after transplantation. Seeking to overcome these challenges, we generated highly purified human cardiomyocytes using a readily scalable system for directed differentiation that relies on activin A and BMP4. We then identified a cocktail of pro-survival factors that limits cardiomyocyte death after transplantation. These techniques enabled consistent formation of myocardial grafts in the infarcted rat heart. The engrafted human myocardium attenuated ventricular dilation and preserved regional and global contractile function after myocardial infarction compared with controls receiving noncardiac hES cell derivatives or vehicle. The ability of hES cell-derived cardiomyocytes to partially remuscularize myocardial infarcts and attenuate heart failure encourages their study under conditions that closely match human disease.     

Partial cleavage of RasGAP by caspases is required for cell survival in mild stress conditions

Tight control of apoptosis is required for proper development and maintenance of homeostasis in multicellular organisms. Cells can protect themselves from potentially lethal stimuli by expressing antiapoptotic factors, such as inhibitors of apoptosis, FLICE (caspase 8)-inhibitory proteins, and members of the Bcl2 family. Here, we describe a mechanism that allows cells to survive once executioner caspases have been activated. This mechanism relies on the partial cleavage of RasGAP by caspase 3 into an amino-terminal fragment called fragment N. Generation of this fragment leads to the activation of the antiapoptotic Akt kinase, preventing further amplification of caspase activity. Partial cleavage of RasGAP is required for cell survival under stress conditions because cells expressing an uncleavable RasGAP mutant cannot activate Akt, cannot prevent amplification of caspase 3 activity, and eventually undergo apoptosis. Executioner caspases therefore control the extent of their own activation by a feedback regulatory mechanism initiated by the partial cleavage of RasGAP that is crucial for cell survival under adverse conditions.     

Phenotypic and genetic characterization of Vibrio cholerae O1 clinical isolates collected through national antimicrobial resistance surveillance network in Nepal

Cholera occurs in sporadic cases and outbreaks in Nepal each year. Vibrio cholerae O1 (n = 522) isolated during 2007-2010 from diarrheal patients at 10 different hospital laboratories in Nepal were characterized. Biochemical and serologic identifications showed that all the isolates belonged to serogroup O1, El Tor biotype. Except 72 isolates of Inaba serotype isolated in the year 2007, all the remaining isolates were of Ogawa serotype. All isolates were resistant to nalidixic acid and furazolidone. Resistance to tetracycline, ciprofloxacin, erythromycin and co-trimoxazole were 21, 4, 16 and 90 % respectively. Seventy-seven of these isolates were selected for further characterization for ctxB gene and MLVA typing. Two different variants of classical type cholera toxin were observed. Ogawa strains from 2007 and 2010-Western Nepal outbreak harbored CTX-3 type cholera toxin, whereas Inaba serotypes in 2007 and the remaining Ogawa serotypes in 2008-2010 harbored CTX 3b-type toxin. MLVA analysis showed circulation of four different groups of altered V. cholerae O1 El Tor strains. Two different profiles were seen among 2007 Inaba (9, 3, 6, x, x) and Ogawa (10, 7, 6, x, x) isolates. The MLVA profile of 2008 and 2009 Ogawa isolates were similar to those of Inaba strains of 2007. Isolates from 2010 also showed three different MLVA profiles; profile 9, 3, 6, x, x in 3 isolates, 11, 7, 6, x, x among 2010 Western Nepal outbreak strains and profile 8, 3, 6, x, x among isolates from Butwal and Kathmandu.     

Pro-inflammatory Genes as Biomarkers and Therapeutic Targets in Oral Squamous Cell Carcinoma

Oral squamous cell carcinoma (OSCC) is a major health problem worldwide, and patients have a particularly poor 5-year survival rate. Thus, identification of the molecular targets in OSCC and subsequent innovative therapies are greatly needed. Prolonged exposure to alcohol, tobacco, and pathogenic agents are known risk factors and have suggested that chronic inflammation may represent a potential common denominator in the development of OSCC. Microarray analysis of gene expression in OSCC cell lines with high basal NF-κB activity and OSCC patient samples identified dysregulation of many genes involved in inflammation, wound healing, angiogenesis, and growth regulation. In particular IL-8, CCL5, STAT1, and VEGF gene expression was up-regulated in OSCC. Moreover, IL-8 protein levels were significantly higher in OSCC cell lines as compared with normal human oral keratinocytes. Targeting IL-8 expression by siRNA significantly reduced the survival of OSCC cells, indicating that it plays an important role in OSCC development and/or progression. Inhibiting the inflammatory pathway by aspirin and the proteasome/NF-κB pathway by bortezomib resulted in marked reduction in cell viability in OSCC lines. Taken together our studies indicate a strong link between inflammation and OSCC development and reveal IL-8 as a potential mediator. Treatment based on prevention of general inflammation and/or the NF-κB pathway shows promise in OSCCs.