Exogenous fluorescent tracer agents based on pegylated pyrazine dyes for real-time point-of-care measurement of glomerular filtration rate

Novel pyrazine carboxamides bearing hydrophilic poly(ethylene glycol) (PEG) moieties were designed, synthesized, and evaluated for use as fluorescent glomerular filtration rate (GFR) tracer agents. Among these, compounds 4d and 5c that contain about 48 ethylene oxide units in the PEG chain exhibited the most favorable physicochemical and renal clearance properties. In vitro studies show that these two compounds have low plasma protein binding, a necessary condition for renal excretion. In vivo animal model results show that 4d and 5c have a higher urine recovery of the injected dose than iothalamate (a commonly considered gold standard GFR agent). Pharmacokinetic studies show that these two compounds exhibit a plasma clearance equivalent to iothalamate, but with a faster (i.e. lower) terminal half-life than iothalamate (possibly from restricted distribution into the extracellular space due to large molecular size and hydrodynamic volume). Furthermore, the plasma clearance of 4d and 5c remained unchanged upon blockage of the tubular secretion pathway with probenecid, a necessary condition for establishment of clearance via glomerular filtration exclusively. Finally, noninvasive real-time monitoring of this class of compounds was demonstrated by pharmacokinetic clearance of 5c by optical measurements in rat model, which correlates strongly with plasma concentration of the tracer. Hence, 4d and 5c are promising candidates for translation to the clinic as exogenous fluorescent tracer agents in real-time point-of-care monitoring of GFR.     

The antibacterial agent,moxifloxacin inhibits virulence factors of <Emphasis Type="Italic">Candida albicans</Emphasis> through multitargeting

Fluoroquinolines are broad spectrum fourth generation antibiotics. Some of the Fluoroquinolines exhibit antifungal activity. We are reporting the potential mechanism of action of a fluoroquinoline antibiotic, moxifloxacin on the growth, morphogenesis and biofilm formation of the human pathogen Candida albicans. Moxifloxacin was found to be Candidacidal in nature. Moxifloxacin seems to inhibit the yeast to Hyphal morphogenesis by affecting signaling pathways. It arrested the cell cycle of C. albicans at S phase. Docking of moxifloxacin with predicted structure of C. albicans DNA Topoisomerase II suggests that moxifloxacin may bind and inhibit the activity of DNA Topoisomerase II in C. albicans. Moxifloxacin could be used as a dual purpose antibiotic for treating mixed infections caused by bacteria as well as C. albicans. In addition chances of developing moxifloxacin resistance in C. albicans are less considering the fact that moxifloxacin may target multiple steps in yeast to hyphal transition in C. albicans.     

Analysis of human syndromes with disordered chromatin reveals the impact of heterochromatin on the efficacy of ATM-dependent G2/M checkpoint arrest

Heterochromatin (HC) poses a barrier to γH2AX focus expansion and DNA double-strand break (DSB) repair, the latter being relieved by ATM-dependent KAP-1 phosphorylation. Using high-resolution imaging, we show here that the HC superstructure markedly restricts ATM signaling to cell cycle checkpoint proteins. The impact of HC is greater than anticipated from the percentage of HC-DNA and, in distinction to DSB repair, ATM only partly overcomes the constraints posed by HC. Importantly, we examine ATM signaling in human syndromes with disordered HC. After depletion of MeCP2 and DNMT3B, proteins defective in the Rett and immunodeficiency with centromere instability and facial anomalies (ICF) syndromes, respectively, we demonstrate enhanced γH2AX signal expansion at HC-chromocenters in mouse NIH 3T3 cells, which have visible HC-chromocenters. Previous studies have shown that the G(2)/M checkpoint is inefficient requiring multiple DSBs to initiate arrest. MeCP2 and DNMT3B depletion leads to hypersensitive radiation-induced G(2)/M checkpoint arrest despite normal DSB repair. Cell lines from Rett, ICF, and Hutchinson-Guildford progeria syndrome patients similarly showed hyperactivated ATM signaling and hypersensitive and prolonged G(2)/M checkpoint arrest. Collectively, these findings reveal that heterochromatin contributes to the previously described inefficient G(2)/M checkpoint arrest and demonstrate how the signaling response can be uncoupled from DSB repair.     

A hybrid approach for the automated finishing of bacterial genomes

Advances in DNA sequencing technology have improved our ability to characterize most genomic diversity. However, accurate resolution of large structural events is challenging because of the short read lengths of second-generation technologies. Third-generation sequencing technologies, which can yield longer multikilobase reads, have the potential to address limitations associated with genome assembly. Here we combine sequencing data from second- and third-generation DNA sequencing technologies to assemble the two-chromosome genome of a recent Haitian cholera outbreak strain into two nearly finished contigs at >99.9% accuracy. Complex regions with clinically relevant structure were completely resolved. In separate control assemblies on experimental and simulated data for the canonical N16961 cholera reference strain, we obtained 14 scaffolds of greater than 1 kb for the experimental data and 8 scaffolds of greater than 1 kb for the simulated data, which allowed us to correct several errors in contigs assembled from the short-read data alone. This work provides a blueprint for the next generation of rapid microbial identification and full-genome assembly.