Method for Label-Free Quantitative Proteomics for <Emphasis Type="Italic">Sorghum bicolor</Emphasis> L. Moench

Sorghum (Sorghum bicolor L. Moench) is a rapidly emerging high biomass feedstock for bioethanol and lignocellulosic biomass production. The robust varietal germplasm of sorghum and its completed genome sequence provide the necessary genetic and molecular tools to study and engineer the biotic/abiotic stress tolerance. Traditional proteomics approaches for outlining the sorghum proteome have many limitations like, demand for high protein amounts, reproducibility and identification of only few differential proteins. In this study, we report a gel-free, quantitative proteomic method for in-depth coverage of the sorghum proteome. This novel method combining phenol extraction and methanol chloroform precipitation gives high total protein yields for both mature sorghum root and leaf tissues. We demonstrate successful application of this method in comparing proteomes of contrasting cultivars of sorghum, at two different phenological stages. Protein identification and relative quantification analyses were performed by a label-free liquid chromatography tandem mass spectrometry (LC/MS-MS) analyses. Several unique proteins were identified respectively from sorghum tissues, specifically 271 from leaf and 774 from root tissues, with 193 proteins common in both tissues. Using gene ontology analysis, the differential proteins identified were finely corroborated with their leaf/root tissue specific functions. This method of protein extraction and analysis would contribute substantially to generate in-depth differential protein data in sorghum as well as related species. It would also increase the repertoire of methods uniquely suited for gel-free plant proteomics that are increasingly being developed for studying abiotic and biotic stress responses.     

Cytotoxic effector T cells elicited by the killed tumor vaccine differ significantly from the effectors generated during active growth of a murine B-cell lymphoma

Active specific immunotherapy of neoplastic diseases is an elusive goal. Using a murine B lymphoma 2C3, we showed that vaccination with the killed tumor cells effectively induces protective immunity and a cytotoxic T cell (CTL) response. Similar protection, however, is rarely observed in mice bearing live tumor cells. These animals usually succumb to the progressively growing tumor. In this study, we inquired whether the splenic CTL induced during tumor progression in mice differ from those evoked by the killed tumor cells. Here we demonstrate that the CTL generated following vaccination are significantly different from those induced in the tumor-bearing hosts. Adding to the complexity, the CTL from the early tumor bearers also differ significantly from those induced at the late stages. These differences are based on their cytotoxic activity, MHC allele specificity, mitogen responsiveness, cytokine secretion profile and T cell receptor Vβ gene expression. The results clearly indicate that passive immunization with killed tumor is most effective, possibly because the CTL induced are not subject to the same regulatory pressure as those induced during active tumor growth. This decreasing effectiveness of CTL could be due to greater variability in antigenic stimulus, less involvement of innate immunity, changes in cytokine milieu and/or costimulatory factors. Received: 5 February 1999 / Accepted: 22 June 1999     

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.     

Modeling metabolic adjustment in <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> upon treatment with isoniazid

Complex biological systems exhibit a property of robustness at all levels of organization. Through different mechanisms, the system tries to sustain stress such as due to starvation or drug exposure. To explore whether reconfiguration of the metabolic networks is used as a means to achieve robustness, we have studied possible metabolic adjustments in Mtb upon exposure to isoniazid (INH), a front-line clinical drug. The redundancy in the genome of M. tuberculosis (Mtb) makes it an attractive system to explore if alternate routes of metabolism exist in the bacterium. While the mechanism of action of INH is well studied, its effect on the overall metabolism is not well characterized. Using flux balance analysis, inhibiting the fluxes flowing through the reactions catalyzed by Rv1484, the target of INH, significantly changes the overall flux profiles. At the pathway level, activation or inactivation of certain pathways distant from the target pathway, are seen. Metabolites such as NADPH are shown to reduce drastically, while fatty acids tend to accumulate. The overall biomass also decreases with increasing inhibition levels. Inhibition studies, pathway level clustering and comparison of the flux profiles with the gene expression data indicate the activation of folate metabolism, ubiquinone metabolism, and metabolism of certain amino acids. This analysis provides insights useful for target identification and designing strategies for combination therapy. Insights gained about the role of individual components of a system and their interactions will also provide a basis for reconstruction of whole systems through synthetic biology approaches.     

Synonymous codon usage pattern among the S,M, and L segments in Crimean-congo hemorrhagic fever virus

Crimean-Congo hemorrhagic fever (CCHF) virus is one among the major zoonosis viral diseases that use the Hyalomma ticks as their transmission vector to cause viral infection to the human and mammalian community. The fatality of infectious is high across the world especially in Africa, Asia, Middle East, and Europe. This study regarding codon usage bias of S, M, and L segments of the CCHF virus pertaining to the host Homo sapiens, reveals in-depth information about the evolutionary characteristics of CCHFV. Relative Synonymous Codon Usage (RSCU), Effective number of codons (ENC) were calculated, to determine the codon usage pattern in each segment. Correlation analysis between Codon adaptation index (CAI), GRAVY (Hydrophobicity), AROMO (Aromaticity), and nucleotide composition revealed bias in the codon usage pattern. There was no strong codon bias found among any segments of the CCHF virus, indicating both the factors i.e., natural selection and mutational pressure shapes the codon usage bias.     

Down-Regulation of Rad51 Activity during Meiosis in Yeast Prevents Competition with Dmc1 for Repair of Double-Strand Breaks

Interhomolog recombination plays a critical role in promoting proper meiotic chromosome segregation but a mechanistic understanding of this process is far from complete. In vegetative cells, Rad51 is a highly conserved recombinase that exhibits a preference for repairing double strand breaks (DSBs) using sister chromatids, in contrast to the conserved, meiosis-specific recombinase, Dmc1, which preferentially repairs programmed DSBs using homologs. Despite the different preferences for repair templates, both Rad51 and Dmc1 are required for interhomolog recombination during meiosis. This paradox has recently been explained by the finding that Rad51 protein, but not its strand exchange activity, promotes Dmc1 function in budding yeast. Rad51 activity is inhibited in dmc1Δ mutants, where the failure to repair meiotic DSBs triggers the meiotic recombination checkpoint, resulting in prophase arrest. The question remains whether inhibition of Rad51 activity is important during wild-type meiosis, or whether inactivation of Rad51 occurs only as a result of the absence of DMC1 or checkpoint activation. This work shows that strains in which mechanisms that down-regulate Rad51 activity are removed exhibit reduced numbers of interhomolog crossovers and noncrossovers. A hypomorphic mutant, dmc1-T159A, makes less stable presynaptic filaments but is still able to mediate strand exchange and interact with accessory factors. Combining dmc1-T159A with up-regulated Rad51 activity reduces interhomolog recombination and spore viability, while increasing intersister joint molecule formation. These results support the idea that down-regulation of Rad51 activity is important during meiosis to prevent Rad51 from competing with Dmc1 for repair of meiotic DSBs.     

Enhancing TB Case Detection: Experience in Offering Upfront Xpert MTB/RIF Testing to Pediatric Presumptive TB and DR TB Cases for Early Rapid Diagnosis of Drug Sensitive and Drug Resistant TB

Background

Diagnosis of pulmonary tuberculosis (PTB) in children is challenging due to difficulties in obtaining good quality sputum specimens as well as the paucibacillary nature of disease. Globally a large proportion of pediatric tuberculosis (TB) cases are diagnosed based only on clinical findings. Xpert MTB/RIF, a highly sensitive and specific rapid tool, offers a promising solution in addressing these challenges. This study presents the results from pediatric groups taking part in a large demonstration study wherein Xpert MTB/RIF testing replaced smear microscopy for all presumptive PTB cases in public health facilities across India.

Methods

The study covered a population of 8.8 million across 18 programmatic sub-district level tuberculosis units (TU), with one Xpert MTB/RIF platform established at each study TU. Pediatric presumptive PTB cases (both TB and Drug Resistant TB (DR-TB)) accessing any public health facilities in study area were prospectively enrolled and tested on Xpert MTB/RIF following a standardized diagnostic algorithm.

Results

4,600 pediatric presumptive pulmonary TB cases were enrolled. 590 (12.8%, CI 11.8–13.8) pediatric PTB were diagnosed. Overall 10.4% (CI 9.5–11.2) of presumptive PTB cases had positive results by Xpert MTB/RIF, compared with 4.8% (CI 4.2–5.4) who had smear-positive results. Upfront Xpert MTB/RIF testing of presumptive PTB and presumptive DR-TB cases resulted in diagnosis of 79 and 12 rifampicin resistance cases, respectively. Positive predictive value (PPV) for rifampicin resistance detection was high (98%, CI 90.1–99.9), with no statistically significant variation with respect to past history of treatment.

Conclusion

Upfront access to Xpert MTB/RIF testing in pediatric presumptive PTB cases was associated with a two-fold increase in bacteriologically-confirmed PTB, and increased detection of rifampicin-resistant TB cases under routine operational conditions across India. These results suggest that routine Xpert MTB/RIF testing is a promising solution to present-day challenges in the diagnosis of PTB in pediatric patients.     

Distinct Types of White Matter Changes Are Observed after Anterior Temporal Lobectomy in Epilepsy

Anterior temporal lobectomy (ATL) is commonly adopted to control medically intractable temporal lobe epilepsy (TLE). Depending on the side of resection, the degree to which Wallerian degeneration and adaptive plasticity occur after ATL has important implications for understanding cognitive and clinical outcome. We obtained diffusion tensor imaging from 24 TLE patients (12 left) before and after surgery, and 12 matched controls at comparable time intervals. Voxel-based analyses were performed on fractional anisotropy (FA) before and after surgery. Areas with postoperative FA increase were further investigated to distinguish between genuine plasticity and processes related to the degeneration of crossing fibers. Before surgery, both patient groups showed bilateral reduced FA in numerous tracts, but left TLE patients showed more extensive effects, including language tracts in the contralateral hemisphere (superior longitudinal fasciculus and uncinate). After surgery, FA decreased ipsilaterally in both ATL groups, affecting the fornix, uncinate, stria terminalis, and corpus callosum. FA increased ipsilaterally along the superior corona radiata in both left and right ATL groups, exceeding normal FA values. In these clusters, the mode of anisotropy increased as well, confirming fiber degeneration in an area with crossing fibers. In left ATL patients, pre-existing low FA values in right superior longitudinal and uncinate fasciculi normalized after surgery, while MO values did not change. Preoperative verbal fluency correlated with FA values in all areas that later increased FA in left TLE patients, but postoperative verbal fluency correlated only with FA of the right superior longitudinal fasciculus. Our results demonstrate that genuine reorganization occurs in non-dominant language tracts after dominant hemisphere resection, a process that may help implement the inter-hemispheric shift of language activation found in fMRI studies. The results indicate that left TLE patients, despite showing more initial white matter damage, have the potential for greater adaptive changes postoperatively than right TLE patients.