Classification of Myopathies on Molecular basis in Drosophila using Raman spectroscopy

Myopathies are muscular diseases in which muscle fibers degenerate due to many factors such as nutrient deficiency, infection and mutations in myofibrillar etc. The objective of this study is to identify the bio-markers to distinguish various muscle mutants in Drosophila (fruit fly) using Raman Spectroscopy. Principal Components based Linear Discriminant Analysis (PC-LDA) classification model yielding >95% accuracy was developed to classify such different mutants representing various myopathies according to their physiopathology.

Sustaining cooperation on networks: an analytical study based on evolutionary game theory

We analytically study the role played by the network topology in sustaining cooperation in a society of myopic agents in an evolutionary setting. In our model, each agent plays the Prisoner's Dilemma (PD) game with its neighbors, as specified by a network. Cooperation is the incumbent strategy, whereas defectors are the mutants. Starting with a population of cooperators, some agents are switched to defection. The agents then play the PD game with their neighbors and compute their fitness. After this, an evolutionary rule, or imitation dynamic is used to update the agent strategy. A defector switches back to cooperation if it has a cooperator neighbor with higher fitness. The network is said to sustain cooperation if almost all defectors switch to cooperation. Earlier work on the sustenance of cooperation has largely consisted of simulation studies, and we seek to complement this body of work by providing analytical insight for the same. We find that in order to sustain cooperation, a network should satisfy some properties such as small average diameter, densification, and irregularity. Real-world networks have been empirically shown to exhibit these properties, and are thus candidates for the sustenance of cooperation. We also analyze some specific graphs to determine whether or not they sustain cooperation. In particular, we find that scale-free graphs belonging to a certain family sustain cooperation, whereas Erdos-Renyi random graphs do not. To the best of our knowledge, ours is the first analytical attempt to determine which networks sustain cooperation in a population of myopic agents in an evolutionary setting.

Structural Insights into Saccharomyces cerevisiae Msh4-Msh5 Complex Function Using Homology Modeling

The Msh4-Msh5 protein complex in eukaryotes is involved in stabilizing Holliday junctions and its progenitors to facilitate crossing over during Meiosis I. These functions of the Msh4-Msh5 complex are essential for proper chromosomal segregation during the first meiotic division. The Msh4/5 proteins are homologous to the bacterial mismatch repair protein MutS and other MutS homologs (Msh2, Msh3, Msh6). Saccharomyces cerevisiae msh4/5 point mutants were identified recently that show two fold reduction in crossing over, compared to wild-type without affecting chromosome segregation. Three distinct classes of msh4/5 point mutations could be sorted based on their meiotic phenotypes. These include msh4/5 mutations that have a) crossover and viability defects similar to msh4/5 null mutants; b) intermediate defects in crossing over and viability and c) defects only in crossing over. The absence of a crystal structure for the Msh4-Msh5 complex has hindered an understanding of the structural aspects of Msh4-Msh5 function as well as molecular explanation for the meiotic defects observed in msh4/5 mutations. To address this problem, we generated a structural model of the S. cerevisiae Msh4-Msh5 complex using homology modeling. Further, structural analysis tailored with evolutionary information is used to predict sites with potentially critical roles in Msh4-Msh5 complex formation, DNA binding and to explain asymmetry within the Msh4-Msh5 complex. We also provide a structural rationale for the meiotic defects observed in the msh4/5 point mutations. The mutations are likely to affect stability of the Msh4/5 proteins and/or interactions with DNA. The Msh4-Msh5 model will facilitate the design and interpretation of new mutational data as well as structural studies of this important complex involved in meiotic chromosome segregation.

Identification of key amino acid residues in the catalytic mechanism of diaminopropionate ammonialyase from Salmonella typhimurium

Diaminopropionate ammonialyase (DAPAL), a fold-typeII pyridoxal 5-phosphate-dependent enzyme, catalyzes the ,-elimination of diaminopropionate (DAP) to pyruvate and ammonia. DAPAL was able to utilize both d- and l-DAP as substrates with almost equal efficiency. Mutational analysis of functionally important residues such as Thr385, Asp125 and Asp194 was carried out to understand the mechanism by which the isomers are hydrolyzed. Further, the putative residues involved in the formation of disulfide bond Cys271 and Cys299 were also mutated. T385S, T385D sDAPAL were as active with dl-DAP as substrate as sDAPAL, whereas the later exhibited a threefold increase in catalytic efficiency with d-Ser as substrate. Further analysis of these mutants suggested that DAPAL might follow an anti-E-2 mechanism of catalysis that does not involve the formation of a quinonoid intermediate. Of the two mutants of Asp125, D125E showed complete loss of activity with d-DAP as substrate, whereas the reaction with l-DAP was not affected significantly, demonstrating that Asp125 was essential for abstraction of protons from the d-isomer. By contrast, mutational analysis of Asp194 showed that the residue may not be directly involved in proton abstraction from l-DAP. sDAPAL does not form a disulfide bond in solution, although the position of Cys299 and Cys271 in the modeled structure of sDAPAL favored the formation of a disulfide bond. Further, unlike eDAPAL, sDAPAL could be activated by monovalent cations. Mutation of the cysteine residues showed that Cys271 may be involved in coordinating the monovalent cation, as observed in the case of other fold-typeII enzymes.

Classification of Myopathies on Molecular basis in Drosophila using Raman spectroscopy

Myopathies are muscular diseases in which muscle fibers degenerate due to many factors such as nutrient deficiency, infection and mutations in myofibrillar etc. The objective of this study is to identify the bio-markers to distinguish various muscle mutants in Drosophila (fruit fly) using Raman Spectroscopy. Principal Components based Linear Discriminant Analysis (PC-LDA) classification model yielding >95% accuracy was developed to classify such different mutants representing various myopathies according to their physiopathology.

Viscoelastic Behavior of Human Lamin A Proteins in the Context of Dilated Cardiomyopathy

Lamins are intermediate filament proteins of type V constituting a nuclear lamina or filamentous meshwork which lines the nucleoplasmic side of the inner nuclear membrane. This protein mesh provides a supporting scaffold for the nuclear envelope and tethers interphase chromosome to the nuclear periphery. Mutations of mainly A-type lamins are found to be causative for at least 11 human diseases collectively termed as laminopathies majority of which are characterised by aberrant nuclei with altered structural rigidity, deformability and poor mechanotransduction behaviour. But the investigation of viscoelastic behavior of lamin A continues to elude the field. In order to address this problem, we hereby present the very first report on viscoelastic properties of wild type human lamin A and some of its mutants linked with Dilated cardiomyopathy (DCM) using quantitative rheological measurements. We observed a dramatic strain-softening effect on lamin A network as an outcome of the strain amplitude sweep measurements which could arise from the large compliance of the quasi-cross-links in the network or that of the lamin A rods. In addition, the drastic stiffening of the differential elastic moduli on superposition of rotational and oscillatory shear stress reflect the increase in the stiffness of the laterally associated lamin A rods. These findings present a preliminary insight into distinct biomechanical properties of wild type lamin A protein and its mutants which in turn revealed interesting differences.

Inactivation of the Bacterial RNA Polymerase Due to Acquisition of Secondary Structure by the omega Subunit

The widely conserved omega subunit encoded by rpoZ is the smallest subunit of Escherichia coli RNA polymerase (RNAP) but is dispensable for bacterial growth. Function of omega is known to be substituted by GroEL in omega-null strain, which thus does not exhibit a discernable phenotype. In this work, we report isolation of omega variants whose expression in vivo leads to a dominant lethal phenotype. Studies show that in contrast to omega, which is largely unstructured, omega mutants display substantial acquisition of secondary structure. By detailed study with one of the mutants, omega(6) bearing N60D substitution, the mechanism of lethality has been deciphered. Biochemical analysis reveals that omega(6) binds to beta ` subunit in vitro with greater affinity than that of omega. The reconstituted RNAP holoenzyme in the presence of omega(6) in vitro is defective in transcription initiation. Formation of a faulty RNAP in the presence of mutant omega results in death of the cell. Furthermore, lethality of omega(6) is relieved in cells expressing the rpoC2112 allele encoding beta ` (2112), a variant beta ` bearing Y457S substitution, immediately adjacent to the beta ` catalytic center. Our results suggest that the enhanced omega(6)-beta ` interaction may perturb the plasticity of the RNAP active center, implicating a role for omega and its flexible state.

A rapid, efficient, and economical inverse polymerase chain reaction-based method for generating a site saturation mutant library

With the development of deep sequencing methodologies, it has become important to construct site saturation mutant (SSM) libraries in which every nucleotide/codon in a gene is individually randomized. We describe methodologies for the rapid, efficient, and economical construction of such libraries using inverse polymerase chain reaction (PCR). We show that if the degenerate codon is in the middle of the mutagenic primer, there is an inherent PCR bias due to the thermodynamic mismatch penalty, which decreases the proportion of unique mutants. Introducing a nucleotide bias in the primer can alleviate the problem. Alternatively, if the degenerate codon is placed at the 5' end, there is no PCR bias, which results in a higher proportion of unique mutants. This also facilitates detection of deletion mutants resulting from errors during primer synthesis. This method can be used to rapidly generate SSM libraries for any gene or nucleotide sequence, which can subsequently be screened and analyzed by deep sequencing. (C) 2013 Elsevier Inc. All rights reserved.

MazF-induced Growth Inhibition and Persister Generation in Escherichia coli

Background: MazEF is a chromosomally encoded bacterial toxin-antitoxin system whose cellular role is controversial. Results: Expression of chromosomal MazF inhibits cell killing by multiple antibiotics in a Lon and ClpP dependent manner. Conclusion: MazF is involved in reversible growth inhibition and bacterial drug tolerance. Significance: Inactive, active-site toxin mutants yield functional insights by selectively activating the corresponding WT toxin in vivo. Toxin-antitoxin systems are ubiquitous in nature and present on the chromosomes of both bacteria and archaea. MazEF is a type II toxin-antitoxin system present on the chromosome of Escherichia coli and other bacteria. Whether MazEF is involved in programmed cell death or reversible growth inhibition and bacterial persistence is a matter of debate. In the present work the role of MazF in bacterial physiology was studied by using an inactive, active-site mutant of MazF, E24A, to activate WT MazF expression from its own promoter. The ectopic expression of E24A MazF in a strain containing WT mazEF resulted in reversible growth arrest. Normal growth resumed on inhibiting the expression of E24A MazF. MazF-mediated growth arrest resulted in an increase in survival of bacterial cells during antibiotic stress. This was studied by activation of mazEF either by overexpression of an inactive, active-site mutant or pre-exposure to a sublethal dose of antibiotic. The MazF-mediated persistence phenotype was found to be independent of RecA and dependent on the presence of the ClpP and Lon proteases. This study confirms the role of MazEF in reversible growth inhibition and persistence.

A Histidine Aspartate Ionic Lock Gates the Iron Passage in Miniferritins from Mycobacterium smegmatis

Background: DNA-binding protein from starved cells (Dps) are nano-compartments that can oxidize and store iron rendering protection from free radicals. Results: A histidine-aspartate ionic cluster in mycobaterial Dps2 modulates the rate of iron entry and exit in these proteins. Conclusion: Substitutions that disrupt the cluster interface alter the iron uptake/release properties with localized structural changes. Significance: Identifying important gating residues can help in designing nano-delivery vehicles. Dps (DNA-binding protein from starved cells) are dodecameric assemblies belonging to the ferritin family that can bind DNA, carry out ferroxidation, and store iron in their shells. The ferritin-like trimeric pore harbors the channel for the entry and exit of iron. By representing the structure of Dps as a network we have identified a charge-driven interface formed by a histidine aspartate cluster at the pore interface unique to Mycobacterium smegmatis Dps protein, MsDps2. Site-directed mutagenesis was employed to generate mutants to disrupt the charged interactions. Kinetics of iron uptake/release of the wild type and mutants were compared. Crystal structures were solved at a resolution of 1.8-2.2 for the various mutants to compare structural alterations vis a vis the wild type protein. The substitutions at the pore interface resulted in alterations in the side chain conformations leading to an overall weakening of the interface network, especially in cases of substitutions that alter the charge at the pore interface. Contrary to earlier findings where conserved aspartate residues were found crucial for iron release, we propose here that in the case of MsDps2, it is the interplay of negative-positive potentials at the pore that enables proper functioning of the protein. In similar studies in ferritins, negative and positive patches near the iron exit pore were found to be important in iron uptake/release kinetics. The unique ionic cluster in MsDps2 makes it a suitable candidate to act as nano-delivery vehicle, as these gated pores can be manipulated to exhibit conformations allowing for slow or fast rates of iron release.

Rad51-Rad52 Mediated Maintenance of Centromeric Chromatin in Candida albicans

Specification of the centromere location in most eukaryotes is not solely dependent on the DNA sequence. However, the non-genetic determinants of centromere identity are not clearly defined. While multiple mechanisms, individually or in concert, may specify centromeres epigenetically, most studies in this area are focused on a universal factor, a centromere-specific histone H3 variant CENP-A, often considered as the epigenetic determinant of centromere identity. In spite of variable timing of its loading at centromeres across species, a replication coupled early S phase deposition of CENP-A is found in most yeast centromeres. Centromeres are the earliest replicating chromosomal regions in a pathogenic budding yeast Candida albicans. Using a 2-dimensional agarose gel electrophoresis assay, we identify replication origins (ORI7-LI and ORI7-RI) proximal to an early replicating centromere (CEN7) in C. albicans. We show that the replication forks stall at CEN7 in a kinetochore dependent manner and fork stalling is reduced in the absence of the homologous recombination (HR) proteins Rad51 and Rad52. Deletion of ORI7-RI causes a significant reduction in the stalled fork signal and an increased loss rate of the altered chromosome 7. The HR proteins, Rad51 and Rad52, have been shown to play a role in fork restart. Confocal microscopy shows declustered kinetochores in rad51 and rad52 mutants, which are evidence of kinetochore disintegrity. CENP-A(CaCse4) levels at centromeres, as determined by chromatin immunoprecipitation (ChIP) experiments, are reduced in absence of Rad51/Rad52 resulting in disruption of the kinetochore structure. Moreover, western blot analysis reveals that delocalized CENP-A molecules in HR mutants degrade in a similar fashion as in other kinetochore mutants described before. Finally, co-immunoprecipitation assays indicate that Rad51 and Rad52 physically interact with CENP-A(CaCse4) in vivo. Thus, the HR proteins Rad51 and Rad52 epigenetically maintain centromere functioning by regulating CENP-A(CaCse4) levels at the programmed stall sites of early replicating centromeres.

Involvement of S-cerevisiae Rpb4 in subset of pathways related to transcription elongation

Yeast Rpb4, a subunit of RNA pol II is not essential for viability but is involved in multiple cellular phenotypes such as temperature sensitivity, enhanced pseudohyphal morphology, and decreased sporulation. Both in vivo and in vitro studies strongly support involvement of Rpb4 in transcription initiation, while its role in transcription elongation is not entirely consistent. Here we show that Rpb4 is not required for recruitment of RNA pol II on the coding region of YLR454w, a representative long gene. Yet we find strong genetic interaction of rpb4 Delta with mutants in many transcription elongation factors such as Paf1, Spt4, Dst1, Elp3 and Rpb9. We demonstrate that, Rpb4 interacts functionally with Paf1 to affect the transcription elongation of the FKS1 gene. Our results suggest that while Rpb4 is not required for general transcription elongation, it could support transcription elongation for specific of class of genes by interaction with other elongation factors. (C) 2014 Elsevier B.V. All rights reserved.

The polycomb group gene EMF2B is essential for maintenance of floral meristem determinacy in rice

Polycomb Repressive Complex 2 (PRC2) represses the transcriptional activity of target genes through trimethylation of lysine 27 of histone H3. The functions of plant PRC2 have been chiefly described in Arabidopsis, but specific functions in other plant species, especially cereals, are still largely unknown. Here we characterize mutants in the rice EMF2B gene, an ortholog of the Arabidopsis EMBRYONIC FLOWER2 (EMF2) gene. Loss of EMF2B in rice results in complete sterility, and mutant flowers have severe floral organ defects and indeterminacy that resemble loss-of-function mutants in E-function floral organ specification genes. Transcriptome analysis identified the E-function genes OsMADS1, OsMADS6 and OsMADS34 as differentially expressed in the emf2b mutant compared with wild type. OsMADS1 and OsMADS6, known to be required for meristem determinacy in rice, have reduced expression in the emf2b mutant, whereas OsMADS34 which interacts genetically with OsMADS1 was ectopically expressed. Chromatin immunoprecipitation for H3K27me3 followed by quantitative (q)RT-PCR showed that all three genes are presumptive targets of PRC2 in the meristem. Therefore, in rice, and possibly other cereals, PRC2 appears to play a major role in floral meristem determinacy through modulation of the expression of E-function genes.

Genome-wide methylation profiling identifies an essential role of reactive oxygen species in pediatric glioblastoma multiforme and validates a methylome specific for H3 histone family 3A with absence of G-CIMP/isocitrate dehydrogenase 1 mutation

Background. Pediatric glioblastoma multiforme (GBM) is rare, and there is a single study, a seminal discovery showing association of histone H3.3 and isocitrate dehydrogenase (IDH) 1 mutation with a DNA methylation signature. The present study aims to validate these findings in an independent cohort of pediatric GBM, compare it with adult GBM, and evaluate the involvement of important functionally altered pathways. Methods. Genome-wide methylation profiling of 21 pediatric GBM cases was done and compared with adult GBM data (GSE22867). We performed gene mutation analysis of IDH1 and H3 histone family 3A (H3F3A), status evaluation of glioma cytosine-phosphate-guanine island methylator phenotype (G-CIMP), and Gene Ontology analysis. Experimental evaluation of reactive oxygen species (ROS) association was also done. Results. Distinct differences were noted between methylomes of pediatric and adult GBM. Pediatric GBM was characterized by 94 hypermethylated and 1206 hypomethylated cytosine-phosphate-guanine (CpG) islands, with 3 distinct clusters, having a trend to prognostic correlation. Interestingly, none of the pediatric GBM cases showed G-CIMP/IDH1 mutation. Gene Ontology analysis identified ROS association in pediatric GBM, which was experimentally validated. H3F3A mutants (36.4%; all K27M) harbored distinct methylomes and showed enrichment of processes related to neuronal development, differentiation, and cell-fate commitment. Conclusions. Our study confirms that pediatric GBM has a distinct methylome compared with that of adults. Presence of distinct clusters and an H3F3A mutation-specific methylome indicate existence of epigenetic subgroups within pediatric GBM. Absence of IDH1/G-CIMP status further indicates that findings in adult GBM cannot be simply extrapolated to pediatric GBM and that there is a strong need for identification of separate prognostic markers. A possible role of ROS in pediatric GBM pathogenesis is demonstrated for the first time and needs further evaluation.

Raman Spectroscopic Studies on Screening of Myopathies

Myopathies are among the major causes of mortality in the world. There is no complete cure for this heterogeneous group of diseases, but a sensitive, specific, and fast diagnostic tool may improve therapy effectiveness. In this study, Raman spectroscopy is applied to discriminate between muscle mutants in Drosophila on the basis of associated changes at the molecular level. Raman spectra were collected from indirect flight muscles of mutants, upheld1 (up1), heldup(2) (hdp(2)), myosin heavy chain7 (Mhc7), actin88F(KM88) (Act88F(KM88)), upheld101 (up101), and Canton-S (CS) control group, for both 2 and 12 days old flies. Difference spectra (mutant minus control) of all the mutants showed an increase in nucleic acid and beta-sheet and/or random coil protein content along with a decrease in a-helix protein. Interestingly, the 12th day samples of up1 and Act88F(KM88) showed significantly higher levels of glycogen and carotenoids than CS. A principal components based linear discriminant analysis classification model was developed based on multidimensional Raman spectra, which classified the mutants according to their pathophysiology and yielded an overall accuracy of 97% and 93% for 2 and 12 days old flies, respectively. The up1 and Act88F(KM88) (nemaline-myopathy) mutants form a group that is clearly separated in a linear discriminant plane from up101 and hdp2 (cardiomyopathy) mutants. Notably, Raman spectra from a human sample with nemaline-myopathy formed a cluster with the corresponding Drosophila mutant (up1). In conclusion, this is the first demonstration in which myopathies, despite their heterogeneity, were screened on the basis of biochemical differences using Raman spectroscopy.