Tracking the processing of damaged DNA double-strand break ends by ligation-mediated PCR: increased persistence of 3′-phosphoglycolate termini in SCAN1 cells

To track the processing of damaged DNA double-strand break (DSB) ends in vivo, a method was devised for quantitative measurement of 3′-phosphoglycolate (PG) termini on DSBs induced by the non-protein chromophore of neocarzinostatin (NCS-C) in the human Alu repeat. Following exposure of cells to NCS-C, DNA was isolated, and labile lesions were chemically stabilized. All 3′-phosphate and 3′-hydroxyl ends were enzymatically capped with dideoxy termini, whereas 3′-PG ends were rendered ligatable, linked to an anchor, and quantified by real-time Taqman polymerase chain reaction. Using this assay and variations thereof, 3′-PG and 3′-phosphate termini on 1-base 3′ overhangs of NCS-C-induced DSBs were readily detected in DNA from the treated lymphoblastoid cells, and both were largely eliminated from cellular DNA within 1 h. However, the 3′-PG termini were processed more slowly than 3′-phosphate termini, and were more persistent in tyrosyl-DNA phosphodiesterase 1-mutant SCAN1 than in normal cells, suggesting a significant role for tyrosyl-DNA phosphodiesterase 1 in removing 3′-PG blocking groups for DSB repair. DSBs with 3′-hydroxyl termini, which are not directly induced by NCS-C, were formed rapidly in cells, and largely eliminated by further processing within 1 h, both in Alu repeats and in heterochromatic α-satellite DNA. Moreover, absence of DNA-PK in M059J cells appeared to accelerate resolution of 3′-PG ends.     

Molecular Evidence of Increased Resistance to Anti-Folate Drugs in Plasmodium falciparum in North-East India: A Signal for Potential Failure of Artemisinin Plus Sulphadoxine-Pyrimethamine Combination Therapy

North-east India, being a corridor to South-east Asia, is believed to play an important role in transmitting drug resistant Plasmodium falciparum malaria to India and South Asia. North-east India was the first place in India to record the emergence of drug resistance to chloroquine as well as sulphadoxine/pyrimethamine. Presently chloroquine resistance is widespread all over the North-east India and resistance to other anti-malarials is increasing. In this study both in vivo therapeutic efficacy and molecular assays were used to screen the spectrum of drug resistance to chloroquine and sulphadoxine/pyrimethamine in the circulating P. falciparum strains. A total of 220 P. falciparum positives subjects were enrolled in the study for therapeutic assessment of chloroquine and sulphadoxine/pyrimethamine and assessment of point mutations conferring resistances to these drugs were carried out by genotyping the isolates following standard methods. Overall clinical failures in sulphadoxine/pyrimethamine and chloroquine were found 12.6 and 69.5% respectively, while overall treatment failures recorded were 13.7 and 81.5% in the two arms. Nearly all (99.0%) the isolates had mutant pfcrt genotype (76T), while 68% had mutant pfmdr-1 genotype (86Y). Mutation in dhps 437 codon was the most prevalent one while dhfr codon 108 showed 100% mutation. A total of 23 unique haplotypes at the dhps locus and 7 at dhfr locus were found while dhps-dhfr combined loci revealed 49 unique haplotypes. Prevalence of double, triple and quadruple mutations were common while 1 haplotype was found with all five mutated codons (F/AGEGS/T) at dhps locus. Detection of quadruple mutants (51I/59R/108N/164L) in the present study, earlier recorded from Car Nicobar Island, India only, indicates the presence of high levels of resistance to sulphadoxine/pyrimethamine in north-east India. Associations between resistant haplotypes and the clinical outcomes and emerging resistance in sulphadoxine/pyrimethamine in relation to the efficacy of the currently used artemisinin combination therapy are discussed.     

Gene Regulation by the LiaSR Two-Component System in Streptococcus mutans

The LiaSR two-component signal transduction system regulates cellular responses to several environmental stresses, including those that induce cell envelope damages. Downstream regulons of the LiaSR system have been implicated in tolerance to acid, antibiotics and detergents. In the dental pathogen Streptococcus mutans, the LiaSR system is necessary for tolerance against acid, antibiotics, and cell wall damaging stresses during growth in the oral cavity. To understand the molecular mechanisms by which LiaSR regulates gene expression, we created a mutant LiaR in which the conserved aspartic acid residue (the phosphorylation site), was changed to alanine residue (D58A). As expected, the LiaR-D58A variant was unable to acquire the phosphate group and bind to target promoters. We also noted that the predicted LiaR-binding motif upstream of the lia operon does not appear to be well conserved. Consistent with this observation, we found that LiaR was unable to bind to the promoter region of lia; however, we showed that LiaR was able to bind to the promoters of SMU.753, SMU.2084 and SMU.1727. Based on sequence analysis and DNA binding studies we proposed a new 25-bp conserved motif essential for LiaR binding. Introducing alterations at fully conserved positions in the 25-bp motif affected LiaR binding, and the binding was dependent on the combination of positions that were altered. By scanning the S. mutans genome for the occurrence of the newly defined LiaR binding motif, we identified the promoter of hrcA (encoding a key regulator of the heat shock response) that contains a LiaR binding motif, and we showed that hrcA is negatively regulated by the LiaSR system. Taken together our results suggest a putative role of the LiaSR system in heat shock responses of S. mutans.     

Comparative evolutionary genomics of <Emphasis Type="Italic">Corynebacterium</Emphasis> with special reference to codon and amino acid usage diversities

The present study has been aimed to the comparative analysis of high GC composition containing Corynebacterium genomes and their evolutionary study by exploring codon and amino acid usage patterns. Phylogenetic study by MLSA approach, indel analysis and BLAST matrix differentiated Corynebacterium species in pathogenic and non-pathogenic clusters. Correspondence analysis on synonymous codon usage reveals that, gene length, optimal codon frequencies and tRNA abundance affect the gene expression of Corynebacterium. Most of the optimal codons as well as translationally optimal codons are C ending i.e. RNY (R-purine, N-any nucleotide base, and Y-pyrimidine) and reveal translational selection pressure on codon bias of Corynebacterium. Amino acid usage is affected by hydrophobicity, aromaticity, protein energy cost, etc. Highly expressed genes followed the cost minimization hypothesis and are less diverged at their synonymous positions of codons. Functional analysis of core genes shows significant difference in pathogenic and non-pathogenic Corynebacterium. The study reveals close relationship between non-pathogenic and opportunistic pathogenic Corynebaterium as well as between molecular evolution and survival niches of the organism.     

Leishmania donovani inhibits ferroportin translation by modulating FBXL5‐IRP2 axis for its growth within host macrophages

Hepcidin mediated ferroportin (Fpn) degradation in macrophages is a well adopted strategy to limit iron availability towards invading pathogens. Leishmania donovani (LD), a protozoan parasite, resides within macrophage and competes with host for availing iron. Using in vitro and in vivo model of infection, we reveal that LD decreases Fpn abundance in host macrophages by hepcidin independent mechanism. Unaffected level of Fpn‐FLAG in LD infected J774 macrophage confirms that Fpn down‐regulation is not due its degradation. While increased Fpn mRNA but decreased protein expression in macrophages suggests blocking of Fpn translation by LD infection that is confirmed by ³⁵S‐methionine labelling assay. We further reveal that LD blocks Fpn translation by induced binding of iron regulatory proteins (IRPs) to the iron responsive element present in its 5′UTR. Supershift analysis provides evidence of involvement of IRP2 particularly during in vivo infection. Accordingly, a significant increase in IRP2 protein expression with simultaneous decrease in its stability regulator F‐box and leucine‐rich repeat Protein 5 (FBXL5) is detected in splenocytes of LD‐infected mice. Increased intracellular growth due to compromised expressions of Fpn and FBXL5 by specific siRNAs reveals that LD uses a novel strategy of manipulating IRP2‐FBXL5 axis to inhibit host Fpn expression.     

Polymerase chain reaction engineering

A mathematical model for polymerase chain reaction (PCR) is developed, taking into account the three steps in this process: melting of DNA; primer annealing; and DNA synthesis (polymerization). Activity and deactivation of the polymerase enzyme as a function of temperature is incorporated in the kinetic model to get a better understanding of the amplification of DNA. Computer simulation of the model is carried out to determine the effects of various parameters, such as the cycle number, initial DNA concentration (copynumber), initial enzyme concentration, extension time, temperature ramp, and enzyme deactivation on the DNA generation. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 359-366, 1997.     

Uranium luminescence in La2Zr2O7: effect of concentration and annealing temperature

The speciation of a particular element in any given matrix is a prerequisite to understanding its solubility and leaching properties. In this context, speciation of uranium in lanthanum zirconate pyrochlore (La₂Zr₂O₇ = LZO), prepared by a low‐temperature combustion route, was carried out using a simple photoluminescence lifetime technique. The LZO matrix is considered to be a potential ceramic host for fixing nuclear and actinide waste products generated during the nuclear fuel cycle. Special emphasis has been given to understanding the dynamics of the uranium species in the host as a function of annealing temperature and concentration. It was found that, in the LZO host, uranium is stabilized as the commonly encountered uranyl species (UO₂²⁺) up to a heat treatment of 500 °C at the surface. Above 500 °C, the uranyl ion is diffused into the matrix as the more symmetric octahedral uranate species (UO₆^6–). The uranate ions thus formed replace the six‐coordinated ‘Zr’ atoms at regular lattice positions. Further, it was observed that concentration quenching takes place beyond 5 mol% of uranium doping. The mechanism of the quenching was found to be a multipolar interaction. Copyright © 2016 John Wiley & Sons, Ltd.     

Finding neoepitopes in mouse models of personalized cancer immunotherapy

Background

Cancer immunotherapy uses one’s own immune system to fight cancerous cells. As immune system is hard-wired to distinguish self and non-self, cancer immunotherapy is predicted to target cancerous cells specifically, therefore is less toxic than chemotherapy and radiation therapy, two major treatments for cancer. Cancer immunologists have spent decades to search for the specific targets in cancerous cells.

Methods

Due to the recent advances in high throughput sequencing and bioinformatics, evidence has merged that the neoantigens in cancerous cells are probably the cancer-specific targets that lead to the destruction of cancer.We will review the transplantable murine tumor models for cancer immunotherapy and the bioinformatics tools used to navigate mouse genome to identify tumor-rejecting neoantigens.

Results

Several groups have independently identified point mutations that can be recognized by T cells of host immune system. It is consistent with the note that the formation of peptide-MHC I-TCR complex is critical to activate T cells. Both anchor residue and TCR-facing residue mutations have been reported. While TCR-facing residue mutations may directly activate specific T cells, anchor residue mutations improve the binding of peptides to MHC I molecules, which increases the presentation of peptides and the T cell activation indirectly.

Conclusions

Our work indicates that the affinity of neoepitopes for MHC I is not a predictor for anti-tumor immune responses in mice. Instead differential agretopic index (DAI), the numerical difference of epitope-MHC I affinities between the mutated and un-mutated sequences is a significant predictor. A similar bioinformatics pipeline has been developed to generate personalized vaccines to treat human ovarian cancer in a Phase I clinical trial.