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AbstractThe olfactory system is capable of detecting and distinguishing thousands of environmental odorants that play a key role in reproduction, social behaviours including pheromones influenced classical events. Membrane secretary odorant binding proteins (OBPs) are soluble lipocalins, localized in the nasal membrane of mammals. They bind and carry odorants within the nasal epithelium to putative olfactory transmembrane receptors (ORs). OBP has not yet been exploited to develop a suitable technique to detect oestrus which is being reported as a difficult task in buffalo. In the present study, using molecular biology and protein engineering approaches, we have cloned six novel OBP isoforms from buffalo nasal epithelium odorant-binding proteins (bnOBPs). Furthermore, 3 D models were developed and molecular-docking, dynamics experiments were performed by in silicoapproaches. In particular, we found four residues (Phe104, Phe134, Phe69 and Asn118) in OBP1a, which contributed to favourable interactions towards two sex pheromones, specifically oleic acid and p-cresol. We expressed this protein in Escherichia colifrom female buffalo urine and validated through fluorescence quenching studies to show similar strong binding affinities of OBP1a to oleic acid and p-cresol. By using structural data, the binding specificity was also verified by site-directed mutagenesis of the four residues followed by in vitrobinding assays. Our results enable us to better understand the functions of different nasal epithelium OBP isoforms in buffaloes. They also lead to improved understanding of the interaction between olfactory proteins and odorants to develop highly selective biosensing devices for non-invasive detection of oestrus in buffaloes. Communicated by Ramaswamy H. Sarma

The ongoing COVID-19 pandemic highlights the need to tackle viral variants, expand the number of antigens, and assess diverse delivery systems for vaccines against emerging viruses. In the present study, a DNA vaccine candidate was generated by combining in tandem envelope protein domain III (EDIII) of dengue virus serotypes 1–4 and a dengue virus (DENV)-2 non-structural protein 1 (NS1) protein-coding region. Each domain was designed as a serotype-specific consensus coding sequence derived from different genotypes based on the whole genome sequencing of clinical isolates in India and complemented with data from Africa. This sequence was further optimized for protein expression. In silicostructural analysis of the EDIII consensus sequence revealed that epitopes are structurally conserved and immunogenic. The vaccination of mice with this construct induced pan-serotype neutralizing antibodies and antigen-specific T cell responses. Assaying intracellular interferon (IFN)-γ staining, immunoglobulin IgG2(a/c)/IgG1 ratios, and immune gene profiling suggests a strong Th1-dominant immune response. Finally, the passive transfer of immune sera protected AG129 mice challenged with a virulent, non-mouse-adapted DENV-2 strain. Our findings collectively suggest an alternative strategy for dengue vaccine design by offering a novel vaccine candidate with a possible broad-spectrum protection and a successful clinical translation either as a stand alone or in a mix and match strategy.

Neurotoxic aggregation of β-amyloid (Aβ) peptides is a hallmark of Alzheimer’s disease and increased reactive oxygen species (ROS) is an associated process. In the present study, we report the neuroprotective effects of disulfide-rich, circular peptides from Clitoria ternatea(C. ternatea) (butterfly pea) on Aβ-induced toxicity in transgenic Caenorhabditis elegans. Cyclotides (∼30 amino acids long) are a special class of cyclic cysteine knot peptides. We show that cyclotide-rich fractions from different plant tissues delay Aβ-induced paralysis in the transgenic CL4176 strain expressing the human muscle-specific Aβ1–42gene. They also improved Aβ-induced chemotaxis defects in CL2355 strain expressing Aβ1–42in the neuronal cells. ROS assay suggests that this protection is likely mediated by the inhibition of Aβ oligomerization. Furthermore, Aβ deposits were reduced in the CL2006 strain treated with the fractions. The study shows that cyclotides from C. ternateacould be a source of a novel pharmacophore scaffold against neurodegenerative diseases.

Background: The eukaryotic voltage-gated sodium channel(e-Nav) is a large asymmetric transmembrane protein with important functions concerning neurological function. No structure has been resolved at high resolution for this protein. Methods: A homology model of the transmembrane and extracellular regions of an Anopheles gambiae para-like channel with emphasis on the pore entrance has been constructed, based upon the templates provided by a prokaryotic sodium channel and a potassium two-pore channel. The latter provides a template for the extracellular regions, which are located above the entrance to the pore, which is likely to open at a side of a dome formed by these loops. Results: A model created with this arrangement shows a structure similar to low-resolution cryoelectron microscope images of a related structure. The pore entrance also shows favorable electrostatic interface. Conclusion: Residues responsible for the negative charge around the pore have been traced in phylogeny to highlight their importance. This model is intended for the study of pore-blocking toxins.

Myotubularin or MTM1 is a lipid phosphatase that regulates vesicular trafficking in the cell. The MTM1gene is mutated in a severe form of muscular disease, X-linked myotubular myopathy or XLMTM, affecting 1 in 50,000 newborn males worldwide. There have been several studies on the disease pathology of XLMTM, but the structural effects of missense mutations of MTM1 are underexplored due to the unavailability of a crystal structure. MTM1 consists of three domains-a lipid-binding N-terminal GRAM domain, the phosphatase domain and a coiled-coil domain which aids dimerisation of Myotubularin homologs. While most mutations reported to date map to the phosphatase domain of MTM1, the other two domains on the sequence are also frequently mutated in XLMTM. To understand the overall structural and functional effects of missense mutations on MTM1, we curated several missense mutations and performed in silicoand in vitrostudies. Apart from significantly impaired binding to substrate, abrogation of phosphatase activity was observed for a few mutants. Possible long-range effects of mutations from non-catalytic domains on phosphatase activity were observed as well. Coiled-coil domain mutants have been characterised here for the first time in XLMTM literature.

Precise functioning and fine-tuning of Toll-like receptor 4 (TLR4) signaling is a critical requirement for the smooth functioning of the innate immune system, since aberrant TLR4 activation causes excessive production of pro-inflammatory cytokines and interferons. This can result in life threatening conditions such as septic shock and other inflammatory disorders. The TRIF-related adaptor molecule (TRAM) adaptor protein is unique to the TLR4 signaling pathway and abrogation of TRAM-mediated TLR4 signaling is a promising strategy for developing therapeutics aimed at disrupting TRAM interactions with other components of the TLR4 signaling complex. The VIPER motif from the vaccinia virus-producing protein, A46 has been reported to disrupt TRAM-TLR4 interactions. We have exploited this information, in combination with homology modeling and docking approaches, to identify a potential binding site on TRAM lined by the BB loop and αC helix. Virtual screening of commercially available small molecules targeting the binding site enabled to short-list 12 small molecules to abrogate TRAM-mediated TLR4 signaling. Molecular dynamics and molecular mechanics calculations have been performed for the analysis of these receptor-ligand interactions.

Peptide-mediated immunity against pathogens in plants can provide information on proteinpeptide interactions and drug discovery in general. The molecular structure of AtPep1, a 23-amino acid signaling peptide isolated from Arabidopsis thaliana leaves and implicated in innate immunity, has evaded structural determination by biophysical methods. The details of molecular interaction of AtPep1 peptide with its receptor (PEPR1), a 170 kDa leucine-rich repeat (LRR) kinase is also unknown. We report a computational approach to the modeling AtPep1 by conformational sampling and its interaction with the receptor PEPR1. Molecular dynamics simulations were employed to sample and cluster energetically favorable conformations of AtPep1 and modeling of PEPR1 through homology. Docking of AtPep1 to PEPR1 and filtering of the biologically relevant poses were facilitated by the computational Ala-scanning mutations and binding energy analysis of the peptide-protein complex. This study provides the first independent in silico validation of the Structure-Activity- Relationship studies carried out on the AtPep1 and provides a molecular mechanism of the peptide-protein complex system.

Chemical recognition plays an important role for the survival and reproduction of many insect species. Odorant binding proteins (OBPs) are the primary components of the insect olfactory mechanism and have been documented to play an important role in the host-seeking mechanism of mosquitoes. They are “transport proteins” believed to transport odorant molecules from the external environment to their respective membrane targets, the olfactory receptors. The mechanism by which this transport occurs in mosquitoes remains a conundrum in this field. Nevertheless, OBPs have proved to be amenable to conformational changes mediated by a pH change in other insect species. In this paper, the effect of pH on the conformational flexibility of mosquito OBPs is assessed computationally using molecular dynamics simulations of a mosquito OBP “CquiOBP1” bound to its pheromone 3OG (PDB ID: 3OGN). Conformational twist of a loop, driven by a set of well-characterized changes in intramolecular interactions of the loop, is demonstrated. The concomitant (i) closure of what is believed to be the entrance of the binding pocket, (ii) expansion of what could be an exit site, and (iii) migration of the ligand towards this putative exit site provide preliminary insights into the mechanism of ligand binding and release of these proteins in mosquitoes. The correlation of our results with previous experimental observations based on NMR studies help us provide a cardinal illustration on one of the probable dynamics and mechanism by which certain mosquito OBPs could deliver their ligand to their membrane-bound receptors at specific pH conditions.

3D domain swapping is a protein structural phenomenon that mediates the formation of the higher order oligomers in a variety of proteins with different structural and functional properties. 3D domain swapping is associated with a variety of biological functions ranging from oligomerization to pathological conformational diseases. 3D domain swapping is realised subsequent to structure determination where the protein is observed in the swapped conformation in the oligomeric state. This is a limiting step to understand this important structural phenomenon in a large scale from the growing sequence data. A new machine learning approach, 3dswap-pred, has been developed for the prediction of 3D domain swapping in protein structures from mere sequence data using the Random Forest approach. 3Dswap-pred is implemented using a positive sequence dataset derived from literature based structural curation of 297 structures. A negative sequence dataset is obtained from 462 SCOP domains using a new sequence data mining approach and a set of 126 sequencederived features. Statistical validation using an independent dataset of 68 positive sequences and 313 negative sequences revealed that 3dswap-pred achieved an accuracy of 63.8. A webserver is also implemented using the 3dswap-pred Random Forest model. The server is available from the URL: http://caps.ncbs.res.in/3dswap-pred

A detailed comparison of protein domains that belong to families and superfamilies shows that structure is better conserved than sequence during evolutionary divergence. Sequence alignments, guided by structural features, permit a better sampling of the protein sequence space and effective construction of libraries for fold recognition. Sequence alignments are useful evolutionary models in defining structure-function relationships for protein superfamilies. The PASS2 database, maintained by the authors, presents alignments of proteins related at the superfamily level and characterised by low sequence similarity. The number of new superfamilies increased to 47% compared with the previous PASS2 version, which shows the crucial importance of updating the PASS2 database. In the current release of the PASS2 database, they align protein superfamilies using a structural alignment protocol. The authors also introduce two alignment assessment methods that depend on the average structural deviations of domains and the extent of conserved secondary structures. They also integrate new and important structural and sequence features at the superfamily level into the database. These features are conserved-unconserved blocks in proteins, spatial distribution of sequences using principal component analysis and a statistical view for each superfamily. The authors suggest that highly structurally deviant superfamily members could be removed as outliers, so that such extreme distant relationships will not obscure the alignment. They report a nearly-automated, updated version of the superfamily alignment database, consisting of 1776 superfamilies and 9536 protein domains, that is in direct correspondence with the SCOP (1.73) database.

AbstractBax is a pro-apoptotic member of the widely studied Bcl-2 family of apoptotic proteins. Its function is regulated by anti-apoptotic members of the same family. Bcl-xLis one such member, which plays a key role in inhibiting the function of Bax. Recent experimental evidences suggest that intra-molecular conformational changes in the all-helical fold of Bax are necessary for it to be amenable to regulation by Bcl-xL, principal among these being proposed interactions between the N-terminus and α5, α6(transmembrane TM1.1 and TM1.2) of Bax. The present study is a detailed molecular dynamics investigation of Bax in an aqueous environment, in order to better understand the nature of intra-molecular conformational changes it undergoes before it translocates and inserts into the mitochondrial membrane. A distinct movement of the N-terminal end is observed in a 100ns production run of Bax. Fluctuations across domains are compared for simulations in full-length and deletion mutants of Bax. A series of hydrogen bonding patterns across N-terminal region and α7,8(BH2 domain) is observed during the simulations. BH2 domain, in turn, acquires new hydrogen bond interactions with TM1 helices. The analysis further revealed other hydrogen bond interactions, across crucial domains in Bax, which are mediated by water molecules across the length of simulation. The structural alliance between N-terminal region and BH2 domain suggests a structural transition cascade leading to the dislocation of TM helices away from hydrophobic interactions that normally prevail with the BH3 domain in the cytosolic form of Bax.

AbstractNeurotransmitter serotonin (5-hydroxytryptamine, 5-HT) an ancient neurotransmitter, involved in several neurophysiological and behavioral functions, acts by interacting with multiple receptors (5-HT1-5-HT7). Alterations in serotonergic signalling have also been implicated in various psychiatric disorders. The availability of the genome data of nonhuman primates permits comparative analysis of human 5-HT receptors with sequences of non-human primates to understand evolutionary divergence. We compared and analyzed serotonergic receptor sequences from human and non-human primates. Phylogenetic analysis by Maximum Likelihood (ML) method classified human and primate 5-HT receptors into six unique clusters. There was considerable conservation of 5-HT receptor sequences between human and non-human primates; however, a greater diversity at the sub-group level was observed. Compared to the other subgroups, larger multiplicity and expansion was seen within the 5-HT4receptor subtype in both human and non-human primates. Analysis of non-synonymous and synonymous substitution ratios (Ka/Ks ratio) using the Nei-Gojobori method suggests that 5-HT receptor sequences have undergone negative (purifying) selection over the course of evolution in human, chimpanzee and rhesus monkey. Abnormal human and non-human primate psychopathalogy and behavior, in the context of these variations is discussed. Analysis of these 5-HT receptors in other species will help understand the molecular evolution of 5-HT receptors, and its possible influence on complex behaviors, and psychiatric disorders.

Purpose: A number of proteome studies have been performed recently to identify pheromone-related protein expression and their molecular function using genetically modified rodents urine. However, no such studies have used Indian commensal rodents; interestingly, in a previous investigation, we confirmed the presence of volatile molecules in commensal rodents urine and these molecules seem to be actively involved in pheromonal communication. Therefore, the present study aims to identify the major urinary protein [MUP] present in commensal rat urine, which will help us to understand the proteins expression pattern and intrinsic properties among the rodents globally. Experimental Design: Initially, the total urinary proteins were separated by 1-D and 2-D electrophoresis and then subsequently analyzed by Matrix Assisted Laser Desorption Ionization-Time of Flight and Mass Spectrometer (MALDITOF/ MS). Furthermore, they were then fragmented with the aid of a Tandem Mass Spectrometer (TOF/TOF) and the identified sequences aligned and confirmed using similarity with the deduced primary structures of members of the lipocalin superfamily. Results: The SDS-PAGE protein profiles showed distinct proteins with molecular masses of 15, 22.4, 25, 28, 42, 50, 55, 68, and 91 kDa. Of these proteins, the 22.4 kDa protein was considered to be target candidate. When 2D electrophoresis was carried out, about ~50 spots were detected with different masses and various pI ranges. The 22.4 kDa protein was found to have a pI of about 4.9. This 22.4 kDa protein spot was digested and subjected to mass spectrometry; it was identified as rat MUP. The fragmented peptides from the rat MUP at 935, 1026, 1192, and 1303 m/z were further fragmented with the aid of MS/MS and generated de novo sequence and this confirmed this protein to be the MUP present in the urine of commensal rats. Conclusion: The present investigation confirms the presence of MUP with a molecular mass of 22.4 kDa in the urine of commensal rats. This protein may be involved in the binding of volatile molecules and opens up a discussion about how volatile and non-volatile molecules in the commensal rats urine may contribute chemo-communic

Abstract: The antagonist-bound conformation of the NR2A and NR2B subunits of N-methyl-D-aspartate (NMDA) ionotropic glutamate receptor are modeled using the crystal structure of the DCKA (5,7-dichloro-kynurenic acid)-bound form of the NR1 subunit ligand-binding core (S1S2). Five different competitive NMDA receptor antagonists [(1) DL-AP5; (2) DL-AP7; (3) CGP-37847; (4) CGP 39551; (5) (RS)-CPP] have been docked into both NR2A and NR2B subunits. Experimental studies report NR2A and NR2B subunits having dissimilar interactions and affinities towards the antagonists. However, the molecular mechanism of this difference remains unexplored. The distinctive features in the antagonist’s interaction with these two different but closely related (~80% sequence identity at this region) subunits are analyzed from the patterns of their hydrogen bonding. The regions directly involved in the antagonist binding have been classified into seven different interaction sites. Two conserved hydrophilic pockets located at both the S1 and S2 domains are found to be crucial for antagonist binding. The positively charged (Lys) residues present at the second interaction site and the invariant residue (Arg) located at the fourth interaction site are seen to influence ligand binding. The geometry of the binding pockets of NR2A and NR2B subunits have been determined from the distance between the C-α atoms in the residues interacting with the ligands. The binding pockets are found to be different for NR2A and NR2B. There are gross dissimilarities in competitive antagonist binding between these two subunits. The binding pocket geometry identified in this study may have the potential for future development of selective antagonists for the NR2A or NR2B subunit. Figure The figure shows the interactions of Drug-E with the NR2A subunit of the NMDA receptor. The amino acids shown here are within 4Å radius of drug. Hydrogen bonds between the drug and receptor are marked in green dotted lines. Drugs are rendered in cpk (ball and stick) and amino acids are shown in blue color (lines). MediaObjects/894_2005_258_Figa_HTML.jpg

AbstractN-Methyl-D-Aspartate (NMDA) receptors are the ligand gated as well as voltage sensitive ionotropic glutamate receptors, widely distributed in the vertebrate central nervous system and they play critical role in the pathogenesis of schizophrenia. Molecular dynamics simulations have been carried out on high resolution crystal structure of NR1 subunit of NMDA receptor ligand binding core (S1S2) in four different conformations. We have investigated consequence of D481N/K483Q double mutation of NR1 subunit from simulation results of (a) glycine bound form (WG), (b) unbound (closed-apo) form (WOG), (c) a double mutated form (DM), and (d) the antagonist (5,7-dichlorokynuric acid) bound form (DCKA). The MD simulations and simulated annealing for 4ns show a distinct conformation for the double mutated conformation that neither follows the antagonist nor apo conformation. There are two distinct sites, loop1 and loop2 where the double mutated structure in its glycine bound form shows significant RMSD deviations as compared to the wild-type. The interactions of glycine with the receptor remain theoretically unchanged in the double mutated structure and there is no detachment of S1S2 domains. The results suggest that separation of S1 and S2 domains may not be essential for channel inactivation. Therefore, it is hypothesized that hypoactivation of NMDA receptor channels may arise out of the conformational changes at non-conserved Loop1 and Loop2 regions observed in the mutated structure. The Loop1 and Loop2 regions responsible for inter-subunit interactions in a functional NMDA receptor, may therefore, render the ligand bound form defunct. This may account for behavioral anomalies due to receptor inactivation seen in grin1 mutated mice.

The ionotropic N-methyl-d-aspartate (NMDA) receptor is of importance in neuronal development, functioning, and degeneration in the mammalian central nervous system. The functional NMDA receptor is a heterotetramer comprising two NR1 and two NR2 or NR3 subunits. We have carried out evolutionary trace (ET) analysis of forty ionotropic glutamate receptor (IGRs) sequences to identify and characterize the residues forming the binding socket. We have also modeled the ligand binding core (S1S2) of NMDA receptor subunits using the recently available crystal structure of NR1 subunit ligand binding core which shares ~40% homology with other NMDA receptor subunits. A short molecular dynamics simulation of the glycine-bound form of wild-type and double-mutated (D481N; K483Q) NR1 subunit structure shows considerable RMSD at the hinge region of S1S2 segment, where pore forming transmembrane helices are located in the native receptor. It is suggested that the disruption of domain closure could affect ion-channel activation and thereby lead to perturbations in normal animal behavior. In conclusion, we identified the amino acids that form the ligand-binding pocket in many ionotropic glutamate receptors and studied their hydrogen bonded and nonbonded interaction patterns. Finally, the disruption in the S1S2 domain conformation (of NR1 subunit- crystal structure) has been studied with a short molecular dynamics simulation and correlated with some experimental observations. Figure The figure shows the binding mechanism of glutamate with NR2B subunit of the NMDA receptor. Glutamate is shown in cpk, hydrogen bonds in dotted lines and amino acids in blue. The amino acids shown here are within a 4-Å radius of the ligand (glutamate)

Protein comparative modeling has useful applications in large-scale structural initiatives and in rational design of drug targets in medicinal chemistry. The reliability of a homology model is dependent on the sequence identity between the query and the structural homologue used as a template for modeling. Here, we present a method for the utilization and conservation of important structural features of template structures by providing additional spatial restraints in comparative modeling programs like MODELLER. We show that root mean square deviation at C a positions between the model and the corresponding experimental structure and the quality of the models can be significantly improved for distantly related systems by utilizing additional spatial restraints of the template structures. We demonstrate the influence of such approaches to homology modeling during distant relationships in understanding functional properties of protein such as ligand binding using cytochrome P450 as an example. Figure Example of a modeling triad. Structures of flavodoxin from Clostridium beijerinckii (PDB code, 5nul) and flavodoxin from Chondrus crispus (PDB code, 2fcr) that are equidistant in sequence identity (Seq. ID.) and structural similarity (RMSD Root mean square deviation ) to the third flavodoxin member (from Desulfovibrio vulgaris, PDB code, 1akr)

Hot water epilepsy (HWE) is a benign and rare form of reflex epilepsy that occurs most commonly in humans. Bdm1 is one of the proteins whose mRNA transcript is overexpressed during HWE in a rat model. We show, by sequence analysis and fold recognition methods, that Bdm1 has strong structural similarities to / hydrolases like the thioesterases. A three-dimensional model derived by comparative modeling methods allowed the search for catalytic residues using a flexible functional template characteristic of these enzymes. We predict that Bdm1 might be regulated by homocysteine levels by means of direct participation in degradation pathways.

Knowledge, both from the three-dimensional structures of homologous proteins and from the general analysis of protein structure, is of value in modeling a protein of known sequence but unknown structure. While many models are still constructed at least in part by manual methods on graphics devices, automated procedures have come into greater use. These procedures include those that assemble fragments of structure from other known structures and those that derive coordinates for the model from the satisfaction of restraints placed on atomic positions.

Genome sequencing has emerged recently as a technology that can be used to address questions regarding the clonal evolution of cancers. This has the potential to be translated into practical applications in a clinical setting. We have undertaken a case study by carrying out whole exome sequencing of a patient with CML since its clinical outcome varies amongst patients during the progression of disease.Our study is an attempt for a better understanding of why patients differ in their response to different dose of drug regimen and to determine its role in clinical outcome. Our case involves a patient with BCR–ABL positive CML. She has been responding to Imatinib, at 200 mg once a day, a dosage lower than the recommended 400mg /day. She has been taking 200 mg / day irregularly for two years and there were frequent interruptions of regular dosage schedule because of severe symptomatic cytopenias requiring blood transfusions. At the end of 2 years, there has been no progression of disease. Her bone marrow aspirate and biopsy have been normal and the BCR-ABL transcript has been below detectable levels.It was by serendipity that we happened to study the whole exome of this patient before we started her on Imatinib. As her dose response was erratic, we decided to do a longitudinal study. We examined the whole exome of the patient with CML at different points during the progression of disease. The aim of this study is to identify novel polymorphisms or variants which might be associated with the case, there by resisting the disease to progress. We were interested in the process of understanding the variability of the clinical outcome to standard treatment from a genomics perspective.The exome sequencing of the bone marrow aspirate was performed at the time of diagnosis and two years post treatment with Imatinib. Matched skin biopsy was used as a control. This study has been approved by the Institutional Ethical Review Board of St. John's Medical college and Hospital.The SNVs of the skin data were used as the control in this experiment to account for germ line mutations. We observe severe genome instability in terms of single nucleotide variants (SNVs) at the time of diagnosis. The counts of SNVs are observed to be drastically reduced in the treated bone marrow sample than at the time of diagnosis. No insertions or deletions were observed in this longitudinal case study. It was also observed that the SNV counts were not dominated in any particular chromosome.The SNVs picked up by exome sequencing were hence contrasted at the stage of diagnosis and post treatment. These SNVs were compared to dbSNP to retain only those which are novel and not previously reported. This finally gave rise to three discreet sets to consider: 1). The SNVs present in the genome of the patient at diagnosis 2). Post treatment, and 3). a subset common to both of them. The non-synonymous coding mutations with single base substitutions were 1,08,436 at the time of diagnosis and reduced to 163 in a stage of remission. The overlaps between these two categories were 169 in number.These hits in respective categories were further filtered based on the score of SNV call, relevance to the disease, presence within domain boundaries and final predicted impact on the function of the coded protein. We have identified novel mutations within the ABL, BCR, Kit and NOTCH genes with high probability of impact on function in the bone marrow sample at diagnosis. These will further be validated experimentally to be confirmed as a probable marker for screening patients. This study will be extended to screen a cohort of patients with similar prognosis as the patient in our case study. The novel mutations will be mapped on the protein structure to show which functional domains could be affected and to provide a structural basis for the deleterious effects of such mutations. We hence attempt to understand this clinical case with an integrated view involving basic experimental biology, bio-informatics and structural biology.These polymorphisms would provide insights on the evolution of CML and eventually allow us to use such readouts as a screen across CML patients to identify cases with better disease prognosis. With the distinct population groups and diverse spectrum of diseases, we believe that our work lays the foundation for larger studies in both CML and other diseases requiring such approaches.No relevant conflicts of interest to declare.

Genome sequencing has emerged recently as a technology that can be used to address questions regarding the clonal evolution of cancers. This has the potential to be translated into practical applications in a clinical setting. We have undertaken a case study by carrying out whole exome sequencing of a patient with CML since its clinical outcome varies amongst patients during the progression of disease.