Sajib Chakraborty

Comparative network clustering of direct repeats (DRs) and cas genes confirms the possibility of the horizontal transfer of CRISPR locus among bacteria

CRISPRs are a diverse family of DNA repeat sequences that are widely distributed among archaea and bacteria. The CRISPR locus is usually composed of three key elements; direct repeats (DRs), spacer sequences and the cas genes. Although recent studies have suggested that spacers may be of extrachromosomal origin, the evolutionary origin of the other two elements of the CRISPR locus has remained unresolved. With the aim to elucidate the evolutionary origin and association of DRs and cas genes of the CRISPR locus, a comparative analysis of the evolutionary network clusters of DRs, cas1 and 16S rRNA genes sequences from 100 different bacteria was conducted. Significant matches of DR and cas1 gene clades imply that these CRISPR components are evolutionary closely linked and potentially evolving simultaneously as a whole locus. On the contrary, the prominent discordance between the CASS (DR and cas1) clades and the 16S rRNA clusters indicates that CRISPR locus has been transferred horizontally as a complete package. Sequence analysis also revealed that DR and cas1 genes are coevolving under analogous evolutionary pressure. This atypical evolutionary pattern also signifies the possibility of horizontal transfer event of CRISPR locus.

In silico predicted mycobacterial epitope elicits in vitro T-cell responses

Epitope-based vaccines permit the selection of only a specific subset of epitopes to induce the necessary immune response, thus providing a rational alternative to conventional design approaches. Using a range of immunoinformatics tools, we identified a novel, contiguous 28 amino acid multi-epitope cluster within the highly conserved secretory protein Ag85B of Mycobacterium tuberculosis, the causative agent of TB. This cluster, named Ep85B, is composed of epitopes which bind to three HLA Class I and 15 Class II molecules, and harbors the potential to generate 99% population coverage in TB-endemic regions. We experimentally evaluated the capacity of Ep85B to elicit T-cell immune responses using whole blood cells and, as predicted, observed significant increases in populations of both CD4+ and memory CD4+ CD45RO+ T-cells. Our results demonstrate the practical utility of an epitope-based design methodology - a strategy that, following further evaluation, may serve as an additional tool for the development of novel vaccine candidates against TB and other diseases.

Assessment of the Evolutionary Origin and Possibility of CRISPR-Cas (CASS) Interference Pathway in Vibrio cholerae O395

Bacteria have developed several defense mechanisms against bacteriophages over evolutionary time, but the concept of prokaryotic RNA interference mediated defense mechanism against phages and other invading genetic elements has emerged only recently. Clustered regularly interspaced short palindromic repeats (CRISPR) together with closely associated genes (cas genes) constitute the CASS system that is believed to provide a RNAi-like defense mechanism against bacteriophages within the host bacterium. However, a CASS mediated RNAi-like pathway in enteric pathogens such as Vibrio cholerae O395 or Escherichia coli O157 have not been reported yet. This study specifically was designed to investigate the possibility and evolutionary origin of CASS mediated RNAi-like pathway in the genome of a set of enteric pathogens, especially V. cholerae. The results showed that V. cholerae O395 and also other related enteric pathogens have the essential CASS components (CRISPR and cas genes) to mediate a RNAi-like pathway. The functional domains of a V. cholerae Cas3 protein, which is believed to act as a prokaryotic Dicer, was revealed and compared with the domains of eukaryotic Dicer proteins. Extensive homology in several functional domains provides significant evidence that the Cas3 protein has the essential domains to play a vital role in RNAi like pathway in V. cholerae. The secondary structure of the pre-siRNA for V. cholerae O395 was determined and its thermodynamic stability also reinforced the previous findings and signifies the probability of a RNAi-like pathway in V. cholerae O395.

Characterization of the Protective HIV-1 CTL Epitopes and the Corresponding HLA Class I Alleles: A Step towards Designing CTL Based HIV-1 Vaccine.

Human immunodeficiency virus (HIV) possesses a major threat to the human life largely due to the unavailability of an efficacious vaccine and poor access to the antiretroviral drugs against this deadly virus. High mutation rate in the viral genome underlying the antigenic variability of the viral proteome is the major hindrance as far as the antibody based vaccine development is concerned. Although the exact mechanism by which CTL epitopes and the restricting HLA alleles mediate their action towards slow disease progression is still not clear, the important CTL restricted epitopes for controlling viral infections can be utilized in future vaccine design. This study was designed for the characterization the HIV-1 optimal CTL epitopes and their corresponding HLA alleles. CTL epitope cluster distribution analysis revealed only two HIV-1 proteins, namely, Nef and Gag, which have significant cluster forming capacity. We have found the role of specific HLA supertypes such as HLA B∗07, HLA B∗58, and HLA A∗03 in selecting the hydrophobic and conserved amino acid positions within Nef and Gag proteins, to be presented as epitopes. The analyses revealed that the clusters of optimal epitopes for Nef and p24 proteins of HIV-1 could potentially serve as a source of vaccine.

Protein abundance of AKT and ERK pathway components governs cell type-specific regulation of proliferation

Signaling through the AKT and ERK pathways controls cell proliferation. However, the integrated regulation of this multistep process, involving signal processing, cell growth and cell cycle progression, is poorly understood. Here, we study different hematopoietic cell types, in which AKT and ERK signaling is triggered by erythropoietin (Epo). Although these cell types share the molecular network topology for pro‐proliferative Epo signaling, they exhibit distinct proliferative responses. Iterating quantitative experiments and mathematical modeling, we identify two molecular sources for cell type‐specific proliferation. First, cell type‐specific protein abundance patterns cause differential signal flow along the AKT and ERK pathways. Second, downstream regulators of both pathways have differential effects on proliferation, suggesting that protein synthesis is rate‐limiting for faster cycling cells while slower cell cycles are controlled at the G1‐S progression. The integrated mathematical model of Epo‐driven proliferation explains cell type‐specific effects of targeted AKT and ERK inhibitors and faithfully predicts, based on the protein abundance, anti‐proliferative effects of inhibitors in primary human erythroid progenitor cells. Our findings suggest that the effectiveness of targeted cancer therapy might become predictable from protein abundance.

The difficulties in cancer treatment

Cancer is clearly the most deadly disease in the developed world as one in three people develop cancer during their lifetime. The cure for cancer is like the Holy Grail since most of the existing treatments are not effective enough to provide full protection from this disease. In recent years the burgeoning of sophisticated genomic, proteomic and bioinformatics techniques has made it possible for us to get a glimpse of the intricate interplay of numerous cellular genes and regulatory genetic elements that are responsible for the manifestation of cancerous phenotypes. With the use of modern genomic technologies we are now beginning to understand the enormous complexity of cancer. However there are few success stories as far as the treatment of cancer is concerned. For instance the treatments of leukemia and lymphoma have been established and proved to be satisfactory. Despite occasional successes the treatment for most cancers is still a long way from reality. In this editorial, we have addressed several reasons for the difficulties in cancer treatment.

Investigation of the Potential Association between Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR) and Antibiotic Resistance Pattern of Bacterial Strains Isolated from Medical Waste and Environmental Water

Enterobacteriaceae are important human pathogens that cause many food and waterborne illness. Rapid emergence of multi-drug resistant bacteria in Bangladesh has become a serious problem. Phage-host interaction is now considered as the major driving force for the conversion of non-pathogenic bacteria to pathogenic ones. Evolution of highly pathogenic and antibiotic resistant bacteria largely depends upon the horizontal gene transfer by means of plasmid, megaplasmid and bacteriophages. Conversely, bacteria may acquire a novel defence mechanism called CRISPR (Clustered regularly interspersed short palindromic repeats) that can restrict horizontal transfer of plasmids and bacteriophages to limit the spread of antibiotic resistance genes among bacterial species. In this study, twenty bacterial strains were isolated from water of different medical waste and Buriganga river. Therefore, CRISPR locus was investigated following various biochemical and molecular analysis of those bacterial isolates. Identification of the bacterial isolates was conducted by Polymerase Chain Reaction (PCR) based assay of 16S rDNA extracted from those isolated strains. Results indicated that most strains were identified as Proteus mirabilis and Citrobacter freundii which mainly cause septicemia, and pneumonia in human. Thereafter, antibiogram of these strains was performed by using 11 different antibiotic discs where bacterial isolates from medical drainage system showed more resistant to antibiotics than the river water. In this study, CRISPR locus was also investigated within the genome of the isolated bacterial stains but unexpectedly, we did not find any CRISPR locus in their genome. In conclusion, we confirm that multi-drug resistant bacterial strains are devoid of CRISPR locus suggesting a possible negative association between CRISPR locus and antibiotic resistance. Further studies to pinpoint are required to elucidate the underlying mechanism of the association between CRISPR and antibiotic resistance in these isolated strains.

Molecular Diagnostic Approach Prevails Superior Over Conventional GelElectrophoresis Method in Detecting Sickle Cell Anemia

Sickle cell anemia is defined as homozygosity caused by the mutation of the glutamic acid residue to valine in the β-globin gene. Sickle cell disease is an increasing global health burden with the estimated number of patients increasing in a concerning manner. Here we report a very interesting and clinically insightful case of hemoglobinopathy which was initially suspected to be Hb S/D Punjab- a rare type of hemoglobinopathy as diagnosed by the hemoglobinelectrophoresis technique. Despite the diagnosis report indicating the rare Hb S/D Punjab, the uncharacteristic clinical presentation of the patient which was not coherent with the classical symptoms of Hb S/D Punjab, forced the clinicians to turn their attention to molecular diagnosis. To clarify the etiology of the clinical case, a sequencing-based molecular diagnosis approach was adopted that revealed the mutational signature of sickle cell anemia (SCA). This case can be regarded as a prominent example where the molecular techniques lead to the correct diagnosis matching with the clinical symptoms while the conventional diagnostic approach failed.

Onco-Multi-OMICS Approach: A New Frontier in Cancer Research

The acquisition of cancer hallmarks requires molecular alterations at multiple levels including genome, epigenome, transcriptome, proteome, and metabolome. In the past decade, numerous attempts have been made to untangle the molecular mechanisms of carcinogenesis involving single OMICS approaches such as scanning the genome for cancer-specific mutations and identifying altered epigenetic-landscapes within cancer cells or by exploring the differential expression of mRNA and protein through transcriptomics and proteomics techniques, respectively. While these single-level OMICS approaches have contributed towards the identification of cancer-specific mutations, epigenetic alterations, and molecular subtyping of tumors based on gene/protein-expression, they lack the resolving-power to establish the casual relationship between molecular signatures and the phenotypic manifestation of cancer hallmarks. In contrast, the multi-OMICS approaches involving the interrogation of the cancer cells/tissues in multiple dimensions have the potential to uncover the intricate molecular mechanism underlying different phenotypic manifestations of cancer hallmarks such as metastasis and angiogenesis. Moreover, multi-OMICS approaches can be used to dissect the cellular response to chemo- or immunotherapy as well as discover molecular candidates with diagnostic/prognostic value. In this review, we focused on the applications of different multi-OMICS approaches in the field of cancer research and discussed how these approaches are shaping the field of personalized oncomedicine. We have highlighted pioneering studies from “The Cancer Genome Atlas (TCGA)” consortium encompassing integrated OMICS analysis of over 11,000 tumors from 33 most prevalent forms of cancer. Accumulation of huge cancer-specific multi-OMICS data in repositories like TCGA provides a unique opportunity for the systems biology approach to tackle the complexity of cancer cells through the unification of experimental data and computational/mathematical models. In future, systems biology based approach is likely to predict the phenotypic changes of cancer cells upon chemo-/immunotherapy treatment. This review is sought to encourage investigators to bring these different approaches together for interrogating cancer at molecular, cellular, and systems levels.

The New Age of Cytopathology: Applications of MALDI Imaging and Super-Resolution Microscopy

The advent of Matrix-assisted laser desorption/ionization (MALDI)-based mass spectrometry imaging uncover new opportunities for the clinicians and pathologists to extract molecular and spatial information from tissues. MALDI time-of-flight (TOF) imaging mass spectrometry (IMS) is an exciting technology which cemented its place in clinical diagnosis especially in oncology. It harnesses the advantages of mass spectrometry in detecting biomolecules with complementary spatial resolution in clinical tissue samples.1 However the challenge remains is this field is the low sensitivity which is characterized by the poor ionization efficiency of numerous bio-molecules that are present in tissue slices.2 A giant leap of this technology came in the form of MALDI-2 which improves the sensitivity of MALDI imaging by up to two orders of magnitude thereby facilitating the detection of bio-molecules of different origins such as protein, lipids, carbohydrates and proteins.2