Differential expression of 9-O-acetylated sialoglycoconjugates on leukemic blasts: a potential tool for long-term monitoring of children with acute lymphoblastic leukemia

Earlier studies have demonstrated overexpression of 9-O-acetylated sialoglycoconjugates (9-O-AcSGs) on lymphoblasts, concomitant with high titers of anti-9-O-AcSG antibodies in childhood acute lymphoblastic leukemia (ALL). Our aim was to evaluate the correlation between expression of different 9-O-AcSGs during chemotherapeutic treatment. Accordingly, expression of 9-O-AcSGs on lymphoblasts of ALL patients (n = 70) were longitudinally monitored for 6 years (1997-2002), using Achatinin-H, a 9-O-acetylated sialic acid (9-O-AcSA) binding lectin with preferential affinity for 9-O-AcSGs with terminal 9-O-AcSAα2→6GalNAc. Western blot analysis of patients (n = 30) showed that 3 ALL-specific 9-O-AcSGs (90, 120 and 135 kDa) were induced at presentation; all these bands disappeared after treatment in patients (n = 22) who had disease-free survival. The 90 kDa band persisted in 8 patients who subsequently relapsed with reexpression of the 120 kDa band. FACS analysis revealed that at presentation (n = 70) 90.1 ± 5.0% cells expressed 9-O-AcSGs, which decreased progressively with chemotherapy, remained <5% during clinical remission and reappeared in relapse (80 ± 10%, n = 18). Early clearance of 9-O-AcSG+ cells, during 4-8 weeks of treatment showed a good correlation with low risk of relapse. Sensitivity of detection of 9-O-AcSG+ cells was 0.1%. Numbers of both high- and low-affinity binding sites were maximum at presentation, decreased with treatment and increased again in clinical relapse. We propose that close monitoring of 90 and 120 kDa 9-O-AcSGs may serve as a reliable index for long-term management of childhood ALL and merits therapeutic consideration

Application of Computational Methods in Planaria Research: A Current Update

Planaria is a member of the Phylum Platyhelminthes including flatworms. Planarians possess the unique ability of regeneration from adult stem cells or neoblasts and finds importance as a model organism for regeneration and developmental studies. Although research is being actively carried out globally through conventional methods to understand the process of regeneration from neoblasts, biology of development, neurobiology and immunology of Planaria, there are many thought provoking questions related to stem cell plasticity, and uniqueness of regenerative potential in Planarians amongst other members of Phylum Platyhelminthes. The complexity of receptors and signalling mechanisms, immune system network, biology of repair, responses to injury are yet to be understood in Planaria. Genomic and transcriptomic studies have generated a vast repository of data, but their availability and analysis is a challenging task. Data mining, computational approaches of gene curation, bioinformatics tools for analysis of transcriptomic data, designing of databases, application of algorithms in deciphering changes of morphology by RNA interference (RNAi) approaches, understanding regeneration experiments is a new venture in Planaria research that is helping researchers across the globe in understanding the biology. We highlight the applications of Hidden Markov models (HMMs) in designing of computational tools and their applications in Planaria decoding their complex biology

Study of Gallbladder cancer in the light of proteomics

Abstract Gallbladder carcinoma (GBC) is a chronic malignancy of the gall bladder and intrahepatic and extrahepatic common bile ducts with a high mortality rate and forms the fifth common cancer of gastrointestinal tract globally. Women remain at higher risk than men and recent studies have reported the highest rate of incidence in women from Delhi, India. GBC treatment suffers from the disadvantage of lack of suitable biomarkers for early diagnosis of the disease. Running title: Proteomic studies in Gallbladder Cancer: Recent Development

New Distribution Record of Octolasion tyrtaeum (Savigny, 1826) and Octolasion cyaneum (Savigny, 1826) (Family: Lumbricidae) Earthworm Species from Leh-Ladakh Union Territory, India

There are five species of the genus Octolasion Örley, [1] in the family Lumbricidae in the world. In India, only two species Octolasion tyrtaeum [2] and Octolasion cyaneum [2], have been recorded. Leh-Ladakh, an Indian territory, has no previous earthworm records. This study reports two species Octolasion tyrtaeum [2] and Octolasion cyaneum [2], as new records from Leh-Ladakh, India

Genome analysis identifies a spontaneous nonsense mutation in ppsD leading to attenuation of virulence in laboratory-manipulated Mycobacterium tuberculosis

Abstract Background A previous laboratory study involving wild type, mutant and devR/dosR complemented strains of Mycobacterium tuberculosis reported the attenuation phenotype of complemented strain, Comp1. This phenotype was intriguing since the parental strain H37Rv, devR mutant (Mut1) and additional complemented strains, Comp9 and Comp11, were virulent in the guinea pig model. Results Towards deciphering the mechanism underlying the attenuation of Comp1, a whole genome sequencing approach was undertaken. Eight Single Nucleotide Polymorphisms (SNPs) unique to the Comp1 strain were identified. Of these, 5 SNPs were non-synonymous and included a G➞A mutation resulting in a W1591Stop mutation in ppsD gene of the phthiocerol dimycocerosate (PDIM) biosynthetic cluster. Targeted sequence analysis confirmed this mutation in only Comp1 strain and not in wild type (H37Rv), devR knockout (Mut1) or other complemented (Comp9 and Comp11) bacteria. Differential expression of the PDIM locus in Comp1 bacteria was observed which was associated with a partial deficiency of PDIM, an increased sensitivity to detergent and a compromised ability to infect human THP-1 cells. Conclusions It is proposed that a spontaneous mutation in the ppsD gene of Comp1 underlies down-modulation of the PDIM locus which is associated with defects in permeability and infectivity as well as virulence attenuation in guinea pigs. Our study demonstrates the value of whole genome sequencing for resolving unexplainable bacterial phenotypes and recommends the assessment of PDIM status while assessing virulence properties of laboratory-manipulated strains of M. tuberculosis

Purification and characterization of 9-O-acetylated sialoglycoproteins from leukemic cells and their potential as immunological tool for monitoring childhood acute lymphoblastic leukemia

Sialic acids as terminal residues of oligosaccharide chains play crucial roles in several cellular recognition events. Exploiting the selective affinity of Achatinin-H toward N-acetyl-9-Oacetylneuraminic acid-a2-6-GalNAc, we have demonstrated the presence of 9-O-acetylated sialoglycoproteins (Neu5,9Ac2- GPs) on lymphoblasts of 70 children with acute lymphoblastic leukemia (ALL) and on leukemic cell lines by fluorimetric HPLC and flow cytometric analysis. This study aims to assess the structural aspect of the glycotope of Neu5,9Ac2-GPsALL and to evaluate whether these disease-specific molecules can be used to monitor the clinical outcome of ALL. The Neu5,9Ac2- GPsALL were affinity-purified, and three distinct leukemiaspecific molecular determinants (135, 120, and 90 kDa) were demonstrated bySDS–PAGE, western blotting, and isoelectric focusing. The carbohydrate epitope of Neu5,9Ac2-GPsALL was confirmed by using synthetic sialic acid analogs. The enhanced presence of anti-Neu5,9Ac2-GPALL antibody in ALL patients prompted us to develop an antigen-ELISA using purified Neu5,9Ac2-GPsALL as coating antigens. Purified antigen was able to detect leukemia-specific antibodies at presentation of disease, which gradually decreased with treatment. Longitudinal monitoring of 18 patients revealed that in the early phase of the treatment patients with lower anti-Neu5,9Ac2-GPs showed a better prognosis. Minimal cross-reactivity was observed in other hematological disorders (n¼50) like chronic myeloid leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, and non-Hodgkin’s lymphoma as well as normal healthy individuals (n¼21). This study demonstrated the potential of purified Neu5,9Ac2-GPsALL as an alternate tool for detection of anti-Neu5,9Ac2-GP antibodies to be helpful for diagnosis and monitoring of childhood ALL patients

Induction of apoptosis by Fe(salen)Cl through caspase-dependent pathway specifically in tumor cells

Iron-based compounds possess the capability of inducing cell death due to their reactivity with oxidant molecules, but their specificity towards cancer cells and the mechanism of action are hitherto less investigated. A Fe(salen)Cl derivative has been synthesized that remains active in monomer form. The efficacy of this compound as an anti-tumor agent has been investigated in mouse and human leukemia cell lines. Fe(salen)Cl induces cell death specifically in tumor cells and not in primary cells. Mouse and human T-cell leukemia cell lines, EL4 and Jurkat cells are found to be susceptible to Fe(salen)Cl and undergo apoptosis, but normal mouse spleen cells and human peripheral blood mononuclear cells (PBMC) remain largely unaffected by Fe(salen)Cl. Fe(salen)Cl treated tumor cells show significantly higher expression level of cytochrome c that might have triggered the cascade of reactions leading to apoptosis in cancer cells. A significant loss of mitochondrial membrane potential upon Fe(salen)Cl treatment suggests that Fe(salen)Cl induces apoptosis by disrupting mitochondrial membrane potential and homeostasis, leading to cytotoxity. We also established that apoptosis in the Fe(salen)Cl-treated tumor cells is mediated through caspase-dependent pathway. This is the first report demonstrating that Fe(salen)Cl can specifically target the tumor cells, leaving the primary cells least affected, indicating an excellent potential for this compound to emerge as a next-generation anti-tumor drug

O-Acetylation of Sialic Acids is Required for the Survival of Lymphoblasts in Childhood Acute Lymphoblastic leukemia (ALL)

Exploiting the selective affinity of Achatinin-H towards 9-O-acetylneuraminic acid(α2-6)GalNAc, we have demonstrated the presence of 9-O-acetylated sialoglycoproteins (Neu5,9Ac2-GPs) on hematopoietic cells of children suffering from acute lymphoblastic leukemia (ALL), indicative of defective sialylation associated with this disease. The carbohydrate epitope of Neu5,9Ac2-GPsALL was confirmed by using several synthetic sialic acid analogues. They are functionally active signaling molecules as demonstrated by their role in mediating lymphoproliferative responses and consequential increased production of IFN-γ due to specific stimulation of Neu5,9Ac2-GPs on PBMCALL with Achatinin-H. Cells devoid of 9-Oacetylations (9-O-AcSA−) revealed decreased nitric oxide production as compared to 9-O-AcSA+ cells on exposure to IFN-γ. Under this condition, a decrease in viability of 9-OAcSA− cells as compared to 9-O-AcSA+ cells was also observed which was reflected from increased caspase 3 activity and apoptosis suggesting the protective role of this glycotope

Disease-associated glycosylated molecular variants of human C-reactive protein activate complement-mediated hemolysis of erythrocytes in tuberculosis and Indian visceral leishmaniasis

Human C-reactive protein (CRP), as a mediator of innate immunity, removed damaged cells by activating the classical complement pathway. Previous studies have successfully demonstrated that CRPs are differentially induced as glycosylated molecular variants in certain pathological conditions. Affinity-purified CRPs from two most prevalent diseases in India viz. tuberculosis (TB) and visceral leishmaniasis (VL) have differential glycosylation in their sugar composition and linkages. As anemia is a common manifestation in TB and VL, we assessed the contributory role of glycosylated CRPs to influence hemolysis via CRP-complement-pathway as compared to healthy control subjects. Accordingly, the specific binding of glycosylated CRPs with erythrocytes was established by flow-cytometry and ELISA. Significantly, deglycosylated CRPs showed a 7–8-fold reduced binding with erythrocytes confirming the role of glycosylated moieties. Scatchard analysis revealed striking differences in the apparent binding constants (104–105M−1) and number of binding sites (106–107sites/erythrocyte) for CRP on patients’ erythrocytes as compared to normal. Western blotting along with immunoprecipitation analysis revealed the presence of distinct molecular determinants on TB and VL erythrocytes specific to disease-associated CRP. Increased fragility, hydrophobicity and decreased rigidity of diseased-erythrocytes upon binding with glycosylated CRP suggested membrane damage. Finally, the erythrocyte-CRP binding was shown to activate the CRP-complement-cascade causing hemolysis, even at physiological concentration of CRP (10μg/ml). Thus, it may be postulated that CRP have a protective role towards the clearance of damaged-erythrocytes in these two disease