Modulation of Malaria Phenotypes by Pyruvate Kinase (PKLR) Variants in a Thai Population

Pyruvate kinase (PKLR) is a critical erythrocyte enzyme that is required for glycolysis and production of ATP. We have shown that Pklr deficiency in mice reduces the severity (reduced parasitemia, increased survival) of blood stage malaria induced by infection with Plasmodium chabaudi AS. Likewise, studies in human erythrocytes infected ex vivo with P. falciparum show that presence of host PK-deficiency alleles reduces infection phenotypes. We have characterized the genetic diversity of the PKLR gene, including haplotype structure and presence of rare coding variants in two populations from malaria endemic areas of Thailand and Senegal. We investigated the effect of PKLR genotypes on rich longitudinal datasets including haematological and malaria-associated phenotypes. A coding and possibly damaging variant (R41Q) was identified in the Thai population with a minor allele frequency of ~4.7%. Arginine 41 (R41) is highly conserved in the pyruvate kinase family and its substitution to Glutamine (R41Q) affects protein stability. Heterozygosity for R41Q is shown to be associated with a significant reduction in the number of attacks with Plasmodium falciparum, while correlating with an increased number of Plasmodium vivax infections. These results strongly suggest that PKLR protein variants may affect the frequency, and the intensity of malaria episodes induced by different Plasmodium parasites in humans living in areas of endemic malaria.     

Sensory Neuron-Derived Eph Regulates Glomerular Arbors and Modulatory Function of a Central Serotonergic Neuron

Olfactory sensory neurons connect to the antennal lobe of the fly to create the primary units for processing odor cues, the glomeruli. Unique amongst antennal-lobe neurons is an identified wide-field serotonergic neuron, the contralaterally-projecting, serotonin-immunoreactive deutocerebral neuron (CSDn). The CSDn spreads its termini all over the contralateral antennal lobe, suggesting a diffuse neuromodulatory role. A closer examination, however, reveals a restricted pattern of the CSDn arborization in some glomeruli. We show that sensory neuron-derived Eph interacts with Ephrin in the CSDn, to regulate these arborizations. Behavioural analysis of animals with altered Eph-ephrin signaling and with consequent arborization defects suggests that neuromodulation requires local glomerular-specific patterning of the CSDn termini. Our results show the importance of developmental regulation of terminal arborization of even the diffuse modulatory neurons to allow them to route sensory-inputs according to the behavioural contexts.     

Isolation and Characterization of Arsenic-Resistant Bacteria from Contaminated Water-Bodies in West Bengal,India

Bengal Basin is known for severe arsenic contamination. In the present study, we have isolated six bacteria from the arsenic contaminated surface water of Bengal Basin. 16S rDNA sequence analysis identified them as Microbacterium oleivorans, Acinetobacter soli, Acinetobacter venetianus, Acinetobacter junii, Acinetobacter baumannii, Acinetobacter calcoaceticus. All the isolates possess arsenic accumulation potential and high molecular weight plasmid (>10 kb). PCR amplification indicated the presence of arsenic-resistance genes (arsB and aoxB) either in the genome or plasmid or in both in the isolated bacteria (except in Acinetobacter venetianus). Exposure to arsenic affected bacterial growth and induced alteration in cytoplasmic membrane integrity.     

Triacylglycerol: nourishing molecule in endurance of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>

The ability of Mycobacterium tuberculosis (M. tuberculosis) to accumulate lipid-rich molecules as an energy source obtained from host cell debris remains interesting. Additionally, the potential of M. tuberculosis to survive under different stress conditions leading to its dormant state in pathogenesis remains elusive. The exact mechanism by which these lipid bodies generated in M. tuberculosis infection and utilized by bacilli inside infected macrophage for its survival is still not understood. In this, during bacillary infection, many metabolic pathways are involved that influence the survival of M. tuberculosis for their own support. However, the exact energy source derived from infecting host cells remain elusive. Therefore, this study highlights several alternative energy sources in the form of triacylglycerol (TAG) and fatty acids, i.e. oleic acids accumulation, which are essential in dormancy-like state under M. tuberculosis infection. The prominent stage in tuberculosis (TB) infection is re-establishment of M. tuberculosis under stress conditions and deployment of a confined strategy to utilize these biomolecules for its persistence survival. So, growing in our understanding of these pathways will help us in accelerating therapies, which could reduce TB prevalence world widely.     

Longitudinal study of Plasmodium falciparum and Plasmodium vivax in a Karen population in Thailand

Background

Pregnancy malaria is caused by Plasmodium falciparum -infected erythrocytes binding the placental receptor chondroitin sulfate A (CSA). This results in accumulation of parasites in the placenta with severe clinical consequences for the mother and her unborn child. Women become resistant to placental malaria as antibodies are acquired which specifically target the surface of infected erythrocytes binding in the placenta. VAR2CSA is most likely the parasite-encoded protein which mediates binding to the placental receptor CSA. Several domains have been shown to bind CSA in vitro; and it is apparent that a VAR2CSA-based vaccine cannot accommodate all the CSA binding domains and serovariants. It is thus of high priority to define minimal ligand binding regions throughout the VAR2CSA molecule.

Methods

To define minimal CSA-binding regions/peptides of VAR2CSA, a phage display library based on the entire var2csa coding region was constructed. This library was screened on immobilized CSA and cells expressing CSA resulting in a limited number of CSA-binding phages. Antibodies against these peptides were affinity purified and tested for reactivity against CSA-binding infected erythrocytes.

Results

The most frequently identified phages expressed peptides residing in the parts of VAR2CSA previously defined as CSA binding. In addition, most of the binding regions mapped to surface-exposed parts of VAR2CSA. The binding of a DBL2X peptide to CSA was confirmed with a synthetic peptide. Antibodies against a CSA-binding DBL2X peptide reacted with the surface of infected erythrocytes indicating that this epitope is accessible for antibodies on native VAR2CSA on infected erythrocytes.

Conclusion

Short continuous regions of VAR2CSA with affinity for multiple types of CSA were defined. A number of these regions localize to CSA-binding domains and to surface-exposed regions within these domains and a synthetic peptide corresponding to a peptide sequence in DBL2 was shown to bind to CSA and not to CSC. It is likely that some of these epitopes are involved in native parasite CSA adhesion. However, antibodies directed against single epitopes did not inhibit parasite adhesion. This study supports phage display as a technique to identify CSA-binding regions of large proteins such as VAR2CSA.     

Mechanistic insights into the inhibition of prostate specific antigen by beta-lactam class compounds

Prostate Specific Antigen (PSA) is a biomarker used in the diagnosis of prostate cancer and to monitor therapeutic response. However, its precise role in prostate carcinogenesis and metastasis remains largely unknown. A number of studies arguing in the favor of an active role of PSA in prostate cancer development and progression have implicated this serine protease in the release and activation of growth factors such as insulin-like growth factor 1 (IGF1) through cleavage of insulin like growth factor binding protein 3 and Transforming Growth Factor beta (TGF-beta) through cleavage of Latent TGF-beta. In contrast, other studies suggest that PSA activity might hinder tumor development and progression. In light of these contradictory findings, efficient inhibitors of PSA are needed for exploring its biological role in tumor development and metastasis. Towards the goal of developing potent inhibitors of PSA, we have explored the molecular mechanism of a series of beta-lactam based compounds on binding to PSA using activity assays, matrix assisted laser desorption ionization with a time-of-flight mass spectrometry, and GOLD docking methodology. The mass spectrometry experiments and the activity assays confirmed the time-dependent and covalent nature of beta-lactam binding. To gain insights on the reaction intermediates at the molecular level, we docked beta-lactam inhibitors to a homology modeled PSA using the GOLD docking program in noncovalent and covalent binding modes. The docking studies elucidated the molecular details of the early noncovalent Michaelis complex, the acyl-enzyme covalent complex, and the nature of conformational reorganization required for the long term stability of the covalent complex. Additionally, the molecular basis for the effect of stereochemistry of the lactam ring on the inhibitory potency was elucidated through docking of beta-lactam enantiomers. As a validation of our docking methodology, two novel enantiomers were synthesized and evaluated for their inhibitory potency using fluorogenic substrate based activity assays. Additionally, cis enantiomers of eight beta-lactam compounds reported in a previous study were docked and their GOLD scores and binding modes were analyzed in order to assess the general applicability of our docking results. The close agreement of our docking results with the experimental data validates the mechanistic insights revealed through the docking studies and paves the way for the design and development of potent and specific inhibitors of PSA.     

Anchorage-independent growth of breast carcinoma cells is mediated by serum exosomes

We hereby report studies that suggest a role for serum exosomes in the anchorage-independent growth (AIG) of tumor cells. In AIG assays, fetal bovine serum is one of the critical ingredients. We therefore purified exosomes from fetal bovine serum and examined their potential to promote growth of breast carcinoma cells in soft agar and Matrigel after reconstituting them into growth medium (EEM). In all the assays, viable colonies were formed only in the presence of exosomes. Some of the exosomal proteins we identified, have been documented by others and could be considered exosomal markers. Labeled purified exosomes were up-taken by the tumor cells, a process that could be competed out with excess unlabeled vesicles. Our data also suggested that once endocytosed by a cell, the exosomes could be recycled back to the conditioned medium from where they can be up-taken by other cells. We also demonstrated that low concentrations of exosomes activate MAP kinases, suggesting a mechanism by which they maintain the growth of the tumor cells in soft agar. Taken together, our data demonstrate that serum exosomes form a growth promoting platform for AIG of tumor cells and may open a new vista into cancer cell growth in vivo.     

Enhanced Oral Bioavailability of Griseofulvin via Niosomes

The aim of the present report was to develop nonionic surfactant vesicles (niosomes) to improve poor and variable oral bioavailability of griseofulvin. Niosomes were prepared by using different nonionic surfactants span 20, span 40, and span 60. The lipid mixture consisted of surfactant, cholesterol, and dicetyl phosphate in the molar ratio of 125:25:1.5, 100:50:1.5, and 75:75:1.5, respectively. The niosomal formulations were prepared by thin film method and ether injection method. The influence of different formulation variables such as surfactant type, surfactant concentration, and cholesterol concentration was optimized for size distribution and entrapment efficiency for both methods. Result indicated that the niosomes prepared by thin film method with span 60 provided higher entrapment efficiency. The niosomal formulation exhibited significantly retarded in vitro release as compared with free drug. The in vivo study revealed that the niosomal dispersion significantly improved the oral bioavailability of griseofulvin in albino rats after a single oral dose. The maximum concentration (C _max) achieved in case of niosomal formulation was approximately double (2.98 μg/ml) as compared to free drug (1.54 μg/ml). Plasma drug profile also suggested that the developed niosomal system also has the potential of maintaining therapeutic level of griseofulvin for a longer period of time as compared to free griseofulvin. The niosomal formulation showed significant increase in area under the curve_0-24 (AUC; 41.56 μg/ml h) as compared to free griseofulvin (22.36 μg/ml h) reflecting sustained release characteristics. In conclusion, the niosomal formulation could be one of the promising delivery system for griseofulvin with improved oral bioavailability and prolonged drug release profiles.