Virander S Chauhan

International Centre for Genetic Engineering and Biotechnology, Trst, Friuli Venezia Giulia, Italy

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Publications (115)372.89 Total impact

  • The Journal of infectious diseases. 09/2014;
  • Jiban Jyoti Panda, Virander Singh Chauhan
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    ABSTRACT: Self-assembly of biomolecules facilitates the creation of a diverse range of hierarchical nanostructures from a wide range of polymeric and non-polymeric materials. Peptides and specifically short peptides are very attractive in this respect due to their unmatched biocompatibility, ease of synthesis, functionability as well as tunable bioactivity along with the availability of rich chemistry for fine-tuning the structure and function of peptides according to environmental conditions. Self-assembled peptide based nanostructures such as tubes, filaments, fibrils, hydrogels, vesicles, and monolayers have been studied by many research groups and found application as three-dimensional cell growing scaffolds, dental implants, neural tissue engineering scaffolds and as carriers for drugs, proteins and genes, and nucleotides. Nanostructures are also being developed from designed or modified amino acids to have enhanced cellular as well as in vivo stability. These modified nanostructures showed enhanced drug delivery properties both under in vivo and in vitro conditions.
    Polym. Chem. 07/2014; 5(15).
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    ABSTRACT: A Plasmodium falciparum chimeric protein, PfMSP-Fu24,was constructed by genetically coupling immunodominant, conserved regions of two merozoite surface proteins, the 19-kDa region C-terminal region of merozoite surface protein 1 (PfMSP-119) and a 11-kDa conserved region of merozoite surface protein 3 (PfMSP-311) to augment the immunogenicity potential of these blood stage malaria vaccine candidates. Here we have described an improved, efficient and scalable process to produce high-quality PfMSP-Fu24. Thechimeric protein was produced in E.coli SHuffle® T7 Express lysY cells that express disulphide isomerase DsbC. A two-step purification process comprising of metal affinity followed by cation exchange chromatography was developed and we were able to obtain PfMSP-Fu24 with purity above 99% and with a considerable yield of 23 mg/L. Immunogenicity of PfMSP-Fu24 formulated with several adjuvants, including Adjuplex, Alhydrogel, Adjuphos, Alhydrogel+glucopyranosyl lipid adjuvant-aqueous (GLA-AF), Adjuphos+GLA-AF, glucopyranosyl lipid adjuvant-stable emulsion (GLA-SE) and Freund's adjuvant was evaluated. PfMSP-Fu24 formulated with GLA-SE and Freund's adjuvant in mice and with Alhydrogel and Freund's adjuvant in rabbits produced high titers of PfMSP-119 and PfMSP-311-specific functional antibodies. Some of the adjuvant formulations induced inhibitory antibody responses and inhibited in vitro growth of P.falciparum parasites in the presence as well as in the absence of human monocytes. These results suggest that PfMSP-Fu24 can form a constituent of a multistage malaria vaccine.
    Clinical and vaccine Immunology: CVI 04/2014; · 2.60 Impact Factor
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    ABSTRACT: Peptide based self assembled nanostructures have attracted growing interest in recent years due to their numerous potential applications particularly in biomedical sciences. Di-peptide Phe–Phe was shown previously to self-assemble into nanotube like structures. In this work, we studied the affect of peptide backbone length and conformational flexibility on the self assembly process by using two dipeptides based on the Phe–Phe backbone (βPhe–Phe and βPhe–ΔPhe): one containing a flexible βPhe amino acid, and the other containing both a flexible βPhe as well as a backbone constraining ΔPhe (α,β-dehydrophenylalanine) amino acid. Electron microscopy and X-ray diffraction experiments revealed that these new di-peptides can self-assemble into nanotubes having different properties than the native Phe–Phe nanotubes. These nanotubes were stable over a broad range of temperatures and the introduction of non-natural amino acids provided them with stability against the action of nonspecific proteases. Moreover, these dipeptides showed no cytotoxicity towards HeLa and L929 cells, and were able to encapsulate small drug molecules. We further showed that anticancerous drug mitoxantrone was more efficient in killing HeLa and B6F10 cells when entrapped in nanotubes as compared to free mitoxantrone. Therefore, these β-phenylalanine and α,β-dehydrophenylalanine containing dipeptide nanotubes may be useful in the development of biocompatible and proteolytically stable drug delivery vehicles.
    J. Mater. Chem. B. 04/2014; 2(20).
  • Akash Saini, Virander Singh Chauhan
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    ABSTRACT: Two highly fibrillogenic peptide sequences (MNFGAFSINP and EDLIIKGISV) were previously reported in the C-terminal fragment (CTF) of TDP-43 (220-414), a protein recently implicated in neuro-degenerative diseases like ALS and FTLD-U. It was observed that the sequences MNFGAFS and EDLIIKG harbor their respective fibrillogenic domains. Here, the self-assembling properties of peptides obtained by systematic deletion of residues from these two sequences were investigated with the help of light scattering, thioflavin T fluorescence, transmission electron microscopy, and CD spectroscopy. It was found that the pentapeptide, NFGAF, and the tetrapeptide, DLII, are the shortest fibrillogenic sequences from MNFGAFS and EDLIIKG, respectively. Structure function studies revealed that self-assembly of the peptides is largely governed by hydrophobic interactions. Both NFGAF and DLII formed hydrogels based on a complex fibrillar network, at significantly low concentrations, and of remarkable strength and stability. Of particular interest was DLII, a rare aliphatic tetrapeptide which formed a hydrogel at a concentration of 1mg/ml in less than an hour. Interestingly, various other tetrapeptides based on DLII (YLII, KLII, NLII and LIID) also formed hydrogels of comparable physical properties, suggesting that an amphipathic peptide design based on the hydrophobic LII motif and a single residue polar terminus is highly favorable for hydrogelation. Peptides discovered in this study, especially DLII and its variants, are some of the shortest ever reported to show such structural and functional features, suggesting that they can be useful templates for the design of peptide based soft materials.
    Langmuir 02/2014; · 4.38 Impact Factor
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    Pinky Kumari, Dinkar Sahal, Virander S Chauhan
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    ABSTRACT: Plasmodium falciparum (Pf) employs a crucial PfHRPII catalyzed reaction that converts toxic heme into hemozoin. Understanding heme polymerization mechanism is the first step for rational design of new drugs, targeting this pathway. Heme binding and hemozoin formation have been ascribed to PfHRPII aspartate carboxylate-heme metal ionic interactions. To investigate, if this ionic interaction is indeed pivotal, we examined the comparative heme binding and β-hematin forming abilities of a wild type dendrimeric peptide BNT1 {harboring the native sequence motif of PfHRPII (AHHAHHAADA)} versus a mutant dendrimeric peptide BNTM {in which ionic Aspartate residues have been replaced by the neutral Asparaginyl residues (AHHAHHAANA)}. UV and IR data reported here reveal that at pH 5, both BNT1 and BNTM exhibit comparable heme binding as well as β-hematin forming abilities, thus questioning the role of PfHRPII aspartate carboxylate-heme metal ionic interactions in heme binding and β-hematin formation. Based on our data and information in the literature we suggest the possible role of weak dispersive interactions like N-H···π and lone-pair···π in heme binding and hemozoin formation.
    PLoS ONE 01/2014; 9(11):e112087. · 3.53 Impact Factor
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    Maryam Imam, Shailja Singh, Naveen Kumar Kaushik, Virander Singh Chauhan
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    ABSTRACT: Merozoite surface protein 3 of Plasmodium falciparum, a 40 kDa protein that also binds heme, has been biophysically characterized, for its tendency to form highly elongated oligomers. The present study aims to systematically analyse the regions in MSP3 sequence involved in oligomerization and correlate its aggregation tendency with its high affinity for binding with heme. Through SEC, DLS and TEM, we have found that MSP3, previously known to form elongated oligomers, actually forms self-assembled filamentous structures that possess amyloid like characteristics. By expressing different regions of MSP3, we observed that the previously described leucine zipper region at the C-terminus of MSP3 may not be the only structural element responsible for oligomerization, and that other peptide segments like MSP3192-196aa (YILGW) may also be required. MSP3 aggregates on incubation were transformed to long unbranched amyloid fibrils. Using immunostaining methods, we found that 5-15 micron size long fibrillar structures stained by anti-MSP3 Abs, were attached to the merozoite surface and also associated with erythrocyte membrane. We also found MSP3 to bind several molecules of heme by UV-spectrophotometry, HPLC and electrophoresis. Present study suggested that its ability to bind heme is somehow related to its inherent characteristics to form oligomers. Moreover, heme interaction with a surface protein like MSP3, which does not participate in hemozoin formation, may suggest a protective role against the heme released from unprocessed hemoglobin released after schizont egress. These studies point out to other roles that MSP3 may play during the blood stages of the parasite, in addition to be an important vaccine candidate.
    Journal of Biological Chemistry 12/2013; · 4.65 Impact Factor
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    ABSTRACT: Plasmodium falciparum reticulocyte binding-like homologous protein 5 (PfRH5) is an essential merozoite ligand that binds with its erythrocyte receptor, Basigin. PfRH5 is an attractive malaria vaccine candidate as it is expressed by a wide number of P. falciparum strains, cannot be genetically disrupted and exhibits limited sequence polymorphisms. Viral vector induced PfRH5 antibodies potently inhibited erythrocyte invasion. However, it has been a challenge to generate full-length recombinant PfRH5 in a bacterial cell based expression system. Here, we have produced full-length recombinant PfRH5 in Escherichia coli that exhibits specific erythrocyte binding similar to that of the native PfRH5 parasite protein and also importantly elicits potent invasion inhibitory antibodies against a number of P. falciparum strains. Anti-Basigin antibodies blocked the erythrocyte binding of both native and recombinant PfRH5 further confirming that they bind with Basigin. We have thus successfully produced full-length PfRH5 as a functionally active erythrocyte binding recombinant protein with a conformational integrity that mimics the native parasite protein and elicits potent strain-transcending parasite neutralizing antibodies. P. falciparum has the capability to develop immune escape mechanisms and thus blood-stage malaria vaccines that target multiple antigens or pathways may prove to be highly efficacious. In this regard, antibody combinations targeting PfRH5 and other key merozoite antigens produced potent additive inhibition against multiple worldwide P. falciparum strains. PfRH5 was immunogenic when immunized with other antigens eliciting potent invasion inhibitory antibody responses with no immune interference. Our results strongly support the development of PfRH5 as a component of a combination blood-stage malaria vaccine.
    Infection and Immunity 10/2013; · 4.07 Impact Factor
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    ABSTRACT: Subtilisin-like proteases of malaria parasite Plasmodium falciparum (PfSUB1, 2 and 3) are expressed at late asexual blood stages. PfSUB1 and 2 are considered important drug targets due to their essentiality for parasite blood stages and role in merozoite egress and invasion of erythrocytes. We have earlier shown the in vitro serine protease activity of PfSUB3 and its localization at asexual blood stages. In this study, we attempted to identify the biological substrate(s) of PfSUB3 and found parasite profilin (PfPRF) as a substrate of PfSUB3. Eukaryotic profilins are multifunctional proteins with primary role in regulation of actin filament assembly. PfPRF possesses biochemical features of eukaryotic profilins and its rodent ortholog is essential in blood stages. Profilin from related apicomplexan parasite Toxoplasma gondii (TgPRF) is known to be involved in parasite motility, host cell invasion, active egress from host cell, immune evasion and virulence in mice. In this study, mature PfSUB3 proteolysed recombinant PfPRF in a dose-dependent manner in in vitro assays. Recombinant PfPRF was assessed for its proinflammatory activity and found to induce high level of TNF-α and low but significant level of IL-12 from mouse bone marrow-derived dendritic cells. Proteolysis of PfPRF by PfSUB3 is suggestive of the probable role of the protease in the processes of motility, virulence and immune evasion.
    Molecular and Biochemical Parasitology 09/2013; · 2.73 Impact Factor
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    ABSTRACT: Current standard of care for sustained back of the eye drug delivery is surgical placement or injection of large, slow release implants using a relatively large 22 gauge needle. We designed novel dipeptide (phenylalanine-α,β-dehydrophenylalanine; Phe-∆Phe) based nanotubes with a diameter of ~15-30nm and a length of ~1500nm that could be injected with a 33 gauge needle for sustained intravitreal delivery of pazopanib, a multi-targeted tyrosine kinase inhibitor. The drug could be loaded during nanotube assembly or post-loaded after nanotube formation, with the former being more efficient at 25% w/w pazopanib loading and ~55% loading efficiency. Plain and peptide loaded nanotubes were non-cytotoxic to retinal pigment epithelial cells even at a concentration of 200μg/ml. Following intravitreal injection of fluorescently labeled nanotubes using a 33 gauge needle in a rat model, the nanotubes persistence and drug delivery were monitored using noninvasive fluorophotometry, electron microscopy and mass spectrometry analysis, respectively. Nanotubes persisted in the vitreous humor during the 15days study and pazopanib levels in the vitreous humor, retina, and choroid-RPE at the end of the study were 4.5, 5, and 2.5-fold higher compared to the plain drug. Thus, Phe-∆Phe nanotubes allow intravitreal injections with a small gauge needle and sustain drug delivery.
    Journal of Controlled Release 09/2013; · 7.63 Impact Factor
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    ABSTRACT: Monomeric hIAPP significantly destabilizes both model and live cell membranes by increasing membrane fluidity. This interaction with membranes happens via carpet formation followed by lipid extraction in a concentration dependent manner and thus we propose that hIAPP aggregation prior to membrane interaction may not be necessary for its cytotoxicity.
    Chemical Communications 08/2013; · 6.38 Impact Factor
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    ABSTRACT: Three de novo designed low molecular weight cationic peptides (IJ2, IJ3 and IJ4) containing an unnatural amino acid α, β - didehydrophenylalanine (∆Phe), exhibited potent antifungal activity against fluconazole (FLC) sensitive and resistant clinical isolates of Candida albicans as well as non-albicans, other yeast and filamentous pathogenic fungi. In present study, their synthesis, susceptibility of different fungi and the mechanism of anti-candidal action have been elucidated. The antimicrobial peptides (AMPs) were synthesized by solid-phase method and checked for antifungal activity against different yeasts and fungi by broth microdilution method. Anti-candidal mode of action of the peptides was investigated through detecting membrane permeabilization by confocal microscopy, reactive oxygen species (ROS) generation by fluorometry, apoptosis and necrosis by flow cytometry and cell wall damage using scanning and transmission electron microscopy. The MIC of the peptides against C. albicans, other yeast and filamentous fungal pathogens ranged between 3.91-250μM. All three peptides exhibited effect on multiple targets in C. albicans including disruption of cell wall structures, compromised cell membrane permeability leading to their enhanced entry into the cells, accumulation of ROS and induction of apoptosis. The peptides also showed synergistic effect when used in combination with fluconazole (FLC) and caspofungin (CAS) against C. albicans. The study suggests that the AMPs alone or in combination with conventional antifungals hold promise for the control of fungal pathogens, and need to be further explored for treatment of fungal infections.
    Biochimica et Biophysica Acta 07/2013; · 4.66 Impact Factor
  • Shaheena Parween, Ashraf Ali, Virander Singh Chauhan
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    ABSTRACT: Peptide based capping agents for gold nanoparticles (GNPs) are possible alternatives for capping and derivatizing GNPs, but suffer from a major disadvantage of sensitivity towards non specific proteases, which may limit their in-vivo utility. Using non-natural analogs of natural α-amino acids offer an attractive alternate strategy to circumvent this potential bottleneck in realizing full potential of peptide based capping gents for GNPs for biological applications. Here, we have designed and developed pentapeptides containing non-natural amino acid (α, β-dehydrophenylalanine and α-aminoisobutyric acid) as capping agents for GNPs. All these peptides were able to efficiently cap GNPs and peptide induced aggregation was not observed. Peptide capped GNPs showed minimal cytotoxicity to mammalian cell lines (HeLa and L929) as well as mice spleenocytes. They encapsulated small drug like molecules and peptide capped GNPs entrapping drugs were more efficient in killing HeLa cells compared to the free drug. Therefore, these non-natural amino acid containing peptide capped GNPs may be further developed as alternate drug delivery vehicles.
    ACS Applied Materials & Interfaces 06/2013; · 5.90 Impact Factor
  • Deepak Gaur, Virander S Chauhan
    The Indian Journal of Pediatrics 04/2013; · 0.72 Impact Factor
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    ABSTRACT: Drug resistant pathogenic fungi use several families of membrane-embedded transporters to efflux antifungal drugs from the cells. The efflux pump Cdr1 (Candida drug resistance 1), belongs to ATP-binding cassette (ABC) superfamily of transporters. Cdr1 is one of the most predominant mechanisms of multidrug resistance (MDR) in azole-resistant (AR) clinical isolates of Candida albicans. Blocking drug efflux represents an attractive approach to combat MDR of this opportunistic human pathogen. In this study, we rationally designed and synthesized transmembrane peptides mimics (TMPMs) of Cdr1 protein (Cdr1p) that correspond to each of the 12 transmembrane helices (TMHs) of the two transmembrane domains (TMDs) of the protein to target the primary structure of the Cdr1p. Several FITC-tagged TMPMs specifically bound to Cdr1p and blocked the efflux of entrapped fluorescent dyes from the AR (Gu5) isolate. These TMPMs did not affect the efflux of entrapped fluorescent dye from cells expressing the Cdr1p homologue Cdr2p or from cells expressing a non- ABC transporter Mdr1p. Notably, the time correlation of single photon counting fluorescence measurements confirmed the specific interaction of FITC-tagged TMPMs with their respective TMH. By using mutant variants of Cdr1p, we show that these TMPM antagonists contain the structural information necessary to target their respective TMHs of Cdr1p and specific binding sites that mediate the interactions between the mimics and its respective helix. Additionally, TMPMs that were devoid of any demonstrable hemolytic, cytotoxic and antifungal activities chemosensitize AR clinical isolates and demonstrated synergy with drugs that further improved the therapeutic potential of fluconazole in vivo.
    Journal of Biological Chemistry 04/2013; · 4.65 Impact Factor
  • Nidhi Hans, Shailja Singh, S K Jain, Virander S Chauhan
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    ABSTRACT: The clinical symptoms of malaria are attributed to the blood stage life cycle of parasite in which merozoite invades erythrocyte, undergoes multiplication and exit to re-invade into new erythrocyte to continue its life cycle. The interaction of repertoire of parasite proteins with host cell receptors is essential for invasion process. Identification, characterization and localization of the proteins involved in invasion will enrich our understanding of this complex process. In the present study we have identified a novel Apical Rhoptry Neck Protein in Plasmodium falciparum, which harbors a predicted signal and transmembrane domain and is conserved across the species. The transcription and translation analysis confirmed its expression in schizont stage of asexual cycle of P. falciparum. Immunoflouresence microscopy in schizonts and merozoites revealed its localization in the neck of rhoptries of P. falciparum. Furthermore, PfARNP has been found at the tight junction during invasion of P. falciparum merozoite to erythrocyte.
    Molecular and Biochemical Parasitology 03/2013; · 2.73 Impact Factor
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    ABSTRACT: hIAPP fibrillization implicated in Type 2 diabetes pathology involves formation of oligomers toxic to insulin producing pancreatic β-cells. We report design, synthesis, 3D structure and functional characterization of dehydrophenylalanine (ΔF) containing peptides which inhibit hIAPP fibrillization. The inhibitor protects β-cells from hIAPP induced toxicity.
    Chemical Communications 03/2013; 49(26):2688-90. · 6.38 Impact Factor
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    ABSTRACT: Background. Antibodies against VAR2CSA, the Plasmodium falciparum variant surface antigen that binds placental chondroitin sulfate A, have been suggested to mediate protection against malaria in pregnancy but also to be markers of infection. Here, we aimed to identify clinically relevant antibody responses taking into consideration variations in parasite exposure and HIV infection.Methods. IgGs against placental and pediatric isolates, VAR2CSA (DBL2X, DBL3X, DBL5ε, DBL6ε domains) and other blood stage antigens (DBLγ, DBLα, MSP119, AMA1, EBA175) were measured in plasmas from 293 Mozambican women at delivery. Associations between antibody responses, factors influencing malaria exposure, HIV infection and pregnancy outcomes were assessed.Results. Maternal antibodies were affected by placental infection, parity, season and neighbourhood of residence. HIV infection modified these associations and attenuated the parity-dependent increase of IgGs. High antibody levels against AMA1, DBL3X, DBL6ε, placental and paediatric isolates were associated with increased weight and gestational age of newborns (p≤0.036) among women with malaria episodes during pregnancy.Conclusions. Anti-parasite IgGs in women at delivery are affected by HIV infection as well as by variations in the exposure to P. falciparum. Heterogeneity of malaria transmission needs to be considered to identify IgGs against VAR2CSA and other parasite antigens associated with improved pregnancy outcomes.
    The Journal of Infectious Diseases 02/2013; · 5.85 Impact Factor
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    ABSTRACT: Aim: Different nanoparticles have been investigated to deliver chemotherapeutic agents, but complex synthesis procedures and biocompatibility issues raise concerns in developing them for safe human usage. The aim of this work is to develop α,β-dehydrophenylalanine-containing, self-assembled, amphipathic dipeptide nanoparticles for tumor-targeted drug delivery and therapy. Material & methods: Solution-phase peptide synthesis was used to synthesize dipeptides. Nanoparticles were prepared by molecular self-assembly. A tumor distribution study was carried out using a radiolabeling method. Tumor regression studies were carried out in murine ascitic tumors in BALB/c mice and breast tumor xenografts in nude mice. Results: Arg-α,β-dehydrophenylalanine formed self-assembled nanoparticles that could be easily derivatized with folic acid. Folic acid-derivatized nanoparticles showed enhanced cellular uptake and, when loaded with doxorubicin, showed enhanced tumor regression compared with underivatized nanoparticles or native drug, without any adverse side effects, both in vitro and in vivo. Original submitted 20 April 2012; Revised submitted 9 November 2012.
    Nanomedicine 02/2013; · 5.26 Impact Factor
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    ABSTRACT: Malaria remains a major health problem worldwide. All clinical symptoms of malaria are attributed to the asexual blood stages of the parasite life cycle. Proteins resident in apical organelles and present on the surface of P. falciparum merozoites are considered promising candidates for the development of blood stage malaria vaccines. In the present study, we have identified and characterized a microneme associated antigen, PfMA [PlasmoDB Gene ID: PF3D7_0316000, PFC0700c]. The gene was selected by applying a set of screening criteria such as transcriptional upregulation at late schizogony, inter-species conservation and the presence of signal sequence or transmembrane domains. The gene sequence of PfMA was found to be conserved amongst various Plasmodium species. We experimentally demonstrated that the transcript for PfMA was expressed only in the late blood stages of parasite consistent with a putative role in erythrocyte invasion. PfMA was localized by immunofluorescence and immuno-electron microscopy to be in the micronemes, an apical organelle of merozoites. The functional role of the PfMA protein in erythrocyte invasion was identified as a parasite adhesin involved in direct attachment with the target erythrocyte. PfMA was demonstrated to bind erythrocytes in a sialic acid independent, chymotrypsin and trypsin resistant manner and its antibodies inhibited P. falciparum erythrocyte invasion. Invasion of erythrocytes is a complex multistep process that involves a number of redundant ligand-receptor interactions many of which still remain unknown and even uncharacterized. Our work has identified and characterized a novel P. falciparum adhesin involved in erythrocyte invasion.
    PLoS ONE 01/2013; 8(9):e74790. · 3.53 Impact Factor

Publication Stats

1k Citations
372.89 Total Impact Points

Institutions

  • 1993–2014
    • International Centre for Genetic Engineering and Biotechnology
      • Plant Biology: Plant Molecular Biology Research Group
      Trst, Friuli Venezia Giulia, Italy
  • 2012
    • CRESIB Barcelona Centre for International Health Research
      • Barcelona Centre for International Health Research
      Barcelona, Catalonia, Spain
  • 2011
    • National Institute for Medical Research (NIMR)
      Dār es Salām, Dar es Salaam, Tanzania
  • 2010
    • IDIBAPS August Pi i Sunyer Biomedical Research Institute
      Barcino, Catalonia, Spain
  • 2009
    • Centro de Investigação em Saúde de Manhiça
      Lourenço Marques, Maputo City, Mozambique
    • Jamia Hamdard University
      New Dilli, NCT, India
  • 2005–2008
    • University of Delhi
      • • Department of Zoology (Faculty of Science)
      • • Department of Chemistry (Faculty of Science)
      Delhi, NCT, India
  • 2006
    • University of Melbourne
      Melbourne, Victoria, Australia
    • Instituto Técnico y Cultural
      Santa Clara de Portugal, Michoacán, Mexico
  • 2003
    • University of California, San Francisco
      • Department of Pediatrics
      San Francisco, CA, United States
  • 2002
    • Indian Institute of Science
      • Bioinformatics Centre
      Bengalore, State of Karnataka, India