Capra cartilage-derived peptide delivery via carbon nano-dots for cartilage regeneration

Targeted delivery of site-specific therapeutic agents is an effective strategy for osteoarthritis treatment. The lack of blood vessels in cartilage makes it difficult to deliver therapeutic agents like peptides to the defect area. Therefore, nucleus-targeting zwitterionic carbon nano-dots (CDs) have immense potential as a delivery vehicle for effective peptide delivery to the cytoplasm as well as nucleus. In the present study, nucleus-targeting zwitterionic CDs have been synthesized as delivery vehicle for peptides while also working as nano-agents towards optical monitoring of cartilage healing. The functional groups of zwitterion CDs were introduced by a single-step microwave assisted oxidation procedure followed by COL II peptide conjugation derived from Capra auricular cartilage through NHS/EDC coupling. The peptide-conjugated CDs (PCDs) allows cytoplasmic uptake within a short period of time (∼30 m) followed by translocation to nucleus after ∼24 h. Moreover, multicolor fluorescence of PCDs improves (blue, green, and read channel) its sensitivity as an optical code providing a compelling solution towards enhanced non-invasive tracking system with multifunctional properties. The PCDs-based delivery system developed in this study has exhibited superior ability to induce ex-vivo chondrogenic differentiation of ADMSCs as compared to bare CDs. For assessment of cartilage regeneration potential, pluronic F-127 based PCDs hydrogel was injected to rabbit auricular cartilage defects and potential healing was observed after 60 days. Therefore, the results confirm that PCDs could be an ideal alternate for multimodal therapeutic agents

Plasmodium vivax Tryptophan-Rich Antigen PvTRAg33.5 Contains Alpha Helical Structure and Multidomain Architecture

Tryptophan-rich proteins from several malarial parasites have been identified where they play an important role in host-parasite interaction. Structural characterization of these proteins is needed to develop them as therapeutic targets. Here, we describe a novel Plasmodium vivax tryptophan-rich protein named PvTRAg33.5. It is expressed by blood stage(s) of the parasite and its gene contains two exons. The exon 1 encodes for a 23 amino acids long putative signal peptide which is likely to be cleaved off whereas the exon 2 encodes for the mature protein of 252 amino acids. The mature protein contains B-cell epitopes which were recognized by the human immune system during P.vivax infection. The PvTRAg33.5 contains 24 (9.5%) tryptophan residues and six motifs whose patterns were similar among tryptophan-rich proteins. The modeled structure of the PvTRAg33.5 consists of a multidomain architecture which is stabilized by the presence of large number of tryptophan residues. The recombinant PvTRAg33.5 showed predominantly α helical structure and alpha helix to beta sheet transition at pH below 4.5. Protein acquires an irreversible non-native state at temperature more than 50°C at neutral pH. Its secondary and tertiary structures remain stable in the presence of 35% alcohol but these structures are destabilized at higher alcohol concentrations due to the disturbance of hydrophobic interactions between tryptophanyl residues. These structural changes in the protein might occur during its translocation to interact with other proteins at its final destination for biological function such as erythrocyte invasion

Defining the erythrocyte binding domains of Plasmodium vivax tryptophan rich antigen 33.5.

Tryptophan-rich antigens play important role in host-parasite interaction. One of the Plasmodium vivax tryptophan-rich antigens called PvTRAg33.5 had earlier been shown to be predominantly of alpha helical in nature with multidomain structure, induced immune responses in humans, binds to host erythrocytes, and its sequence is highly conserved in the parasite population. In the present study, we divided this protein into three different parts i.e. N-terminal (amino acid position 24-106), middle (amino acid position 107-192), and C-terminal region (amino acid position 185-275) and determined the erythrocyte binding activity of these fragments. This binding activity was retained by the middle and C-terminal fragments covering 107 to 275 amino acid region of the PvTRAg33.5 protein. Eight non-overlapping peptides covering this 107 to 275 amino acid region were then synthesized and tested for their erythrocyte binding activity to further define the binding domains. Only two peptides, peptide P4 (at 171-191 amino acid position) and peptide P8 (at 255-275 amino acid position), were found to contain the erythrocyte binding activity. Competition assay revealed that each peptide recognizes its own erythrocyte receptor. These two peptides were found to be located on two parallel helices at one end of the protein in the modelled structure and could be exposed on its surface to form a suitable site for protein-protein interaction. Natural antibodies present in the sera of the P. vivax exposed individuals or the polyclonal rabbit antibodies against this protein were able to inhibit the erythrocyte binding activity of PvTRAg33.5, its fragments, and these two synthetic peptides P4 and P8. Further studies on receptor-ligand interaction might lead to the development of the therapeutic reagent

Variations in the mitochondrial DNA markers in the Anopheles (Cellia) sundaicus population from the Andaman and Nicobar Islands, India

Four sibling species in the Anopheles sundaicus complex have earlier been reported, including species D from the Andaman and Nicobar Islands of India where it constitutes 58% of all Anopheles population and is a major malaria vector. Earlier, we have reported the identical sequences for ribosomal DNA markers among the specimens of A. sundaicus from Andaman and Nicobar islands irrespective of their habitat. These ITS2 sequences were also identical to the reported sequence of variant III of Southeast Asian A. sundaicus. In the present study, we describe variations in three mitochondrial DNA markers among these specimens from Andaman and Nicobar islands. There were two different genotypes for each locus of COI and COII, and three genotypes for cytochrome-b (Cyt-b) locus resulting in three different combined genotypes (genotypes I, II and III) in the population. Specimens with combined genotype I (59%, n=100) were found only among the A. sundaicus population breeding in fresh water whereas two different multi-loci genotypes i.e. genotype II (25%, n=100) and genotype III (16%, n=100) were present in the population breeding in brackish water. Thus, the A. sundaicus population breeding in fresh water was homogenous with single multi-loci genotype and can be distinguished from the heterogenous mosquito population breeding in brackish water with these markers. These Cyt-b and COI sequences of A. sundaicus species D were also different from the reported Southeast Asian species of A. sundaicus

High immunogenecity and erythrocyte-binding activity in the tryptophan-rich domain (TRD) of the 74-kDa Plasmodium vivax alanine-tryptophan-rich antigen (PvATRAg74)

Plasmodium vivax is the most widespread species of human malaria parasite affecting 70-80 million people worldwide each year. In recent years, some potential vaccine candidate antigens from P. vivax have been identified including tryptophan-rich antigens PvTRAg and PvTARAg55. We report here the identification and partial characterization of a 74 kDa P. vivax alanine-tryptophan-rich antigen (PvATRAg74) which is expressed by all asexual blood stages of the parasite. This protein contains two major domains, i.e. alanine-rich domain (ARD) in N-terminal region and the tryptophan-rich domain (TRD) at C-terminus. PvATRAg74 also contains variable numbers of octa-peptide repeats in the ARD region. The C-terminal PvATRAg74 containing TRD was highly conserved among 32 P. vivax isolates while N-terminal ARD showed genetic polymorphisms. The 36 kDa TRD was expressed in E. coli and named here as His<SUB>6</SUB>-TRD. The purified recombinant His<SUB>6</SUB>-TRD showed binding with uninfected human erythrocytes. This antigen was also recognized by all 38 P. vivax patients' sera on ELISA thus showing a very high seropositivity rates. In vitro stimulation of lymphocytes with purified His<SUB>6</SUB>-TRD indicated that it induced T cell immune response in majority (94%, n=16) of P. vivax exposed individuals. The stimulated T cells produced higher amount of IL-4 and IL-10 than IFN-γ, TNF-α, and IL-12 indicating a Th2 type of response bias. Unlike PvTARAg55, this antigen is more immunogenic in humans and possesses the erythrocyte-binding activity. Immunogenecity of PvATRAg74 is similar to PvTRAg whose erythrocyte-binding activity still remains unknown

Determination of erythrocyte binding regions of PvTRAg33.5.

<p>(<b>A</b>) Schematic representation of PvTRAg33.5. Exon 1 encodes for a 23 amino acid signal peptide. Wavy lines indicate the intron. Exon 2 (shaded grey) encodes the mature protein which was fragmented in to three parts i.e. N-PvTRAg33.5, M-PvTRAg33.5 and C-PvTRAg33.5. (<b>B</b>) Cell-ELISA showing erythrocyte binding of Histidine-tagged PvTRAg33.5 and its three fragments with human erythrocytes (C-PvTRAg33.5 was GST-tagged). Increasing concentrations of the purified recombinant proteins were allowed to bind with ∼1 million erythrocytes in a microtiter plate and reacted with primary anti-His₆ or anti GST antibody and HRP conjugated secondary antibody. Recombinant Histidine-tagged thioredoxin from <i>D. desulfuricans</i> was used as negative control. Error bar indicates the standard deviation of mean from three experiments. Int, intron.</p

Location of erythrocyte binding peptide domains on modeled PvTRAg33.5.

<p>PvTRAg33.5 has three subdomains. Subdomain 1 (blue), Subdomain 2 (red), and subdomain 3 (green). The helices of different subdomains are shown in respective colors and coiled structure as loops. Erythrocyte binding peptides P4 (in helix 6) and P8 (in helix 8) are shown in cyan color.</p

Inhibition of erythrocyte binding of PvTRAg33.5 derived fragments and peptides by rabbit anti-PvTRAg33.5 antibody.

<p>The tagged recombinant PvTRAg33.5, its fragments, or synthetic peptides were mixed with different dilutions of polyclonal antisera raised in rabbit against PvTRAg33.5 before adding to the microtiter plate coated with erythrocytes. Further steps of color development were same as in Fig. 1 and 2B. Binding in the absence of antibody was taken as percentage control. Error bar indicates the standard deviation of mean from three experiments. No Ab, no antibody; PIS, pre-immune rabbit sera.</p

Determination of erythrocyte peptide binding domains of PvTRAg33.5.

<p>(<b>A</b>) Schematic representation of eight non-overlapping peptides designed from M-PvTRAg33.5 and C-PvTRAg33.5 fragments. Amino acid sequence and name of each peptide along with residue numbers is shown. (<b>B</b>) Cell-ELISA showing erythrocyte binding affinity of these synthetic non overlapping peptides. Increasing concentrations of these peptides were allowed to bind with erythrocytes. Reaction with primary and secondary antibodies was carried out as described in Fig. 1B. Recombinant thioredoxin from <i>D. desulfuricans</i> was used as negative controls. Mean± SD value of absorbance from three experiments is plotted. SP, signal peptide; Int, intron.</p

Inhibition of erythrocyte binding of PvTRAg33.5 derived fragments and peptides by <i>P. vivax</i> patients’ sera.

<p>Pooled <i>P. vivax</i> infected patients’ sera (<b>A</b>) and by purified anti-PvTRAg33.5 antibodies from these pooled patients sera (<b>B</b>) were used for these antibody mediated inhibition assays. The tagged recombinant PvTRAg33.5, its fragments, or synthetic peptides were mixed with different dilutions of <i>P.vivax</i> patients’ sera or purified antibodies before adding to the microtiter plate coated with one million erythrocytes. Further steps of color development were same as in Fig. 1 and 2B. Binding in the absence of antibody is taken as percentage control. Error bar indicates the standard deviation of mean from three experiments. No Ab, no antibody; HCS, healthy human control sera.</p