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BACKGROUND: Tryptophan-rich antigens of malarial parasites have been proposed to be the potential vaccine candidate antigens. Plasmodium vivax contains the largest number of such antigens, which need to be evaluated for their immune responses. METHODS: Recombinant proteins of 15 P. vivax tryptophan-rich antigens (PvTRAgs) were expressed, purified, and used for the human humoral and cellular immune responses. Genetic polymorphism of these 15 genes was also determined among clinical P. vivax isolates. RESULTS: The T lymphocytes of P. vivax exposed individuals expressed higher level of CD69 against all 15 PvTRAgs. These antigens also activated the large population of CD4(+) T cells and produced higher level of intracellular IL-2, INF-γ and IL-4. Although there was a mixed Th1 and Th2 response against these antigens, this response was biased toward Th2. The majority of P. vivax patients (75.7%-100%, n = 33) produced IgG antibodies against these antigens. Most of these antigens showed conserved T- and B-cell epitopes in the parasite population. CONCLUSIONS: These results suggest the presence of memory T cells in humans against these antigens to generate faster and more specific immune responses to minimize the P. vivax infection. Further characterization of these PvTRAgs may lead to the identification of a potential therapeutic target.
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Anticuerpos Antiprotozoarios/biosíntesis , Antígenos de Protozoos/inmunología , Malaria Vivax/inmunología , Plasmodium vivax/inmunología , Triptófano/inmunología , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Secuencia de Bases , Secuencia Conservada , Humanos , Inmunidad Celular/inmunología , Inmunidad Humoral/inmunología , Inmunoglobulina G/biosíntesis , India/epidemiología , Interferón gamma/metabolismo , Interleucina-2/metabolismo , Interleucina-4/metabolismo , Malaria Vivax/epidemiología , Datos de Secuencia Molecular , Plasmodium vivax/genética , Polimorfismo Genético , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADN , Linfocitos T/inmunología , Células TH1/inmunología , Células Th2/inmunologíaRESUMEN
The quest for an ideal wound dressing material has been a strong motivation for researchers to explore novel biomaterials for this purpose. Such explorations have led to the extensive use of silk fibroin (SF) as a suitable polymer for several applications over the years. Unfortunately, another major silk protein-sericin has not received its due attention yet in spite of having favorable biological properties. In this study, we report an approach of blending SF and silk sericin (SS) without the usage of chemical crosslinkers is made possible by the usage of formic acid which evaporates to induceß-sheets formation to form cytocompatible films. Raman spectroscopy confirms the presence of SF/SS components in blend and formation ofß-sheet in films.In situ, gelation kinetics studies were conducted to understand the change in gelation properties with addition of sericin into SF. Methyl thiazolyl tetrazolium and live/dead assays were performed to study cellular attachment, viability and proliferation on SF/SS films. The antibacterial properties of SF/SS films were tested using Gram-negative and Gram-positive bacteria. The re-structured SF/SS films were stable, transparent, show good mechanical properties, antibacterial activity and cytocompatibility, therefore can serve as suitable biomaterial candidates for skin regeneration applications.
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Fibroínas , Sericinas , Sericinas/química , Fibroínas/química , Ingeniería de Tejidos , Materiales Biocompatibles/química , AntibacterianosRESUMEN
Natural materials derived/extracted Ceramics is an excellent material for developing ceramic-based orthopedic implants. Recently, we have demonstrated an easily scalable, energy-efficient green method to extract ceramic particles from bio-waste i.e. chicken bone. Though the chicken bone extract (CBE) has good biocompatibility, it lacks good mechanical properties in the 3D printed condition as that of human bones. Here, we have reinforced CBE with different weight proportions of silicon carbide to improve the mechanical characteristics of the composite. The hybrid of CBE (oxide) and carbide (SiC) is sintered at different temperatures to understand the effect of the interface of the two ceramics. It is observed that temperature has minimal effect and composition has a noticeable effect on mechanical strength as well as bio-toxicity. The toughness (â¼3.58 MJ/m3) and compressive strength (â¼64.64 MPa) of the 90:10 composition sintered at 1250 °C show the maximum optimum values. A mathematical model has also been developed to predict and correlate the toughness with porosity, volumetric loading, and elastic modulus of the 3D-printed ceramic composite.
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Óxidos , Prótesis e Implantes , Humanos , Ensayo de Materiales , Porosidad , Impresión Tridimensional , CerámicaRESUMEN
Chronic wounds suffer from impaired healing due to microbial attack and poor vascular growth. Thermoresponsive hydrogels gained attention in wound dressing owing to their gelation at physiological temperature enabling them to take the shape of asymmetric wounds. The present study delineates the development of thermoresponsive hydrogel (MCK), from hair-derived keratin (K) and methylcellulose (MC) in the presence of sodium sulfate. The gelation temperature (Tg) of this hydrogel is in the range of 30 °C to 33 °C. Protein-polymer interaction leading to thermoreversible sol-gel transition involved in MCK blends has been analyzed and confirmed by FTIR, XRD, and thermal studies. Keratin, has introduced antioxidant properties to the hydrogel imparted cytocompatibility towards human dermal fibroblasts (HDFs) as evidenced by both MTT and live dead assays. In vitro wound healing assessment has been shown by enhanced migration of HDFs in the presence of MCK hydrogel compared to the control. Also, CAM assay and CD31 expression by the Wistar rat model has shown increased blood vessel branching after the implantation of MCK hydrogel. Further, in vivo study, demonstrated MCK efficacy of hydrogel in accelerating full-thickness wounds with minimal scarring in Wistar rats, re-epithelialization, and reinstatement of the epidermal-dermal junction thereby exhibiting clinical relevance for chronic wounds.
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Queratinas , Repitelización , Ratas , Animales , Humanos , Queratinas/farmacología , Hidrogeles/farmacología , Metilcelulosa , Ratas Wistar , Cicatrización de HeridasRESUMEN
Chronic wounds are the outcome of an imbalanced inflammatory response caused by sustenance of immune microenvironment. In this context, tissue engineered graft played great role in healing wounds but faced difficulty in scar remodelling, immune rejection and poor vascularization. All the limitations faced are somewhere linked with the immune cells involved in healing. In this consideration, immunomodulatory biomaterials bridge a large gap with the delivery of modulating factors for triggering key inflammatory cells responsible towards interplay in the wound micro-environment. Inherent physico-chemical properties of biomaterials substantially determine the nature of cell-materials interaction thereby facilitating differential cytokine gradient involved in activation or suppression of inflammatory signalling pathways, and followed by surface marker expression. This review aims to systematically describe the interplay of immune cells involved in different phases in the wound microenvironment and biomaterials. Additionally, it also focuses on modulating innate immune cell responses in the context of triggering the halted phase of the wound healing, i.e., inflammatory phase. The various strategies are highlighted for modulation of wound microenvironment towards wound regeneration including stem cells, cytokines, growth factors, vitamins, and anti-inflammatory agents to induce interactive ability of biomaterials with immune cells. The last section focuses on prospective approaches and current potential strategies for wound regeneration. This includes the development of different models to bridge the gap between mouse models and human patients. Emerging new tools to study inflammatory response owing to biomaterials and novel strategies for modulation of monocyte and macrophage behaviour in the wound environment are also discussed.
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Materiales Biocompatibles , Interacción Gen-Ambiente , Animales , Ratones , Humanos , Materiales Biocompatibles/metabolismo , Macrófagos/metabolismo , Cicatrización de Heridas , Citocinas/metabolismo , InmunomodulaciónRESUMEN
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 (â¼30 m) followed by translocation to nucleus after â¼24 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.
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With the fast changing lifestyle, vitamin D deficiency is becoming extremely common. Therefore, development of economical, efficient, and fast sensors for vitamin D is the need of the hour. Carbon-based nanomaterials are extensively explored in sensing of variety of biomolecules. In the present study, an antibody-free, highly sensitive, carbon-nanotube-based, highly responsive vitamin D3 sensor is reported. Nitrogen-doped carbon nanotubes are utilized to overcome the limiting factor of hydrophobic character of pure carbon. The synthesized N-doped CNTs showed a specific surface area of 24 m2/g. The surface charges of vitamin D3 and the vitamin D3/NCNT complex are found to be -20 and -6.4 mV, respectively, by zeta potential measurements. The sensor is able to deliver high performance in the concentration range of 0-10 nM, with a limit of detection of 16 pM. The response study indicated the sensitivity value as 0.000495 mA/cm2 nM. The sensor is also able to show a higher selectivity toward vitamin D3 in comparison to other biomolecules. The long-term stability, reproducibility, good linear range, and ultralow detection capability of the sensor are also reported.
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Nanoestructuras , Nanotubos de Carbono , Colecalciferol , Nanotubos de Carbono/química , Nitrógeno/química , Reproducibilidad de los ResultadosRESUMEN
We describe here an approximately 40-kDa Plasmodium vivax tryptophan-rich antigen (PvTRAg40) which contains 321 amino acids and 11.4% tryptophan residues. This protein shows 65% homology (35% identity) with the previously described PvTRAg, besides sharing 23 of 27 positionally conserved tryptophan residues and similar genomic organization. The nucleotide sequence of the entire tryptophan-rich domain of PvTRAg40 was identical among 35 P. vivax clinical isolates. The protein is expressed by ring, trophozoite, and schizont stages of the parasite. The cDNA covering exon 2 of PvTRAg40 was cloned and expressed in the pPROEXHTa vector, and recombinant protein was purified. A high humoral immune response (90.7% seropositivity; n = 43) against this recombinant protein was detected in humans during the course of natural P. vivax infection. Eighty percent of the total of 20 P. vivax-exposed individuals exhibited lymphoproliferative responses against this antigen. The T cells of these individuals produced larger amounts of interleukin-12 (IL-12), IL-4, and IL-10 than gamma interferon and tumor necrosis factor alpha cytokines in response to the recombinant protein. Production of Th2-biased cytokines, conserved T- and B-cell epitopes, and an enhanced humoral immune response indicate that PvTRAg40 could possibly induce antibody-mediated immune protection against infection.
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Antígenos de Protozoos/química , Antígenos de Protozoos/inmunología , Plasmodium vivax/inmunología , Triptófano/química , Secuencia de Aminoácidos , Animales , Anticuerpos Antiprotozoarios/biosíntesis , Antígenos de Protozoos/genética , Antígenos de Protozoos/metabolismo , Secuencia de Bases , Citocinas/biosíntesis , Regulación de la Expresión Génica , Humanos , Inmunoglobulina G/biosíntesis , Activación de Linfocitos , Malaria Vivax/inmunología , Datos de Secuencia Molecular , Plasmodium vivax/genética , Plasmodium vivax/metabolismo , Alineación de Secuencia , Linfocitos T/inmunologíaRESUMEN
Present study describes the characterization of apical membrane antigen 1 (PvAMA1) polymorphisms among Indian Plasmodium vivax isolates. The partial PvAMA1 gene (covering domain I and domain II regions) sequenced from sixty-one (n=61) isolates in this study resulted into 49 haplotypes. Comparison with the previously available PvAMA1 sequences in the GenBank database revealed that 45 of these were new haplotypes that have never been reported till date. For further analyses, we also included 11 previously reported PvAMA1 sequences from India available in the database. Thus genetic diversity and effect of natural selection were analyzed both at domain I and domain II of this promising malaria vaccine candidate among 72 Indian P. vivax isolates. Non-synonymous mutations were found at 25 codons (16 at domain I and 9 at domain II) where 17 codons were dimorphic while rest of them (8 codons) were trimorphic. Thus codon polymorphisms were observed to be more at domain I as compared to domain II. Although the difference between the rate of non-synonymous (dN) and synonymous (dS) mutations was positive (dN-dS, 0.002+/-0.004SE) at domain II, it was not significantly different from each other (P=0.272), indicating tendency of stronger diversifying selection at this domain. The dN-dS difference for domain I (-0.006+/-0.009SE, P=0.268) and for entire 900 bp region (-0.002+/-0.005E, P=0.320) being negative and statistically insignificant suggests the role of both positive as well as purifying selection. Three-dimensional distributions of all polymorphic residues were mapped on a modeled PvAMA1 structure. Results suggested that almost all of the observed polymorphisms were located at one surface of the antigen. In conclusion, PvAMA1 antigen displays high diversity among Indian isolates with more diversifying selection at domain II. The result has significant value in malaria vaccine development using this antigen.
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Antígenos de Protozoos/química , Antígenos de Protozoos/genética , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Plasmodium vivax/genética , Polimorfismo Genético , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Selección Genética , Secuencia de Aminoácidos , Animales , Codón/química , Haplotipos , Humanos , India , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Recombinación Genética , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
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.
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Eritrocitos/metabolismo , Eritrocitos/parasitología , Plasmodium vivax/metabolismo , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Triptófano , Secuencia de Aminoácidos , Animales , Humanos , Malaria Vivax/sangre , Modelos Moleculares , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Plasmodium vivax/fisiología , Unión Proteica , Estructura Terciaria de Proteína , Receptores de Superficie Celular/metabolismoRESUMEN
RTS,S is the most advanced malaria vaccine candidate, currently under phase-III clinical trials in Africa. This Plasmodium falciparum vaccine contains part of the central repeat region and the complete C-terminal T cell epitope region (Th2R and Th3R) of the circumsporozoite protein (CSP). Since naturally occurring polymorphisms at the vaccine candidate loci are critical determinants of the protective efficacy of the vaccines, it is imperative to investigate these polymorphisms in field isolates. In this study we have investigated the genetic diversity at the central repeat, C-terminal T cell epitope (Th2R and Th3R) and N-terminal T cell epitope regions of the CSP, in P. falciparum isolates from Madhya Pradesh state of India. These isolates were collected through a 5-year prospective study aimed to develop a well-characterized field-site for the future evaluation of malaria vaccine in India. Our results revealed that the central repeat (63 haplotypes, nâ=â161) and C-terminal Th2R/Th3R epitope (24 haplotypes, n = 179) regions were highly polymorphic, whereas N-terminal non-repeat region was less polymorphic (5 haplotypes, n = 161) in this population. We did not find any evidence of the role of positive natural selection in maintaining the genetic diversity at the Th2R/Th3R regions of CSP. Comparative analysis of the Th2R/Th3R sequences from this study to the global isolates (n = 1160) retrieved from the GenBank database revealed two important points. First, the majority of the sequences (~61%, n = 179) from this study were identical to the Dd2/Indochina type, which is also the predominant Th2R/Th3R haplotype in Asia (~59%, n = 974). Second, the Th2R/Th3R sequences in Asia, South America and Africa are geographically distinct with little allele sharing between continents. In conclusion, this study provides an insight on the existing polymorphisms in the CSP in a parasite population from India that could potentially influence the efficacy of RTS,S vaccine in this region.
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Variación Genética , Vacunas contra la Malaria/genética , Plasmodium falciparum/genética , Proteínas Protozoarias/genética , Adolescente , Adulto , Secuencia de Aminoácidos , Niño , Epítopos de Linfocito T/inmunología , Femenino , Genotipo , Geografía , Haplotipos , Humanos , India/epidemiología , Vacunas contra la Malaria/inmunología , Malaria Falciparum/epidemiología , Malaria Falciparum/inmunología , Malaria Falciparum/parasitología , Masculino , Datos de Secuencia Molecular , Filogenia , Plasmodium falciparum/inmunología , Plasmodium falciparum/aislamiento & purificación , Polimorfismo Genético , Estudios Prospectivos , Proteínas Protozoarias/clasificación , Proteínas Protozoarias/inmunología , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Adulto JovenRESUMEN
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.
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Antígenos de Protozoos/química , Plasmodium vivax/química , Proteínas Protozoarias/química , Transporte Biológico , Epítopos de Linfocito B/inmunología , Humanos , Concentración de Iones de Hidrógeno , Inmunidad , Datos de Secuencia Molecular , Plasmodium vivax/inmunología , Estructura Secundaria de Proteína , Proteínas Protozoarias/inmunología , Triptófano/químicaRESUMEN
OBJECTIVE: Glial cells missing 2 (GCM2) gene encodes a parathyroid-specific transcription factor. We assessed GCM2 gene sequence in patients with isolated hypoparathyroidism (IH). DESIGN: Case-control study. METHODS: Complete DNA sequencing of the GCM2 gene including its exons, promoter, and 5' and 3' UTRs was performed in 24/101 patients with IH. PCR-restriction fragment length polymorphism was used to detect a novel R110W mutation in all 101 IH patients and 655 healthy controls. Significance of the mutation was assessed by electrophoretic mobility shift assay (EMSA) and nuclear localization on transfection. RESULTS: A heterozygous R110W mutation was present in DNA-binding domain in 11/101 patients (10.9%) and absent in 655 controls (P<10(-7)). Four of 13 nonaffected first-degree relatives for five of these index cases had R110W mutation. Four heterozygous single nucleotide polymorphisms were found in the 5' region. One of the 11 patients with R110W also had T370M change in compound heterozygous form. Mutant R110W and T370M GCM2 proteins showed decreased binding with GCM recognition elements on EMSA indicating loss of function. Both wild-type and R110W mutant GCM2 proteins showed nuclear localization. CONCLUSIONS: The present study indicates a significant association of R110W variant with IH. Absence of effect of heterozygous R110W mutation on DNA binding and presence of the same mutation in asymptomatic family members indicate that additional genetic (akin to T370M change) or nongenetic factors might contribute to the expression of diseases in IH. Alternatively, it is possible that association of R110W with IH could be due to linkage disequilibrium with the unidentified relevant genes in IH.
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Hipoparatiroidismo/epidemiología , Hipoparatiroidismo/genética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Mutación Puntual , Factores de Transcripción/química , Factores de Transcripción/genética , Estudios de Casos y Controles , Análisis Mutacional de ADN , Salud de la Familia , Femenino , Predisposición Genética a la Enfermedad/epidemiología , Células HeLa , Heterocigoto , Humanos , Masculino , Linaje , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Polimorfismo de Nucleótido Simple , Prevalencia , Estructura Terciaria de ProteínaRESUMEN
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.
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Anopheles/genética , ADN Mitocondrial/genética , Polimorfismo Genético , Animales , Secuencia de Bases , Citocromos b/genética , ADN Mitocondrial/química , Complejo IV de Transporte de Electrones/genética , Genotipo , India , Datos de Secuencia Molecular , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
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 His6-TRD. The purified recombinant His6-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 His6-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-gamma, TNF-alpha, 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.
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Antígenos de Protozoos/inmunología , Antígenos de Protozoos/metabolismo , Plasmodium vivax/inmunología , Adulto , Animales , Anticuerpos Antiprotozoarios/sangre , Antígenos de Protozoos/química , Antígenos de Protozoos/genética , Proliferación Celular , Niño , Secuencia Conservada , ADN Protozoario/química , ADN Protozoario/genética , Ensayo de Inmunoadsorción Enzimática , Eritrocitos/metabolismo , Escherichia coli/genética , Femenino , Expresión Génica , Humanos , Linfocinas/metabolismo , Masculino , Datos de Secuencia Molecular , Peso Molecular , Plasmodium vivax/genética , Polimorfismo Genético , Unión Proteica , Estructura Terciaria de Proteína , Secuencias Repetitivas de Aminoácido , Análisis de Secuencia de ADN , Linfocitos T/inmunologíaRESUMEN
Anopheles (Cellia) stephensi Liston 1901 is one of the major malaria vectors in the Indian subcontinent, Iran, and the Middle East. Three races in this species, namely A. stephensi stephensi (type form), A. stephensi variety mysorensis, and A. stephensi intermediate form, have earlier been reported by several investigators. We describe here the sequencing of the ribosomal DNA internal transcribed spacer 2 (ITS2) and domain-3 (D3) loci of the A. stephensi type and variety mysorensis forms. We also sequenced field-collected adult specimens of this mosquito from three different regions of India. Both forms of A. stephensi showed identical ITS2 and D3 sequences. We did not find any intraspecies sequence variation among the 70 specimens sequenced in this study. In contrast to the eight ITS2 haplotypes observed among Iranian A. stephensi population, we found only one ITS2 haplotype in India. This is the first time to our knowledge that the sequence of the D3 locus of A. stephensi is being reported here. In conclusion, the type and variety mysorensis forms of A. stephensi exhibit identical nucleotide sequences at their ITS2 and D3 loci.
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Anopheles/clasificación , Anopheles/genética , ADN Espaciador Ribosómico/genética , Animales , Secuencia de Bases , India , Datos de Secuencia MolecularRESUMEN
The antifolate drugs sulfadoxine and pyrimethamine are commonly used to treat Plasmodium falciparum malaria. However, they can also affect the Plasmodium vivax parasite if it coexists with P. falciparum, as both species have common drug targets. Resistance to the antifolate drugs arises due to point mutations in the target enzymes of the respective parasite. To assess the cross-species impact of antifolate drug treatment, we describe here the dihydrofolate reductase (DHFR) mutations among field isolates of P. vivax and P. falciparum. The overall DHFR mutation rate for P. vivax was lower than that for P. falciparum. However, both species of Plasmodium followed similar trends of DHFR mutations. Similar to P. falciparum, the DHFR mutation rate of P. vivax also varied from region to region. It was lower in P. vivax-dominant regions but higher in the P. falciparum-dominated areas and highest where antifolates are used as the first line of antimalarial treatment. In conclusion, the antifolate treatment of falciparum malaria is proportionately affecting the DHFR mutations of P. vivax, suggesting that the drug should be used with caution to minimize the development of cross-species resistance in the field.