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Molecular mechanisms of red cell invasion by the Plasmodium vivax parasite remain obscure since information on receptor-ligand interaction is scarce. Our lab had identified Basigin as receptor molecule for one of the important members of Pvfam “a’ family protein i.e., PvTRAg38. Here, we demonstrate by using solid phase binding assay, and Surface Plasmon Resonance that seven out of ten-erythrocyte binding Pvfam ‘a’ family proteins of P. vivax interact with erythrocyte receptor Basigin. This receptor-ligand interaction seems to be important for parasite’s survival since each of these proteins interfered with the parasite growth in a heterologous culture system. Furthermore, a higher parasite growth inhibition rate was achieved with combination of proteins suggesting the significance of multiple parasite ligands interaction with the same erythrocyte receptor during invasion process. These results will be helpful in understanding P. vivax biology and developing the therapeutics for vivax malaria.

This research work has designed an algorithm to identify islanding events using the current signals in a distribution grid interfaced with renewable energy (RE) sources situated in remote areas. A median-based islanding recognition factor (MIRF) is designed by processing the current signal using Stockwell transform (ST). A current rate of change of islanding recognition factor (CRCIRF) is computed by differentiating the root mean square (RMS) current concerning time. The MIRF and CRCIRF are multiplied element by element to calculate the current-based islanding recognition factor (IRFC) used to recognize islanding events and non-islanding events. Simple decision rules are used to discriminate Islanding events from the faulty and the operational events by comparing peak magnitude of IRFC with pre-set threshold values. This IDM effectively recognizes islanding events in the presence of noise with 10 dB signal-to-noise ratio (SNR) level. The performance of IDM is established on a practical distribution feeder. Developed work is executed in MATLAB/Simulink.

Background Crucial gaps in our understanding of Plasmodium vivax reticulocyte invasion and protective immunity have hampered development of vivax vaccines. P. vivax exclusively invades reticulocytes that is mediated by the P. vivax reticulocyte-binding proteins (PvRBPs) specifically PvRBP2c and PvRBP1a. Vivax infections in Duffy-null individuals have suggested the evolution of alternate invasion pathways that may be mediated by the PvRBPs. Thus, PvRBPs appear as potential targets for efficacious P. vivax neutralization. However, there are limited data validating their vaccine efficacy. In the absence of vivax invasion assays, binding-inhibitory activity of antibodies has been reported to be associated with protection and a measure of vaccine potential. Methods -based analysis was performed of the PvRBP reticulocyte-binding properties and binding-inhibitory activity of specific anti-PvRBP2c/PvRBP1a human antibodies. Results PvRBP2c and PvRBP1a displayed a distinct reticulocyte-binding specificity, and their specific reticulocyte-binding domains were mapped within their N-terminal regions. Importantly, naturally acquired antibodies against the reticulocyte-binding domains efficaciously blocked reticulocyte binding of native PvRBPs, suggesting that the human immune system produced functional binding-inhibitory antibodies through exposure to vivax malaria. Conclusions Reticulocyte-binding domains of PvRBP2c/PvRBP1a are targets of naturally acquired binding-inhibitory antibodies, substantiating their promise as candidate antigens against which vaccine-inducible immunity could potentially be boosted through natural infections.

Elucidating the molecular mechanisms of the host-parasite interaction during red cell invasion by Plasmodium is important for developing newer antimalarial therapeutics. Recently, we have characterized a Plasmodium vivax tryptophan-rich antigen PvTRAg38, which is expressed by its merozoites, binds to host erythrocytes, and interferes with parasite growth. Interaction of this parasite ligand with the host erythrocyte occurs through its two regions present at amino acid positions 167–178 (P2) and 197–208 (P4). Each region recognizes its own erythrocyte receptor. Previously, we identified band 3 as the chymotrypsin-sensitive erythrocyte receptor for the P4 region, but the other receptor, binding to P2 region, remained unknown. Here, we have identified basigin as the second erythrocyte receptor for PvTRAg38, which is resistant to chymotrypsin. The specificity of interaction between PvTRAg38 and basigin was confirmed by direct interaction where basigin was specifically recognized by P2 and not by the P4 region of this parasite ligand. Interaction between P2 and basigin is stabilized through multiple amino acid residues, but Gly-171 and Leu-175 of P2 were more critical. These two amino acids were also critical for parasite growth. Synthetic peptides P2 and P4 of PvTRAg38 interfered with the parasite growth independently but had an additive effect if combined together indicating involvement of both the receptors during red cell invasion. In conclusion, PvTRAg38 binds to two erythrocyte receptors basigin and band 3 through P2 and P4 regions, respectively, to facilitate parasite growth. This advancement in our knowledge on molecular mechanisms of host-parasite interaction can be exploited to develop therapeutics against P. vivax malaria.

Plasmodium tryptophan rich proteins play important role in host-parasite interaction. Earlier, we have described that one of the merozoite expressed Plasmodium vivax tryptophan-rich antigen PvTRAg35.2 binds to the host erythrocytes, have conserved sequences in parasite population, and generates humoral as well as cellular immune responses in humans during this parasitic infection. Here, we show that PvTRAg35.2 interferes with the parasite growth in a heterologous Plasmodium falciparum culture system. This probably suggests the recognition of the common erythrocyte receptor(s) by certain merozoite ligands of these two parasite species. We have mapped the erythrocyte binding activity of PvTRAg35.2 to its two different regions positioned at amino acid residues 155-190 and 263-283. Binding of these peptide domains to the erythrocytes was inhibited by anti-PvTRAg35.2 antibodies either raised in rabbit or produced by the P. vivax patients. The cross-competition between peptides of PvTRAg35.2 and PvTRAg33.5 or PvTRAg38 during erythrocyte binding assay suggested sharing of host cell receptors by these PvTRAgs. Further studies on these receptor-ligand interactions may lead to the development of therapeutic agents for P. vivax malaria.

Tryptophan-rich antigens of malarial parasites interact with host molecules and play an important role in parasite survival. Merozoite expressed Plasmodium vivax tryptophan-rich antigen PvTRAg38 binds to human erythrocytes and facilitates parasite growth in a heterlologous Plasmodium falciparum culture system. Recently, we identified band 3 in human erythrocytes as one of its receptors, although the receptor-ligand binding mechanisms remain unknown. In the present study, using synthetic mutated peptides of PvTRAg38, we show that multiple amino acid residues of its 12 amino acid domain (KWVQWKNDKIRS) at position 197-208 interact with three different ectodomains of band 3 receptor on human erythrocytes. Our findings may help in the design of new therapeutic approaches for malaria.

Clearance of non-infected red blood cells (nRBCs) is one of the main components of anemia associated with Plasmodium vivax malaria. Recently, we have shown that anemic patients with P. vivax infection had elevated levels of anti-RBCs antibodies, which could enhance in vitro phagocytosis of nRBCs and decrease their deformability. Using immunoproteomics, here we characterized erythrocytic antigens that are differentially recognized by autoantibodies from anemic and non-anemic patients with acute vivax malaria. Protein spots exclusively recognized by anemic P. vivax-infected patients were identified by mass spectrometry revealing band 3 and spectrin as the main targets. To confirm this finding, antibody responses against these specific proteins were assessed by ELISA. In addition, an inverse association between hemoglobin and anti-band 3 or anti-spectrin antibodies levels was found. Anemic patients had higher levels of IgG against both band 3 and spectrin than the non-anemic ones. To determine if these autoantibodies were elicited because of molecular mimicry, we used in silico analysis and identified P. vivax proteins that share homology with human RBC proteins such as spectrin, suggesting that infection drives autoimmune responses. These findings suggest that band 3 and spectrin are potential targets of autoantibodies that may be relevant for P. vivax malaria-associated anemia.

BACKGROUND Plasmodium vivax synthesizes the largest number of 36 tryptophan-rich proteins belonging to the Pv-fam-a family. These parasite proteins need to be characterized for their biological function because tryptophan-rich proteins from other Plasmodium species have been proposed as vaccine candidates. METHODS Recombinant P. vivax tryptophan-rich antigens (PvTRAgs) were used to determine their erythrocyte-binding activity by a cell-based enzyme-linked immunosorbent assay, flow cytometry, and a rosetting assay. RESULTS Only 4 (PvTRAg26.3, PvTRAg34, PvTRAg36, and PvTRAg36.6) of 21 PvTRAgs bind to host erythrocytes. The cross-competition data indicated that PvTRAg36 and PvTRAg34 share their erythrocyte receptors with previously described proteins PvTRAg38 and PvTRAg33.5, respectively. On the other hand, PvTRAg26.3 and PvTRAg36.6 cross-compete with each other and not with any other PvTRAg, indicating that these 2 proteins bind to the same but yet another set of erythrocyte receptor(s). Together, 10 of 36 PvTRAgs possess erythrocyte-binding activity in which each protein recognizes >1 erythrocyte receptor. Further, each erythrocyte receptor is shared by >1 PvTRAg. CONCLUSIONS This redundancy may be useful for the parasite to invade red blood cells and cause disease pathogenesis, and it can be exploited to develop therapeutics against P. vivax malaria.

We demonstrate polarization-dependent photo-response enhancement in metamaterial-coupled quantum dots-in-a-well infrared detectors. A gold split-ring resonator metamaterial layer was patterned by electron-beam lithography in the detector aperture. In this integrated structure, the detector spectral response is given by the convolution of the metamaterial field enhancement and the original detector response. Our polarization-resolved measurement unambiguously shows that the spectral response can be strongly modified by metamaterial patterning. When the metamaterial resonance matches the QD absorption peak, we obtain a clear enhancement of generated photocurrent. Various metamaterial designs can be employed to implement multi-functional detector structures.

Objectives Human Plasmodium knowlesi infections have been reported from several South-East Asian countries, excluding India, but its drug susceptibility profile in mixed-infection cases remains unknown. Methods The chloroquine resistance transporter (CRT) and dihydrofolate reductase (DHFR) genes of P. knowlesi and other Plasmodium species were sequenced from clinical isolates obtained from malaria patients living in the Andaman and Nicobar Islands, India. The merozoite surface protein-1 and 18S rRNA genes of P. knowlesi were also sequenced from these isolates. Results Among 445 samples analysed, only 53 of them had P. knowlesi-specific gene sequences. While 3 of the 53 cases (5.66%) had P. knowlesi monoinfection, the rest were coinfected with Plasmodium falciparum (86.79%, n = 46) or Plasmodium vivax (7.55%, n = 4), but none with Plasmodium malariae or Plasmodium ovale. There was discordance in the drug resistance-associated mutations among the coinfecting Plasmodium species. This is because the P. knowlesi isolates contained wild-type sequences, while P. falciparum isolates had mutations in the CRT and DHFR marker genes associated with a higher level of chloroquine and antifolate drug resistance, respectively. The mutation pattern indicates that the same patient, having a mixed infection, may be harbouring the drug-susceptible P. knowlesi parasite and a highly drug-resistant P. falciparum parasite. Conclusions A larger human population in South-East Asia may be at risk of P. knowlesi infection than reported so far. The different drug susceptibility genotypes of P. knowlesi from its coinfecting Plasmodium species in mixed infections adds a new dimension to the malaria control programme, requiring formulation of an appropriate drug policy.

Mutations in the chloroquine resistance (CQR) transporter gene of Plasmodium falciparum (Pf crt ; chromosome 7) play a key role in CQR, while mutations in the multidrug resistance gene (Pf mdr1 ; chromosome 5) play a significant role in the parasite's resistance to a variety of antimalarials and also modulate CQR. To compare patterns of genetic variation at Pf crt and Pf mdr1 loci, we investigated 460 blood samples from P. falciparum -infected patients from four Asian, three African, and three South American countries, analyzing microsatellite (MS) loci flanking Pf crt (five loci [∼40 kb]) and Pf mdr1 (either two loci [∼5 kb] or four loci [∼10 kb]). CQR Pf mdr1 allele-associated MS haplotypes showed considerably higher genetic diversity and higher levels of subdivision than CQR Pf crt allele-associated MS haplotypes in both Asian and African parasite populations. However, both Pf crt and Pf mdr1 MS haplotypes showed similar levels of low diversity in South American parasite populations. Median-joining network analyses showed that the Pf crt MS haplotypes correlated well with geography and CQR Pf crt alleles, whereas there was no distinct Pf mdr1 MS haplotype that correlated with geography and/or CQR Pf mdr1 alleles. Furthermore, multiple independent origins of CQR Pf mdr1 alleles in Asia and Africa were inferred. These results suggest that variation at Pf crt and Pf mdr1 loci in both Asian and African parasite populations is generated and/or maintained via substantially different mechanisms. Since Pf mdr1 mutations may be associated with resistance to artemisinin combination therapies that are replacing CQ, particularly in Africa, it is important to determine if, and how, the genetic characteristics of this locus change over time.

Elucidation of molecular mechanisms of receptor-ligand biology during host-parasite interaction helps in developing therapeutic targets. Several Pv-fam-a family proteins of Plasmodium vivax bind to host erythrocytes but their erythrocyte receptors remains to be explored. Here, we show that three merozoite proteins (PvTRAg36, PvATRAg74, and PvTRAg38) of this family interact with Band 3 on human erythrocytes through its three exofacial loops (loop 1, loop 3, and loop 6). These parasite proteins also interfered with the parasite growth in in-vitro, and the inhibition rate seems to be associated with their binding affinity to Band 3. This redundancy in receptor-ligand interaction could be one of the probable mechanism parasite utilizes to invade the host erythrocyte more efficiently.

Plasmodium vivax is most wide spread and a neglected malaria parasite. There is a lack of information on parasite biology of this species. Genome of this parasite encodes for the largest number of tryptophan-rich proteins belonging to ‘Pv-fam-a’ family and some of them are potential drug/vaccine targets but their functional role(s) largely remains unexplored. Using bacterial and yeast two hybrid systems, we have identified the interacting partners for two of the P. vivax tryptophan-rich antigens called PvTRAg36.6 and PvTRAg56.2. The PvTRAg36.6 interacts with early transcribed membrane protein (ETRAMP) of P.vivax. It is apically localized in merozoites but in early stages it is seen in parasite periphery suggesting its likely involvement in parasitophorous vacuole membrane (PVM) development or maintenance. On the other hand, PvTRAg56.2 interacts with P.vivax merozoite surface protein7 (PvMSP7) and is localized on merozoite surface. Co-localization of PvTRAg56.2 with PvMSP1 and its molecular interaction with PvMSP7 probably suggest that, PvTRAg56.2 is part of MSP-complex, and might assist or stabilize the protein complex at the merozoite surface. In conclusion, the PvTRAg proteins have different sub cellular localizations and specific associated functions during intra-erythrocytic developmental cycle.

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.

We report on a systematic study of the effect of barriers on quantum dots-in-a-well infrared photodetectors. Four devices are fabricated and characterized with varying composition for barriers adjacent to quantum dots and away from quantum dots. Effects of these "proximity" and "remote" barriers are studied by comparing photoluminescence, responsivity, dark current, background-limited operating temperature, activation energy, and detectivity. The growth mechanism for a conformal coverage of quantum dots with proximity barriers is described and supported with reflection high-energy electron diffraction and transmission electron microscopy images. It is shown that proximity barriers and remote barriers influence the characteristics of the detector very differently, with increases in proximity barrier energy leading to higher responsivity and lower dark current, while remote barriers reduce the responsivity and dark currents simultaneously. It is demonstrated that confinement enhancing barriers as proximity barriers optimize the SNR at low bias range, suitable for focal plane array applications.

Objectives: To study the origin and spread of the chloroquine-resistant Plasmodium falciparum population in the Indian subcontinent. Methods: Fourteen microsatellites spanning a ∼120 kb region, flanking the P. falciparum chloroquine resistance transporter (pfcrt) gene, were analysed in 185 parasite isolates. Results: The Indian P. falciparum population exhibited a selective valley of reduced genetic variation in the flanking microsatellites of the mutant pfcrt alleles (up to ±29 kb) as compared with the wild-type allele. This valley is much narrower than the ±200 kb valley reported from African and South-East Asian countries. The majority of the isolates showed asymmetry in the selective valley, where upstream microsatellites showed less genetic variation than the downstream microsatellites. Regional variation in the width and symmetry of the selective valley was noticed, which seems to be related to the number of pfcrt alleles present in the parasite population of a region. Forty-six different microsatellite haplotypes were observed among the P. falciparum isolates containing mutant pfcrt alleles. Parasite populations from different regions of mainland India shared microsatellite haplotypes between them, but they shared none with the isolates from the Andaman and Nicobar Islands, and vice versa. Indian isolates shared microsatellite haplotypes with the isolates from Papua New Guinea and Thailand. Conclusions: With regard to chloroquine there is regional variation in the selection pressure on the P. falciparum population in India. These findings will help the regional implementation of drug policy in India's malaria control programme.

Reduction in the dark current and improvement in signal to noise ratio in the quantum dots in a well infrared photodetectors using resonant tunneling barriers have been demonstrated. Ultra-low dark current levels and high detectivity of 3×1010 cm.Hz1/2/W at 77K for f/2 optics has been obtained for longwave infrared detection. In another experiment, the ability to control the excited state in the DWELL has been demonstrated by systematically varying the quantum well thickness. These detectors demonstrate high operating temperature with high detectivity values, even for high operating temperatures.

Our previous research has reported on the development of the first generation of quantum dots-in-a-well (DWELL) focal plane arrays (FPAs), which are based on InAs quantum dots (QDs) embedded in an InGaAs well having GaAs barriers, which have demonstrated spectral tunability via an externally applied bias voltage. More recently, technologies in DWELL devices have been further advanced by embedding InAs QDs in InGaAs and GaAs double wells with AlGaAs barriers, leading to a less strained InAs/InGaAs/GaAs/AlGaAs heterostructure. These lower strain quantum dots-in-a-double-well devices exhibit lower dark current than the previous generation DWELL devices while still demonstrating spectral tunability. This paper compares two different configurations of double DWELL (DDWELL) FPAs to a previous generation DWELL detector and to a commercially available quantum well infrared photodetector (QWIP). All four devices are 320 × 256 pixel FPAs that have been fabricated and hybridized with an Indigo 9705 read-out integrated circuit. Radiometric characterization, average array responsivity, array uniformity and measured noise equivalent temperature difference for all four devices is computed and compared at 60 K. Overall, the DDWELL devices had lower noise equivalent temperature difference and higher uniformity than the first-generation DWELL devices, although the commercially available QWIP has demonstrated the best performance.

With the spread of chloroquine (CQ)-resistant malaria in India, sulfadoxine-pyrimethamine (SP) alone or in combination with artesunate is used as an alternative antimalarial drug. Due to continuous drug pressure, the Plasmodium falciparum parasite is exhibiting resistance to antifolates because of mutations in candidate genes dihydrofolate reductase ( dhfr ) and dihydropteroate synthetase ( dhps ). Our earlier study on flanking microsatellite markers of dhfr mutant alleles from India had shown a single origin of the pyrimethamine resistance and some minor haplotypes which shared haplotypes with Southeast Asian (Thailand) strains. In the present study, we have analyzed 193 of these Indian P. falciparum isolates for 15 microsatellite loci around dhps to investigate the genetic lineages of the mutant dhps alleles in different parts of the country. Eighty-one of these samples had mutant dhps alleles, of which 62 were from Andaman and Nicobar Islands and the remaining 19 were from mainland India. Of 112 isolates with a wild-type dhps allele, 109 were from mainland India and only 3 were from Andaman and Nicobar Islands. Consistent with the model of selection, the mean expected heterozygosity ( H e ) around mutant dhps alleles ( H e = 0.55; n = 81) associated with sulfadoxine resistance was lower ( P ≤ 0.05) than the mean H e around the wild-type dhps allele ( H e = 0.80; n = 112). There was more genetic diversity in flanking microsatellites of dhps than dhfr among these isolates, which confirms the assertion that dhps mutations are at a very early stage of fixation in the parasite population. Microsatellite haplotypes around various mutant dhps alleles suggest that the resistant dhps alleles have multiple independent origins in India, especially in Andaman and Nicobar Islands. Determining the genetic lineages of the resistant dhps alleles on Andaman and Nicobar Islands and mainland India is significant, given the role of Asia in the intercontinental spread of chloroquine- and pyrimethamine-resistant parasites in the past.

Mid-wave and long-wave infrared (IR) quantum-dots-in-a-well (DWELL) focal plane arrays (FPAs) are promising technology for multispectral (MS) imaging and sensing. The DWELL structure design provides the detector with a unique property that allows the spectral response of the detector to be continuously, albeit coarsely, tuned with the applied bias. In this paper, a MS classification capability of the DWELL FPA is demonstrated. The approach is based upon: 1) imaging an object repeatedly using a sequence of bias voltages in the tuning range of the FPA and then 2) applying a classification algorithm to the totality of readouts, over multiple biases, at each pixel to identify the “class” of the material. The approach is validated for two classification problems: separation among different combinations of three IR filters and discrimination between rocks. This work is the first demonstration of the MS classification capability of the DWELL FPA.

Systematic study of various types of intersubband transitions in the quantum dots-in-a-well (DWELL) infrared photodetectors has been presented. By changing the thickness of the quantum well, the excited state energy can be tuned with respect to the barrier, without altering the quantum dot ground state. Bound to continuum transitions offer very high extraction probability for photoexcited electrons but poor absorption coefficient, while the bound to bound transitions have higher absorption but poorer extraction probability. Bound to quasi-bound transition is optimum for intermediate values of electric fields with superior signal to noise ratio. The bound to quasi-bound device has the detectivity of 4 × 10 11 cm Hz 1/2 W −1 (+3 V, f /2 optics) at 77 K and 7.4 × 10 8 cm Hz 1/2 W −1 at 200 K, which is highest reported detectivity at 200 K for detector with long wave cutoff wavelength. High performance focal plane arrays have been fabricated with noise equivalent temperature difference of 44 mK at 80 K for 6.1 μm peak wavelength.

Need for malaria vaccine necessitates the characterization of potential antigens of the Plasmodium parasite. Recently, we have identified several Plasmodium vivax tryptophan-rich antigens (PvTRAgs). Here, we describe the immunological characterization of hitherto undescribed two such antigens PvTRAg 35.2 and PvTRAg 80.6 which are respective homologue of Plasmodium falciparum merozoite associated tryptophan-rich antigen (PfMaTrA) and P. falciparum tryptophan and threonine rich antigen (PfTryThrA) involved in erythrocyte invasion. Each of the pvtrag genes is comprised of two exons where exon 2 encodes for major part of the protein. PvTRAg 35.2 and PvTRAg 80.6 showed 97.06% and 94.12% (n = 34) seropositivity rates, and 92.3% (n = 13) and 100% (n = 29) lymphoproliferative responses, respectively, among P. vivax exposed individuals. Geometric mean values of IL-12, IFN-γ, TNF-α, IL-4 and IL-10 in PBMC culture supernatants of P. vivax exposed individuals were 182.02, 60.3, 62.84, 196.01 and 177.17 pg/ml against PvTRAg 35.2 and 185.27, 58.15, 64.56, 142.01 and 157.2 pg/ml against PvTRAg 80.6 showing mixed immune response with distinct biased towards anti-inflammatory Th2 phenotype. The pvtrag 35.2 gene was highly conserved in the parasite population whereas pvtrag 80.6 showed minor variations in the N-terminal region but highly conserved in the C-terminal region containing tryptophan-rich domain.

We report on the comparison of mesa sidewall profiles of InAs/GaSb strained-layer superlattice (SLS) detector structures (λ 50% cutoff ≈ 14 μm at V bias = 0 V and T = 30 K) obtained after (a) a conventional BCl3-based inductively coupled plasma etch, (b) a chemical etch (H2O2:HCl:H2O, 1:1:4), and (c) a combination of both etches. We found that the smoothest sidewall profile with reasonable undercut (~5 μm) was obtained after chemical etch only. The chemical etch was optimized primarily using an n-type GaSb substrate. During this process, numerous chemical etchants were examined. GaSb n-type substrates were chosen for this study in preference over InAs substrates due to their high chemical reactivity and the complicated composition of the native oxide. In addition, SLS detectors are usually grown on GaSb substrates and, after hybridization of the focal-plane array to the readout integrated circuit, the GaSb substrate is etched away using a combination of wet and dry etching techniques. We found that H2O2:HCl:H2O (1:1:4) etching solution provided the smoothest etched surface of GaSb, with a root-mean-square roughness of 1.59 nm.

ObjectiveGlial cells missing 2 (GCM2) gene encodes a parathyroid-specific transcription factor. We assessed GCM2 gene sequence in patients with isolated hypoparathyroidism (IH).DesignCase–control study.MethodsComplete 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.ResultsA heterozygous R110W mutation was present in DNA-binding domain in 11/101 patients (10.9%) and absent in 655 controls (P−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.ConclusionsThe 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.

We have investigated the electrical and optical properties of an nBn based Type-II InAs/GaSb strained layer superlattice detector as a function of absorber region background carrier concentration. Temperature-dependent dark current, responsivity and detectivity were measured. At T = 77 K and Vb = 0.1 V, with two orders of magnitude change in doping concentration, the dark current density increased from ∼0.3 mA/cm2 to ∼0.3 A/cm2. We attribute this to a depletion region that exists at the AlGaSb barrier and the SLS absorber interface. The device with non-intentionally doped absorption region demonstrated the lowest dark current density (0.3 mA/cm2 at 0.1 V) with a specific detectivity D∗ at zero bias equal to 1.2 × 1011 Jones at 77 K. The D∗ value decreased to 6 × 1010 cm Hz1/2/W at 150 K. This temperature dependence is significantly different from conventional PIN diodes, in which the D∗ decreases by over two orders of magnitude from 77 K to 150 K, making nBn devices a promising alternative for higher operating temperatures.

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.

Infrared detectors based on quantum wells and quantum dots have attracted a lot of attention in the past few years. Our previous research has reported on the development of the first generation of quantum dots-in-a-well (DWELL) focal plane arrays, which are based on InAs quantum dots embedded in an InGaAs well having GaAs barriers. This focal plane array has successfully generated a two-color imagery in the mid-wave infrared (i.e. 3–5 μm) and the long-wave infrared (i.e. 8–12 μm) at a fixed bias voltage. Recently, the DWELL device has been further modified by embedding InAs quantum dots in InGaAs and GaAs double wells with AlGaAs barriers, leading to a less strained InAs/InGaAs/GaAs/AlGaAs heterostructure. This is expected to improve the operating temperature while maintaining a low dark current level. This paper examines 320 × 256 double DWELL based focal plane arrays that have been fabricated and hybridized with an Indigo 9705 read-out integrated circuit using Indium-bump (flip-chip) technology. The spectral tunability is quantified by examining images and determining the transmittance ratio (equivalent to the photocurrent ratio) between mid-wave and long-way infrared filter targets. Calculations were performed for a bias range from 0.3 to 1.0 V. The results demonstrate that the mid-wave transmittance dominates at these low bias voltages, and the transmittance ratio continuously varies over different applied biases. Additionally, radiometric characterization, including array uniformity and measured noise equivalent temperature difference for the double DWELL devices is computed and compared to the same results from the original first generation DWELL. Finally, higher temperature operation is explored. Overall, the double DWELL devices had lower noise equivalent temperature difference and higher uniformity, and worked at higher temperature (70 K and 80 K) than the first generation DWELL device.

This paper reports on a comparison between a commercially available quantum-well infrared focal plane array (FPA) and a custom quantum-dot (QD)-in-a-well (DWELL) infrared FPA in the long-wave infrared (LWIR). The DWELL detectors consist of an active region composed of InAs QDs embedded in In0.15Ga0.85As quantum wells. DWELL samples were grown using molecular beam epitaxy and fabricated into 320 times 256 pixels FPA with a flip-chip indium bump technique. Both the DWELL and QmagiQ commercial quantum-well detector were hybridized to an Indigo ISC9705 readout circuit and tested in the same camera system. Calibrated blackbody measurements at a device temperature of 60 K with LWIR optics yield a noise equivalent change in temperature of 17 mK and 91 mK for quantum-well and DWELL FPAs operating at 0.95- and 0.58-V biases, respectively. The comparison of the DWELL and quantum-well FPA when imaging a 35degC black body showed that the DWELL had a signal-to-noise ratio of 124 while the quantum-well FPA showed 1961. As well, the quantum-well FPA showed a higher collection efficiency of 1.3 compared to the DWELL.

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.

We describe here an ∼40-kDaPlasmodium vivaxtryptophan-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 35P. vivaxclinical 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 naturalP. vivaxinfection. Eighty percent of the total of 20P. 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.

SUMMARY Plasmodium vivax, the most widespread species of human malaria parasite responsible for 70–80 million cases each year requires a vaccine. In recent years, many potential vaccine candidate antigens have been identified from P. vivax including PvTRAg. We describe here cellular immune response to recombinant PvTRAg expressed in Escherichia coli. The in vitro stimulation of PBMCs derived from P. vivax-exposed individuals (n = 16) showed strong proliferative response (SI > 2·2) to PvTRAg as compared to PBMCs from normal healthy controls (n = 8). Although both Th1 (IFN-γ, TNF-α and IL-12) and Th2 (IL-4 and IL-10) cytokines were secreted by the PBMCs of the P. vivax-exposed individuals in response to PvTRAg, the overall response was more inclined towards Th2. In conclusion, recombinant PvTRAg was found to elicit strong cellular immune response among the P. vivax-exposed individuals.

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.

The nonmevalonate pathway of isoprenoid biosynthesis in the apicoplast of the human malaria parasite Plasmodium falciparum is distinct from the mevalonate-dependent pathway of humans and thus a good drug target. We describe here the hammerhead ribozyme based cleavage of the ispH (lytB) gene transcript involved in the last step of this nonmevalonate pathway. Using RNA folding program, three hammerhead ribozymes named as RZ(876), RZ(1260), and RZ(1331) were predicted against ispH (lytB) mRNA. Messenger walk screening (RNaseH) assay confirmed the target accessibility for these ribozymes. All three ribozymes cleaved the target RNA in vitro but RZ(876) exhibited the highest catalytic potential (62.92%). Therefore, RZ(876) was chemically synthesized with appropriate chemical modifications to protect it from nuclease attack while using it for in vitro parasite growth inhibition assay. This ribozyme RZ(876) was able to inhibit 87.36% parasite growth at 30 microM concentration compared to the untreated culture. However, an absolute inhibition of 29.41% was achieved compared to the control ribozyme (RZ(ctrl)). Nonetheless, the growth inhibition effect was found to be sequence-specific as indicated by the decreased level of ispH (lytB) transcript after ribozyme treatment. In conclusion, we have identified the ispH (lytB) as a potential target whose transcript can be cleaved by a ribozyme RZ(876).

Summary The Anopheles annularis group of mosquitoes is widely distributed in Southeast Asia and may be locally important as malaria vectors. Members of this group are morphologically very similar and often difficult to distinguish, particularly A. nivipes and A. philippinensis. We report the sequence analysis of the rDNA internal transcribed spacer 2 (ITS2) and Domain-3 (D3) regions of the four members of the A. annularis group – A. nivipes, A. philippinensis, A. annularis and A. pallidus – and a method for their molecular identification. No intraspecies sequence variation was detected among the specimens, while interspecific sequence differences were greater for ITS2 than the D3 regions. Comparison of the D3 sequences of the four species revealed two SmaI restriction sites in A. nivipes, but only one site in A. philippinensis, A. annularis and A. pallidus. The ApaI site was present in both A. philippinensis and A. pallidus, while an NcoI site was present in A. pallidus only. Restriction digestion of the PCR products of D3 fragment individually with SmaI, ApaI and NcoI produced a distinctive pattern for all the four species. We present, for the first time, a PCR-RFLP method to distinguish the four members of the A. annularis group of mosquitoes.

Plasmodium tryptophan-rich proteins are involved in host-parasite interaction and thus potential drug/vaccine targets. Recently, we have described several P. vivax tryptophan-rich antigens (PvTRAgs), including merozoite expressed PvTRAg38, from this noncultivable human malaria parasite. PvTRAg38 is highly immunogenic in humans and binds to host erythrocytes, and this binding is inhibited by the patient sera. This binding is also affected if host erythrocytes were pretreated with chymotrypsin. Here, Band 3 has been identified as the chymotrypsin-sensitive erythrocyte receptor for this parasite protein. Interaction of PvTRAg38 with Band 3 has been mapped to its three different ectodomains (loops 1, 3, and 6) exposed at the surface of the erythrocyte. The binding region of PvTRAg38 to Band3 has been mapped to its sequence, KWVQWKNDKIRSWLSSEW, present at amino acid positions 197-214. The recombinant PvTRAg38 was able to inhibit the parasite growth in in vitro Plasmodium falciparum culture probably by competing with the ligand(s) of this heterologous parasite for the erythrocyte Band 3 receptor. In conclusion, the host-parasite interaction at the molecular level is much more complicated than known so far and should be considered during the development of anti-malarial therapeutics.

Anopheles (Cellia) annularis Van der Wulp is a complex of two sibling species provisionally designated as species A and B and can only be differentiated on the basis of the paracentric inversion in the ovarian polytene chromosomes. To analyze the distribution of these two species and to develop a molecular method for the identification of these two cryptic species, we sequenced the ribosomal DNA internal transcribed spacer 2 (ITS2) and domain 3 (D3) of A. annularis specimens collected from Sonapur (Assam), Jabalpur (Madhya Pradesh), Ranchi (Jharkhand), and Ghaziabad (Uttar Pradesh). We did not find any sequence variation among the specimens collected from Assam, Madhya Pradesh, and Jharkhand states, whereas two types of sequences were obtained from the specimens collected from the state of Uttar Pradesh, which correspond to species A and B of the A. annularis complex. Species A was more prevalent among the all four regions studied. The ITS2 sequence of species A showed unique restriction sites for MvaI and Eco24I, while species B displayed HinfI and NruI sites. Similarly, the D3 sequence of species A showed unique restriction site for Alw26I, while species B showed a unique KpnI site. In this study, we report for the first time the development of ribosomal DNA polymerase chain reaction-restriction fragment length polymorphism methods for identifying these two cryptic species of the Annularis complex.

Summary Background Sporadic idiopathic hypoparathyroidism (SIH) is the most common cause of hypoparathyroidism. While calcium sensing receptor (CaSR) autoantibodies are observed in 49% of cases, aetiopathogenetic mechanisms in others are under investigation. Mutations in the PTH gene including its 3′ untranslated region, autoimmune regulator gene and lead CTLA-4 gene single nucleotide polymorphism (SNPs) are not associated with the disease. There are reports of de novo activating mutations of the CaSR gene in a few patients with SIH. Objective To assess the frequency of CaSR mutations in patients with SIH. Subjects and methods DNA sequencing of all six translating exons and nine of 12 intron/exon boundaries of the CaSR gene was performed by Sangers dideoxy chain termination method using an automated sequencer in 39 patients with SIH. Spot urinary calcium/creatinine ratio in the fasting state and ultrasonography of the abdomen was performed to assess hypercalciuria and nephrolithiasis. The PCR-RFLP analysis was performed using Hin1II restriction endonuclease in 32 additional patients with SIH and 90 healthy controls to further assess the prevalence of a novel missense SNP observed in the DNA sequencing. Results Nucleotide sequence analysis revealed the presence of a wild type CaSR gene in all subjects, except in one patient who showed a missense mutation (Val621Met) due to substitution of base G→A in the heterozygous state at position 79877 in exon 7 (codon 621) coding for the first transmembrane loop of the CaSR. The V621M polymorphism was confirmed by PCR-RFLP and was due to a maternal allele. However, the mother and brother of this patient with the same SNP were asymptomatic and had normal serum chemistry indicating the functionally inert nature of the polymorphism. None of the additional 32 patients with SIH and 90 controls showed V621M SNP. The urinary calcium/creatinine ratio and ultrasonography were normal in all patients with SIH. Conclusion De novo activating mutation of the CaSR gene typical of familial hypoparathyroidism is not common among patients with SIH in India.

Metal corroding sulfate reducing bacteria have been poorly characterized at molecular level due to difficulties pertaining to isolation and handling of anaerobes. We report here for the first time the presence and characterization of thioredoxin 2 in an obligate anaerobic dissimilatory sulfate reducing bacterium Desulfovibrio desulfuricans. In silico analysis of the D. desulfuricans genome revealed the presence of thioredoxin 1 (dstrx1), thioredoxin 2 (dstrx2) and thioredoxin reductase (dstrxR) genes. These genes were found to be actively expressed by the bacteria under the anaerobic growth conditions. We have overexpressed the anaerobic thioredoxin genes in E. coli to produce functionally active recombinant proteins. Recombinant DsTrxR recognized both DsTrx1 and DsTrx2 as its substrate. Mutation studies revealed that the activity of DsTrx2 can be completely abolished with a single amino acid mutation (C69A) in the signature motif ‘WCGPC’. Furthermore, the N-terminal domain of DsTrx2 containing two extra CXXC motifs was found to have a negative regulation on its biochemical activity. In conclusion, we have shown the presence of thioredoxin 2 for the first time in an obligate anaerobe which in this anaerobe may be required for its survival under either oxidative stress conditions or metal ion hemostasis.

Effective malaria control programs require continuous monitoring of drug pressure in the field, using molecular markers.We used sequence analysis to investigate the pfcrt and pfmdr1 mutations in Indian Plasmodium falciparum isolates. To evaluate the chloroquine drug pressure in the field, isolates were collected from 5 different areas at 2 time points, with an interval of 2 years.In 265 P. falciparum isolates, pfcrt mutations were observed at codons 72, 74, 75, 76, and 220, resulting in 8 different genotypes: SMNTS (61.89%), CIETS (12.08%), CMNKS (0.38%), CMNTA (2.64%), CMNTS (4.91%), SMNTA (0.38%), CIDTS (2.26%), and wild-type CMNKA (15.47%). During the 2-year period, there was a significant decrease in the number of isolates with the SMNTS genotype and an increase in the number of isolates with the highly chloroquine-resistant pfcrt genotype CIETS (P.05). The N86Y mutation was less prevalent (30.13%) than the Y184F mutation (99.16%) in the pfmdr1 gene in 239 isolates, but the number of isolates with the N86Y mutation increased significantly during the 2-year period (P.05). The number of isolates with higher total numbers of pfcrt and pfmdr1 2-loci mutations, therefore, increased significantly during this period. There was a regional bias in the mutation rate of these genes, because isolates from areas where chloroquine resistance was high had higher numbers of 2-loci mutations, and areas where chloroquine resistance was low had isolates with lower numbers of 2-loci mutations.There was a temporal increase in the number of pfcrt and pfmdr1 2-loci mutations, and this led to the higher level of chloroquine resistance. This is a cause for concern for the antimalarial drug policy in India.

Anopheles (Cellia) sundaicus (Rodenwaldt) is an important malaria vector in the Andaman and Nicobar islands of India where it breeds in freshwater as well as in brackish water. To establish the molecular identity of An. sundaicus on these islands we analyzed samples from four geographically isolated areas-Teressa, Nancowry, Car Nicobar and Katchal islands. PCR-amplification and nucleotide sequence analysis were performed for internal transcribed spacer 2 (ITS2) and domain-3 (D3) of 28S rRNA. The ITS2 region of An. sundaicus from all four islands was identical but different from An. sundaicus A of Vietnam and An. sundaicus s.s of Malaysia. Furthermore, freshwater and brackish water forms of An. sundaicus did not reveal any sequence variation. Similarly, the D3 sequences were identical among all An. sundaicus samples from the four islands. D3 sequences for a species of the Sundaicus Complex are reported here for the first time and thus could not be compared with other regional isolates of this species. In conclusion, probably only one member of the Sundaicus Complex exists on the Andaman and Nicobar islands, which breeds in freshwater as well as in brackish water and is different from the An. sundaicus A and Malaysian An. sundaicus s.s. The identification of a new sibling species of the Sundaicus Complex in these islands is significant from the viewpoint of vector control strategies.

Tryptophan-rich proteins play important biological functions for the Plasmodium parasite. Plasmodium vivax contains remarkably large numbers of such proteins belonging to the “Pv-fam-a” family that need to be characterized. Earlier, we reported the presence of memory T cells and naturally acquired antibodies against 15 of these proteins in P. vivax malaria-exposed individuals (M. Zeeshan, H. Bora, and Y. D. Sharma, J Infect Dis 207: 175–185, 2013, http://dx.doi.org/10.1093/infdis/jis650 ). Here, we sought to characterize and ascertain the cross talk between effector responses of T and B cells in malarial patients against all Pv-fam-a family proteins. Therefore, we expressed the remaining 21 of these proteins in Escherichia coli and studied the humoral and cellular immune responses based on the same parameters used in our previous study. Naturally acquired IgG antibodies were detected against all 21 antigens in P. vivax patient sera (37.7 to 94.4% seropositivity). These antigens were able to activate the lymphocytes of P. vivax -exposed individuals, and the activated CD4 + T lymphocytes produced higher levels of Th1 (interleukin-2 [IL-2] and gamma interferon [IFN-γ]) and Th2 (IL-4 and IL-10) cytokines than the healthy controls, but the response was Th2 biased. The combined results of present and previous studies seem to suggest a striking link between induction of the CD4 + T cell response and naturally acquired antibodies against all 36 proteins of the Pv-fam-a family, the majority of them having conserved sequences in the parasite population. Further work is required to utilize this information to develop immunotherapeutic treatments for this disease.

Histones play important role in DNA packaging, replication and gene expression. Here, we describe the isolation and characterization of histone 2B (PvH2B) gene from the most common but non-cultivable human malaria parasite Plasmodium vivax. The isolated cDNA clone of PvH2B was allowed to express in Escherichia coli and the recombinant protein was purified by affinity chromatography. The expressed PvH2B protein showed DNA-binding properties on the South-Western analysis and the confocal microscopy localized it in the parasite nucleus. This gene is actively expressed during blood stages of the parasite and all P. vivax patients produced antibodies against the protein. The mRNA of PvH2B was found to contain a poly(A) tail at its 3' end, unlike abundant mRNA of human H2B. The encoded polypeptide is 118 amino acid long contains a nuclear targeting site, a signature motif of H2B and showed 74% homology to its host molecule. The structure of PvH2B showed that it has certain differences from that of its host at critical functional sites (viz acetylation, methylation, trypsin cleavage, DNA-binding and inter-histone interaction) which are required for general gene expression and DNA packaging. The distinctive structural features of P. vivax H2B described here may help in designing the specific antimalarial drugs.

Our recent studies have focused on the identification and characterization of the tryptophan-rich proteins of the Plasmodium vivax parasite where their role in the elicitation of humoral and cellular responses and erythrocyte-binding activity was investigated. Here, we report the humoral responses of a 32.4-kDa P. vivax tryptophan-rich antigen (PvTRAg32.4) among the sera of P. vivax-infected patients. PvTRAg32.4 also contains an unusually high percentage of tryptophan residues (10.7 %) that are positionally conserved with its orthologues in Plasmodium yoelii (PypAg1 and PypAg2) and Plasmodium falciparum (PfTryThrA and PfMATRA). Thirty-four of the 40 (85.0 %) P. vivax isolates showed seropositivity to recombinant PvTRAg32.4 by ELISA. The mean ± SD values of optical density (OD) for P. vivax subjects and naive individuals were 1.02 ± 0.36 and 0.26 ± 0.11, respectively. In the Western blot analysis, majority of the subjects studied (n = 44) showed reactivity to the recombinant, purified PvTRAg32.4. This antigen does not show binding to the erythrocytes, but the immunofluorescence data reveals that it is expressed in the erythrocytic stages of the parasite. Sequence analysis of the clinical isolates from various parts of the country shows that PvTRAg32.4 is highly conserved. Functional in-depth characterization of more such type of novel proteins in the parasite is warranted for the development of successful malaria intervention methods.

The cg2 gene of Plasmodium falciparum has been proposed to be associated with chloroquine resistance. Here we describe PCR amplification and sequencing of all the four repeat regions (kappa (kappa), gamma (gamma), psi (psi) and omega (omega)) of this gene, from Indian isolates. There were variant forms for each of these repeat regions (two for kappa and gamma, and three for psi and omega) among the 123 Indian isolates of P. falciparum. Among these isolates certain forms of psi and omega repeats were uniquely present while some of the reported forms of the kappa and omega repeats were absent. The pattern of combination of all four repeat regions of cg2 gene (genotype) was analysed from 52 isolates. A total of 11 different genotypes were observed among these cases, of which 10 were unique to Indian isolates. Certain genotypes were more common than others. The nucleotide sequencing of all the four repeat regions revealed that Indian isolates have some unique repeating units within the gamma and omega domains. Altogether, the PCR and sequencing results showed that there was an unrelatedness between cg2 repeats and chloroquine resistance.

We present here computer generated model of N-terminal fragment, amino acids (aa) 36-245, of a Plasmodium vivax heat shock metalloprotease called PVHSP28, whose gene was cloned and characterised earlier. The fragment showed homology with HSPs from many organisms, including Escherichia coli and Haemophilus influenzae. PVHSP28 had the signature sequence 'HEXXH' and 'EXXXD' of Zinc metalloproteases. Being the first malarial HSP possessing metalloprotease activity, PVHSP28 is an ideal target for the design of new anti-malarial drugs. However, except for a small region (aa 62-132) which had 24.6% sequence similarity with 1TAQ (a DNA polymerase), it did not show sequence similarity with any published structures in protein data bank. Hence it could not be modelled using any automated modeling programs. We modelled 36-245 aa of PVHSP28 using predicted secondary structure as well as experimentally determined and predicted properties of the protein on the basis of its amino acid sequence, using various Internet tools and in-house package MODEL. The model was energy minimised using Sander's module of AMBER 5.0, working on a Silicon Graphics machine, with all atom force field.