Seasonal and annual variations of CO2 and CH4 at Shadnagar, a semi-urban site.

Carbon dioxide (CO2) and methane (CH4) are the most important greenhouse gases (GHGs) due to their significant role in anthropogenic global climate change. The spatio-temporal variations of their concentration are characterized by the terrestrial biosphere, seasonal weather patterns and anthropogenic emissions. Hence, to understand the variability in regional surface GHG fluxes, high precision GHGs measurements were initiated by the National Remote Sensing Center (NRSC) of India. We report continuous CO2 and CH4measurements during 2014 to 2017 for the first time from Shadnagar, a suburban site in India. Annual mean CO2 and CH4 concentrations are 399.56 ± 5.46 ppm and 1.929 ± 0.09 ppm, respectively, for 2017. After the strong El Niño of 2015-2016, an abnormal rise in CO2 growth rate of 5.5 ppm year-1 was observed in 2017 at the study site, compared to 3.03 ppm year-1 at Mauna Loa. Thus, the repercussion of the El Niño effect diminishes the net uptake by the terrestrial biosphere accompanied by increased soil respiration. Seasonal tracer to tracer correlation between CO2 and CH4 was also analyzed to characterize the possible source-sink relationship between the species. We compared CO2 and CH4 concentrations to simulations from an atmospheric chemistry transport model (ACTM). The seasonal phases of CH4 were well captured by the ACTM, whereas the seasonal cycle amplitude of CO2 was underestimated by about 30%.

Evaluation of weather-based rice yield models in India.

The objective of this study was to compare two different rice simulation models--standalone (Decision Support System for Agrotechnology Transfer [DSSAT]) and web based (SImulation Model for RIce-Weather relations [SIMRIW])--with agrometeorological data and agronomic parameters for estimation of rice crop production in southern semi-arid tropics of India. Studies were carried out on the BPT5204 rice variety to evaluate two crop simulation models. Long-term experiments were conducted in a research farm of Acharya N G Ranga Agricultural University (ANGRAU), Hyderabad, India. Initially, the results were obtained using 4 years (1994-1997) of data with weather parameters from a local weather station to evaluate DSSAT simulated results with observed values. Linear regression models used for the purpose showed a close relationship between DSSAT and observed yield. Subsequently, yield comparisons were also carried out with SIMRIW and DSSAT, and validated with actual observed values. Realizing the correlation coefficient values of SIMRIW simulation values in acceptable limits, further rice experiments in monsoon (Kharif) and post-monsoon (Rabi) agricultural seasons (2009, 2010 and 2011) were carried out with a location-specific distributed sensor network system. These proximal systems help to simulate dry weight, leaf area index and potential yield by the Java based SIMRIW on a daily/weekly/monthly/seasonal basis. These dynamic parameters are useful to the farming community for necessary decision making in a ubiquitous manner. However, SIMRIW requires fine tuning for better results/decision making.

Genetic fingerprinting and identification of differentially expressed genes in isolates of Leishmania donovani from Indian patients of post-kala-azar dermal leishmaniasis.

Post-kala-azar dermal leishmaniasis (PKDL) is an unusual dermatosis that develops as a sequel in 5-15% of cured cases of kala-azar (KA) after months or years of treatment in India. Molecular differences are reported to exist between the KA and PKDL isolates which may underlie the diversity in clinical manifestations of the disease. Here, arbitrary primed-PCR (AP-PCR) has been used for genetic fingerprinting of parasite isolates from dermal lesions of PKDL patients (n=14) and compared with bone-marrow derived parasites from KA patients (n=3). All isolates showed an identical AP-PCR pattern with 4 arbitrary primers. Further, AP-PCR was exploited to identify the stage regulated genes of the parasite. Six polymorphic fragments were identified in PKDL in comparison with KA isolates, and were subjected to Northern blot analysis. Five polymorphic fragments represented transcribed sequences; 4 out of 5 drew differential expression in pro- and amastigote stages, although the expression was comparable between PKDL and KA isolates. The study led to the identification of genes, which exhibit stage-regulated expression in Leishmania donovani derived from PKDL or KA patients, including a putative phosphodiesterase, DEAD box RNA helicase, iron superoxide dismutase b (fesodb) and a hypothetical protein. Demonstration of transcripts of DEAD box RNA helicase in PKDL and KA diseased tissues implicates its role in disease pathogenesis.

Genetically modified live attenuated parasites as vaccines for leishmaniasis.

Leishmaniasis causes significant morbidity and mortality worldwide and is an important public health problem. Even though it is endemic in developing countries in tropical regions of the world,in recent years economic globalization and increased travel has extended its reach to people in developed countries. Leishmania is usually spread by the bite of the female sandfly. In addition, naïve populations can be exposed to Leishmania infection through transfusion of blood and blood products from infected asymptomatic individuals. There are several clinical forms of leishmaniasis caused by different species of the parasite. In some cases, the only possible cure for this disease is drug treatment. However, prolonged use of such drugs has led to parasite drug resistance. At present there are no effective vaccines against Leishmania. Many vaccine strategies have been pursued, including the use of whole cell lysate, killed, avirulent or irradiated parasites. Additionally, DNA vaccines and purified or recombinant parasite antigens have also been tested. Most of these strategies have shown some degree of effectiveness in animal models but little or no protection in humans. There is now a general consensus among Leishmania vaccine researchers that parasite persistence may be important for effective protective response and could be achieved by live attenuated parasite immunization. In this article we reviewed the efforts in developing genetically defined live attenuated Leishmania parasites as vaccine candidates with the goal of achieving a low level of parasite persistence without being virulent in the host and inducing protective immunity.

Arbitrary-primed PCR for genomic fingerprinting and identification of differentially regulated genes in Indian isolates of Leishmania donovani.

The arbitrary-primed PCR (AP-PCR) technique was employed with the twin goals of identifying genetic polymorphisms within the Indian isolates and to identify differentially expressed gene sequences. The parasite isolates from Indian Kala-azar patients could be differentiated from Leishmania donovani isolates from distinct geographic regions. Moreover, differences within the Indian isolates could also be identified. A majority (17/19) of the Indian isolates gave identical AP-PCR pattern, while two isolates gave consistently divergent pattern. The distinctive AP-PCR fragments obtained with Indian isolates were used as probes in Northern blot analysis. Three such fragments were found to represent transcribed sequences that were differentially expressed in the two stages of the parasite. These sequences led to cloning and characterization of Leishmania Centrin gene and a novel gene termed A-1 that is over-expressed in amastigote stage of the parasite. The study demonstrates the utility of random genome sampling methods in genomic fingerprinting and in identifying differentially transcribed sequences that could potentially contribute to parasite virulence.

Parasite detection in patients with post kala-azar dermal leishmaniasis in India: a comparison between molecular and immunological methods.

AIMS: To evaluate the sensitivity and specificity of serological, immunohistochemical, and molecular methods in the diagnosis of post kala-azar dermal leishmaniasis (PKDL). METHODS: Twenty five patients with confirmed PKDL and 25 controls were included in the study. G2D10, a monoclonal antibody against Leishmania, was used for the immunohistochemical (IHC) staining of lesion sections to visualise anti-Leishmania donovani antibodies. The diagnostic usefulness of IHC was compared with enzyme linked immunosorbent assay (ELISA) with a recombinant (rk39) antigen, and a species specific polymerase chain reaction (PCR) assay, amplifying a kinetoplast minicircle DNA sequence. RESULTS: IHC detected 22 of 25 PKDL cases, giving a sensitivity of 88%. The diagnostic sensitivity of both the ELISA and PCR tests was higher (96%). All of the 25 controls examined were negative in PCR, indicating 100% specificity of the test, whereas ELISA showed 96% specificity. CONCLUSIONS: IHC with G2D10 significantly enhances the sensitivity of detection of PKDL over routine haematoxylin and eosin staining. ELISA with a recombinant antigen is an economical and practical assay. PCR is the most sensitive and specific diagnostic method for PKDL. The tests described would facilitate the recognition of patients with PKDL, enabling timely treatment, which would contribute greatly to the control of kala-azar.

Evaluation of enzyme-linked immunosorbent assay for diagnosis of post-kala-azar dermal leishmaniasis with crude or recombinant k39 antigen.

The diagnosis of post-kala-azar dermal leishmaniasis (PKDL), a dermatosis that provides the only known reservoir for the parasite Leishmania donovani in India, remains a problem. Timely recognition and treatment of PKDL would contribute significantly to the control of kala-azar. We evaluated here the potential of the enzyme-linked immunosorbent assay (ELISA) as a diagnostic tool for PKDL. Antigen prepared from promastigotes and axenic amastigotes with parasite isolates that were derived from skin lesions of a PKDL patient gave sensitivities of 86.36 and 92%, respectively, in the 88 PKDL cases examined. The specificity of the ELISA test was examined by testing groups of patients with other skin disorders (leprosy and vitiligo) or coendemic infections (malaria and tuberculosis), as well as healthy controls from areas where this disease is endemic or is not endemic. A false-positive reaction was obtained in 14 of 144 (9.8%) of the controls with the promastigote antigen and in 14 of 145 (9.7%) of the controls with the amastigote antigen. Evaluation of the serodiagnostic potential of recombinant k39 by ELISA revealed a higher sensitivity (94.5%) and specificity (93.7%) compared to the other two antigens used. The data demonstrate that ELISA with crude or recombinant antigen k39 provides a relatively simple and less-invasive test for the reliable diagnosis of PKDL.

Diagnosis of visceral leishmaniasis: comparative potential of amastigote antigen, recombinant antigen and PCR.

Development of simple, economical and non-invasive tests for the early diagnosis of visceral leishmaniasis (VL) or kala-azar (KA) remains a challenge, and serological studies based on antigen prepared from the amastigote stage of Leishmania donovani, the stage that causes infection, are lacking. In the present study, circulating antibodies to total antigen isolated from the promastigote and amastigote stages of the parasite, as well as to recombinant K39 (rK39) antigen, are measured by enzyme-linked immunosorbent assay (ELISA) and the results compared with a polymerase chain reaction (PCR) test for KA diagnosis. In 116 samples of KA examined, the amastigote antigen gave significantly higher mean absorbance values in ELISA than did the promastigote antigen. The sensitivity for KA detection was significantly higher using the amastigote antigen (94%) than the promastigote antigen (90.5%). Analysis in 91 controls showed that specificity was higher with amastigote antigen (92.3%) than with promastigote antigen (86.8-89.0%). Reliability of ELISA diagnosis with amastigote antigen was only marginally lower than that with rK39 ELISA or with the PCR test. Easy availability and low cost of indigenous amastigote antigen, together with the simplicity of ELISA compared with PCR, make ELISA based on amastigote antigen a promising choice for the diagnosis of KA.

Diagnosis of visceral leishmaniasis: comparative potential of amastigote antigen, recombinant antigen and PCR.

Development of simple, economical and non-invasive tests for the early diagnosis of visceral leishmaniasis (VL) or kala-azar (KA) remains a challenge, and serological studies based on antigen prepared from the amastigote stage of Leishmania donovani, the stage that causes infection, are lacking. In the present study, circulating antibodies to total antigen isolated from the promastigote and amastigote stages of the parasite, as well as to recombinant K39 (rK39) antigen, are measured by enzyme-linked immunosorbent assay (ELISA) and the results compared with a polymerase chain reaction (PCR) test for KA diagnosis. In 116 samples of KA examined, the amastigote antigen gave significantly higher mean absorbance values in ELISA than did the promastigote antigen. The sensitivity for KA detection was significantly higher using the amastigote antigen (94%) than the promastigote antigen (90.5%). Analysis in 91 controls showed that specificity was higher with amastigote antigen (92.3%) than with promastigote antigen (86.8-89.0%). Reliability of ELISA diagnosis with amastigote antigen was only marginally lower than that with rK39 ELISA or with the PCR test. Easy availability and low cost of indigenous amastigote antigen, together with the simplicity of ELISA compared with PCR, make ELISA based on amastigote antigen a promising choice for the diagnosis of KA.

A simple and sensitive test for field diagnosis of post kala-azar dermal leishmaniasis.

BACKGROUND: Current methods for diagnosis of post kala-azar dermal leishmaniasis (PKDL) do not offer adequate sensitivity and specificity. OBJECTIVES: To develop a simple and sensitive test for field diagnosis of PKDL. METHODS: Immunochromatographic nitrocellulose strips precoated with recombinant k39 antigen were evaluated for the detection of circulating antibodies to leishmanial k39 in PKDL sera. A drop of serum applied to the strip followed by buffer led to the development of two visible bands indicating the presence of anti-k39 IgG. RESULTS: The strip test was able to detect cases of PKDL with 91% sensitivity. The specificity of the test was evaluated using controls with other skin diseases, other common infections and healthy persons from endemic and non-endemic regions. Of 125 controls examined, all were negative on the strip test, indicating 100% specificity of the test. CONCLUSIONS: The immunochromatographic nitrocellulose strips provide a non-invasive, rapid and accurate method for diagnosing PKDL.

Expression of a mutant form of Leishmania donovani centrin reduces the growth of the parasite.

Leishmania donovani, a protozoan parasite, causes visceral disease in humans. To identify genes that control growth, we have isolated for the first time in the order Kinetoplastida a gene encoding for centrin from L. donovani. Centrin is a calcium-binding cytoskeletal protein essential for centrosome duplication or segregation. Protein sequence similarity and immunoreactivity confirmed that Leishmania centrin is a homolog of human centrin 2. Immunofluorescence analysis localized the protein in the basal body. Calcium binding analysis revealed that its C-terminal Ca(2+) binding domain binds 16-fold more calcium than the N-terminal domain. Electrophoretic mobility shift of centrin treated with EGTA and abrogation of the shift in its mutants lacking a Ca(2+) binding site suggest that Ca(2+) binding to these regions may have a role in the protein conformation. The levels of centrin mRNA and protein were high during the exponential growth of the parasite in culture and declined to a low level in the stationary phase. Expression of N-terminal-deleted centrin in the parasite significantly reduces its growth rate, and it was found that significantly more cells are arrested in the G(2)/M stage than in control cells. These studies indicate that centrin may have a functional role in Leishmania growth.

Development of a species-specific PCR assay for detection of Leishmania donovani in clinical samples from patients with kala-azar and post-kala-azar dermal leishmaniasis.

We have developed a PCR assay that is capable of amplifying kinetoplast DNA (kDNA) of Leishmania donovani in a species-specific manner among Old World leishmanias. With Indian strains and isolates of L. donovani the assay was sensitive enough to detect kDNA in an amount equivalent to a single parasite or less. The extreme sensitivity of the assay was reflected in its ability to detect parasite DNA from small volumes of peripheral blood of patients with kala-azar (KA) and from skin lesions from patients with post-KA dermal leishmaniasis (PKDL). A total of 107 clinical leishmaniasis samples were analyzed. Of these 102 (95.3%) were positive by PCR. The test provided a diagnosis of KA with 96% sensitivity using patient whole-blood samples instead of bone marrow or spleen aspirates that are obtained by invasive procedures. The assay was also successful in the diagnosis of 45 of 48 PKDL cases (93.8%). Cross-reactions with pathogens prevalent in the area of endemicity, viz., Mycobacterium tuberculosis, Mycobacterium leprae, and Plasmodium spp., could be ruled out. Eighty-one control samples, including dermal scrapings from healthy portions of skin from patients with PKDL were all negative. Two of twenty controls from the area of endemicity were found positive by PCR assay; however, there was a good possibility that these two were asymptomatic carriers since they were serologically positive for KA. Thus, this PCR assay represents a tool for the diagnosis of KA and PKDL in Indian patients in a noninvasive manner, with simultaneous species identification of parasites in clinical samples.

Heat-stress induced modulation of protein phosphorylation in virulent promastigotes of Leishmania donovani.

In parasites such as Leishmania, the study of molecular events induced in response to heat stress is of immense interest since temperature increase is an integral part of the life cycle. Protein phosphorylation is known to control major steps of proliferation and differentiation in eukaryotic cells. Studies on intracellular signaling systems in protozoa are relatively recent. We have examined the effect of heat shock on the protein phosphorylation status in promastigotes of Leishmania donovani. The patterns of total protein phosphorylation and specific phosphorylation at tyrosine residues were examined using [32P]-orthophosphate labelling of the parasites and immunoblotting with a monoclonal anti-phosphotyrosine antibody. The major proteins of L. donovani that were phosphorylated at 24 degrees C had apparent molecular weights of 110, 105, 66-68, 55, 36-40 and 20 kDa. Heat shock (from 24 to 37 degrees C) led to a significant decrease in phosphorylation of the majority of phosphoproteins in the virulent promastigotes. On the other hand, the avirulent promastigotes did not show any decrease in protein phosphorylation on exposure to heat stress. Predominant phosphorylation at tyrosine residues was detectable in proteins of putative size 105-110 kDa in both virulent and avirulent parasites. Heat shock led to a reduction in the level of phosphotyrosine in both these proteins in the case of virulent parasites, while no such reduction was detectable in avirulent parasites. Significant modifications in the phosphorylation status of proteins in response to heat stress including that of tyrosine containing proteins, observed exclusively in virulent parasites, suggest that modulation of protein phosphorylation/dephosphorylation may play a role in signal transduction pathways in the parasite upon heat shock encountered on entering the mammalian host.

Fabrication and characterization of sputtered-carbon microelectrode arrays.

This paper describes a robust and reliable process for fabricating a novel sputter-deposited, thin-film carbon microelectrode array using standard integrated circuit technologies and silicon micromachining. Sputter-deposited carbon films were investigated as potential candidates for microelectrode materials. The surface properties and cross section of the microelectrode arrays were studied by atomic force microscopy and scanning electron microscopy, respectively. Electrical site impedance, crosstalk, and lifetime (dielectric integrity) of microelectrodes in the array were characterized. Electrochemical response of the microelectrodes to hexaammineruthenium(III) chloride and dopamine were investigated by fast-scan cyclic voltammetry and high-speed, computer-based chronoamperometry; results show that thin-film carbon microelectrodes are well-behaved electrochemically. The thin carbon films offer extremely good electrical, mechanical, and chemical properties and thus qualify as viable candidates for various electroanalytical applications, particularly acute neurophysiological studies.