Realization of a DNA biosensor using inverted Lamb wave MEMS resonator based on ZnO/SiO2/Si/ZnO membrane.

A novel technique based on inverted Lamb wave MEMS resonator has been exploited for the realization of a DNA biosensor. Zinc oxide based Lamb wave MEMS resonator in the inverted configuration of ZnO/SiO2/Si/ZnO is fabricated for label free and efficient detection of Neisseria meningitidis, responsible for bacterial meningitis. Meningitis remains a devastating endemic in sub-Saharan Africa. Its early detection can prevent the spread and its lethal complications. The developed biosensor shows a very high sensitivity of 310 Hz(ngµl-1)-1 and very low detection limit of 82 pgµl-1 for symmetric mode of the Lamb wave device while the antisymmetric mode shows a sensitivity of 202 Hz(ngµl-1)-1 and the limit of detection of 84 pgµl-1. This very high sensitivity and very low detection limit of the Lamb wave resonator can be attributed to very high mass loading effect on the membranous structure of Lamb wave device, unlike the bulk substrate based devices. The indigenously developed MEMS based inverted Lamb wave biosensor shows high selectivity, long shelf life and good reproducibility. The ease of operation, low processing time and possibility of wireless integration of the of the Lamb wave DNA sensor paves a path towards the promising application in the field of meningitidis detection. The use of fabricated biosensor can be extended to other viral and bacterial detection applications as well.

Type II growing skull fracture in a 12-year-old girl.

A 12-year-old female patient came to the Neurology Outpatient Clinic with the complaining of headache, frequent episodes of abnormal body movements and swelling in the right frontal scalp region. Her parents gave remote history of head trauma. History of trauma in a paediatric patient followed by the onset of gradually progressive swelling in the scalp along with radiological findings of calvarial defect and protrusion of gliotic brain tissue through it led to the diagnosis of growing skull fracture. Her parents were counselled about the surgical management for which they agreed. The scalp defect was repaired followed by uneventful post-operative period. She is on regular anti-epileptics for the episodes of seizers she had at the time of presentation and is on regular follow-up.

Comparative assessment of canine-origin Lactobacillus johnsonii CPN23 and dairy-origin Lactobacillus acidophillus NCDC 15 for nutrient digestibility, faecal fermentative metabolites and selected gut health indices in dogs.

The present experiment was undertaken to validate a probiotic of canine origin for its potential use in dogs. A total of fifteen adult female Labrador dogs were allocated to three equal groups and fed a basal diet without probiotic (control) or with probiotic of either canine (Lactobacillus johnsonii CPN23; cPRO) or dairy (L. acidophilus NCDC 15; dPRO) origin for 9 weeks. The digestibility of most macronutrients remained similar among the groups; however, fibre digestibility was improved (P = 0·034) in dogs receiving cPRO. The faecal fermentative metabolites ammonia (P < 0·05) and lactate (P = 0·094) were altered favourably, indicating a positive influence of both probiotics. Faecal concentrations of acetate, propionate and butyrate were increased (P < 0·01) in both probiotic groups. However, improvements were higher in cPRO v. dPRO. The delayed-type hypersensitivity reaction to intradermal inoculation of phytohaemagglutinin-P was higher (P = 0·053) in cPRO as compared with control. The antibody response to sheep erythrocytes was, however, similar across the three groups. Overall, in dogs, the canine-origin probiotic was superior when compared with the dairy-origin probiotic.

An insight into death wish among patients with amyotrophic lateral sclerosis in India using "Wish-to-Die Questionnaire".

AIMS: In amyotrophic lateral sclerosis (ALS), death wish is expressed in a varying proportion of patients in different countries. In this first study from India, influence of belief system of religion/spirituality and attitude towards death, widely prevalent in the country, in decision making, was evaluated. MATERIAL AND METHODS: Twenty ALS patients were assessed using 'Wish-to-Die Questionnaire' (WDQ) developed to reflect seven domains, namely religion/spirituality, belief in karma, meaning of life, hope, family support, financial support and death wish. Functional impairment, depression, hopelessness and suicidal ideation were assessed by ALS Functional Rating Scale, Beck's Depression Inventory, Beck Hopelessness Scale and The Scale of Suicidal Ideation, respectively. RESULTS: On WDQ, all the 20 patients had belief in religion/spirituality, had hope and family support. Nineteen patients (95%) believed in karma, 16 (80%) still found life meaningful and 15 (75%) had financial support. Six patients (30%) had mild to moderate depression; hopelessness was present in 6 (30%) and suicidal ideation was present in one (5%). The 5 (25%) patients who expressed death wish did not significantly differ from others in 6 domains (religion/spirituality, belief in karma, meaning of life, hope, family support, financial support) of WDQ. The main reason in 3 patients who expressed death wish was lack of financial support. The fourth patient could not find meaning of life after the onset of illness, and the fifth wished to end his life since he had satisfactorily fulfilled all his responsibilities. CONCLUSION: Smaller proportion of patients of ALS expressed death wish in India compared to the Western countries. This may be attributed to belief in religion/spirituality and karma, having meaning of life and family support. As this is the first report from India, useful information may be obtained if similar studies are done on a larger sample.

Comparative Analysis of Cellular Immune Responses in Treated Leishmania Patients and Hamsters against Recombinant Th1 Stimulatory Proteins of Leishmania donovani.

Our prior studies demonstrated that cellular response of T helper 1 (Th1) type was generated by a soluble antigenic fraction (ranging from 89.9 to 97.1 kDa) of Leishmania donovani promastigote, in treated Leishmania patients as well as hamsters and showed significant prophylactic potential against experimental visceral leishmaniasis (VL). Eighteen Th1 stimulatory proteins were identified through proteomic analysis of this subfraction, out of which 15 were developed as recombinant proteins. In the present work, we have evaluated these 15 recombinant proteins simultaneously for their comparative cellular responses in treated Leishmania patients and hamsters. Six proteins viz. elongation factor-2, enolase, aldolase, triose phosphate isomerase, protein disulfide isomerase, and p45 emerged as most immunogenic as they produced a significant lymphoproliferative response, nitric oxide generation and Th1 cytokine response in PBMCs and lymphocytes of treated Leishmania patients and hamsters respectively. The results suggested that these proteins may be exploited for developing a successful poly-protein and/or poly-epitope vaccine against VL.

Characterization of glycolytic enzymes--rAldolase and rEnolase of Leishmania donovani, identified as Th1 stimulatory proteins, for their immunogenicity and immunoprophylactic efficacies against experimental visceral leishmaniasis.

Th1 immune responses play an important role in controlling Visceral Leishmaniasis (VL) hence, Leishmania proteins stimulating T-cell responses in host, are thought to be good vaccine targets. Search of such antigens eliciting cellular responses in Peripheral blood mononuclear cells (PBMCs) from cured/exposed/Leishmania patients and hamsters led to the identification of two enzymes of glycolytic pathway in the soluble lysate of a clinical isolate of Leishmania donovani--Enolase (LdEno) and aldolase (LdAld) as potential Th1 stimulatory proteins. The present study deals with the molecular and immunological characterizations of LdEno and LdAld. The successfully cloned and purified recombinant proteins displayed strong ability to proliferate lymphocytes of cured hamsters' along with significant nitric-oxide production and generation of Th1-type cytokines (IFN-γ and IL-12) from stimulated PBMCs of cured/endemic VL patients. Assessment of their prophylactic potentials revealed ∼ 90% decrease in parasitic burden in rLdEno vaccinated hamsters against Leishmania challenge, strongly supported by an increase in mRNA expression levels of iNOS, IFN-γ, TNF-α and IL-12 transcripts along with extreme down-regulation of TGF-ß, IL-4 and IL-10. However, animals vaccinated with rLdAld showed comparatively lesser prophylactic efficacy (∼ 65%) with inferior immunological response. Further, with a possible implication in vaccine design against VL, identification of potential T-cell epitopes of both the proteins was done using computational approach. Additionally, in-silico 3-D modelling of the proteins was done in order to explore the possibility of exploiting them as potential drug targets. The comparative molecular and immunological characterizations strongly suggest rLdEno as potential vaccine candidate against VL and supports the notion of its being effective T-cell stimulatory protein.

Leishmania donovani triose phosphate isomerase: a potential vaccine target against visceral leishmaniasis.

Visceral leishmaniasis (VL) is one of the most important parasitic diseases with approximately 350 million people at risk. Due to the non availability of an ideal drug, development of a safe, effective, and affordable vaccine could be a solution for control and prevention of this disease. In this study, a potential Th1 stimulatory protein- Triose phosphate isomerase (TPI), a glycolytic enzyme, identified through proteomics from a fraction of Leishmania donovani soluble antigen ranging from 89.9-97.1 kDa, was assessed for its potential as a suitable vaccine candidate. The protein- L. donovani TPI (LdTPI) was cloned, expressed and purified which exhibited the homology of 99% with L. infantum TPI. The rLdTPI was further evaluated for its immunogenicity by lymphoproliferative response (LTT), nitric oxide (NO) production and estimation of cytokines in cured Leishmania patients/hamster. It elicited strong LTT response in cured patients as well as NO production in cured hamsters and stimulated remarkable Th1-type cellular responses including IFN-ã and IL-12 with extremely lower level of IL-10 in Leishmania-infected cured/exposed patients PBMCs in vitro. Vaccination with LdTPI-DNA construct protected naive golden hamsters from virulent L. donovani challenge unambiguously (∼90%). The vaccinated hamsters demonstrated a surge in IFN-ã, TNF-á and IL-12 levels but extreme down-regulation of IL-10 and IL-4 along with profound delayed type hypersensitivity and increased levels of Leishmania-specific IgG2 antibody. Thus, the results are suggestive of the protein having the potential of a strong candidate vaccine.

A novel recombinant Leishmania donovani p45, a partial coding region of methionine aminopeptidase, generates protective immunity by inducing a Th1 stimulatory response against experimental visceral leishmaniasis.

The development of a vaccine against visceral leishmaniasis (VL) conferring long-lasting immunity remains a challenge. Identification and proteomic characterization of parasite proteins led to the detection of p45, a member of the methionine aminopeptidase family. To our knowledge the present study is the first known report that describes the molecular and immunological characterization of p45. Recombinant Leishmania donovani p45 (rLdp45) induced cellular responses in cured hamsters and generated Th1-type cytokines from peripheral blood mononuclear cells of cured/endemic VL patients. Immunization with rLdp45 exerted considerable prophylactic efficacy (∼85%) supported by an increase in mRNA expression of iNOS, IFN-γ, TNF-α and IL-12 and decrease in TGF-ß and IL-4, indicating its potential as a vaccine candidate against VL.

Evaluation of Leishmania donovani protein disulfide isomerase as a potential immunogenic protein/vaccine candidate against visceral Leishmaniasis.

In Leishmania species, Protein disulfide isomerase (PDI)--a redox chaperone, is reported to be involved in its virulence and survival. This protein has also been identified, through proteomics, as a Th1 stimulatory protein in the soluble lysate of a clinical isolate of Leishmania donovani (LdPDI). In the present study, the molecular characterization of LdPDI was carried out and the immunogenicity of recombinant LdPDI (rLdPDI) was assessed by lymphocyte proliferation assay (LTT), nitric oxide (NO) production, estimation of Th1 cytokines (IFN-γ and IL-12) as well as IL-10 in PBMCs of cured/endemic/infected Leishmania patients and cured L. donovani infected hamsters. A significantly higher proliferative response against rLdPDI as well as elevated levels of IFN-γ and IL-12 were observed. The level of IL-10 was found to be highly down regulated in response to rLdPDI. A significant increase in the level of NO production in stimulated hamster macrophages as well as IgG2 antibody and a low level of IgG1 in cured patient's serum was observed. Higher level of IgG2 antibody indicated its Th1 stimulatory potential. The efficacy of pcDNA-LdPDI construct was further evaluated for its prophylactic potential. Vaccination with this construct conferred remarkably good prophylactic efficacy (∼90%) and generated a robust cellular immune response with significant increases in the levels of iNOS transcript as well as TNF-α, IFN-γ and IL-12 cytokines. This was further supported by the high level of IgG2 antibody in vaccinated animals. The in vitro as well as in vivo results thus indicate that LdPDI may be exploited as a potential vaccine candidate against visceral Leishmaniasis (VL).

Treatment of Leishmania donovani-infected hamsters with miltefosine: analysis of cytokine mRNA expression by real-time PCR, lymphoproliferation, nitrite production and antibody responses.

OBJECTIVES: Miltefosine, an orally effective antileishmanial drug, works directly on the parasite by impairing membrane synthesis and subsequent apoptosis of the parasite and has also been reported to have macrophage-activating functions that aid parasite killing. We investigated the type of immunological responses generated in miltefosine-treated Leishmania donovani-infected hamsters, which simulate the clinical situation of human kala-azar. METHODS: Twenty-five-day-old infected hamsters, treated with miltefosine at 40 mg/kg for 5 consecutive days, were euthanized on days 30 and 45 post treatment (p.t.) and checked for parasite clearance and for real-time analysis of mRNAs of the Th1/Th2 cytokines interferon-γ (IFN-γ), interleukin-12 (IL-12), tumour necrosis factor-α (TNF-α), inducible nitric oxide synthase (iNOS), IL-4, IL-10 and transforming growth factor-ß (TGF-ß), nitric oxide (NO) production, the lymphocyte transformation test (LTT) and antibody responses. Responses were compared with the normal and Leishmania-infected groups at the same time points. RESULTS: By day 45 p.t. there was a significant increase in the mRNA expression of iNOS, IFN-γ, IL-12 and TNF-α, whereas there were significant decreases in IL-4, IL-10 and TGF-ß in cured hamsters as compared with their infected counterparts. In vitro stimulation of lymphocytes with concanavalin A and soluble Leishmania donovani antigen showed a maximum LTT response and there was a gradual increase in the NO level (∼7-fold compared with infected counterparts). Anti-Leishmania IgG and IgG1 levels, found to be elevated in the infected group, decreased significantly after treatment but there was a significant increase in IgG2 isotype. CONCLUSIONS: Treatment of Leishmania-infected hamsters with miltefosine reverses the Th2-type response into a strong Th1-type immune response.

Elongation factor-2, a Th1 stimulatory protein of Leishmania donovani, generates strong IFN-γ and IL-12 response in cured Leishmania-infected patients/hamsters and protects hamsters against Leishmania challenge.

In visceral leishmaniasis, Th1 types of immune responses correlate with recovery from and resistance to disease, and resolution of infection results in lifelong immunity against the disease. Leishmanial Ags that elicit proliferative and cytokine responses in PBMCs from cured/exposed/Leishmania patients have been characterized through proteomic approaches, and elongation factor-2 is identified as one of the potent immunostimulatory proteins. In this study, we report the cloning and expression of Leishmania donovani elongation factor-2 protein (LelF-2) and its immunogenicity in PBMCs of cured/exposed Leishmania-infected patients and hamsters (Mesocricetus auratus). Leishmania-infected cured/exposed patients and hamsters exhibited significantly higher proliferative responses to recombinant Lelf-2 (rLelF-2) than those with L. donovani-infected hosts. The soluble L. donovani Ag stimulated PBMCs of cured/exposed and Leishmania patients to produce a mixed Thl/Th2-type cytokine profile, whereas rLelF-2 stimulated the production of IFN-γ, IL-12, and TNF-α but not IL-4 or IL-10. Further, rLelF-2 downregulated LPS-induced IL-10 as well as soluble L. donovani Ag-induced IL-4 production by Leishmania patient PBMCs. The immunogenicity of rLelF-2 was also checked in hamsters in which rLelF-2 generates strong IL-12- and IFN-γ-mediated Th1 immune response. This was further supported by a remarkable increase in IgG2 Ab level. We further demonstrated that rLelF-2 was able to provide considerable protection (∼65%) to hamsters against L. donovani challenge. The efficacy was supported by the increased inducible NO synthase mRNA transcript and Th1-type cytokines IFN-γ, IL-12, and TNF-α and downregulation of IL-4, IL-10, and TGF-ß. Hence, it is inferred that rLelF-2 elicits a Th1 type of immune response exclusively and confers considerable protection against experimental visceral leishmaniasis.

16alpha-Hydroxycleroda-3,13 (14)Z-dien-15,16-olide from Polyalthia longifolia: a safe and orally active antileishmanial agent.

BACKGROUND AND PURPOSE: New antileishmanials from natural products are urgently needed due to the emergence of drug resistance complicated by severe cytotoxic effects. 16alpha-Hydroxycleroda-3,13 (14)Z-dien-15,16-olide (Compound 1) from Polyalthia longifolia was found to be a potential antileishmanial and non-cytotoxic, as evidenced by long-term survival (>6 months) of treated animals. This prompted us to determine its target and, using molecular modelling, identify the interactions responsible for its specific antileishmanial activity. EXPERIMENTAL APPROACH: In vitro activity of compound was assessed using intracellular transgenic green fluorescent protein-stably expressed Leishmania donovani parasites. In vivo activity and survival of animals post-treatment were evaluated in L. donovani-infected hamsters. Known property of clerodane diterpenes as potent human DNA topoisomerase inhibitors led us to evaluate the inhibition of recombinant L. donovani topoisomerase I using relaxation assay. Mode of cell death induced by Compound 1 was assessed by phosphotidylserine exposure post-treatment. Molecular modelling studies were conducted with DNA topoisomerase I to identify the binding interactions responsible for its activity. KEY RESULTS: Bioassay-guided fractionation led to isolation of Compound 1 as a non-cytotoxic, orally active antileishmanial. Compound 1 inhibited recombinant DNA topoisomerase I which, ultimately, induced apoptosis. Molecular docking studies indicated that five strong hydrogen-bonding interactions and hydrophobic interactions of Compound 1 with L. donovani DNA-topoisomerase are responsible for its antileishmanial activity. CONCLUSIONS AND IMPLICATIONS: The data reveal Compound 1 is a potent and safe antileishmanial. The study further exploited the structural determinants responsible for its non-cytotoxic and potent activity, to raise the feasibility of specifically targeting the target enzyme responsible for its activity through rational drug design.

Reporter genes facilitating discovery of drugs targeting protozoan parasites.

Transfection of protozoan parasites, such as Plasmodium, Leishmania, Trypanosoma and Toxoplasma, with various reporter gene constructs, has revolutionized studies to understand the biology of the host-parasite interactions at the cellular level. It has provided impetus to the development of rapid and reliable drug screens both for established drugs and for new molecules against different parasites and other pathogens. Furthermore, reporter genes have proved to be an excellent and promising tool for studying disease progression. Here, we review the recent advances made by using reporter genes for in vitro and in vivo drug screening, high-throughput screening, whole-animal non-invasive imaging for parasites and for the study of several aspects of host-parasite interactions.

Transgenic Leishmania donovani clinical isolates expressing green fluorescent protein constitutively for rapid and reliable ex vivo drug screening.

OBJECTIVES: Several Leishmania strains with episomal expression of green fluorescent protein (GFP) require constant drug pressure for its continuous expression and hence limit its use in ex vivo or in vivo systems. The aim of this study was to alleviate this problem by stably integrating the GFP gene into the parasite genome, so as to use these transfectants for ex vivo and in vivo drug screening. METHODS: The GFP gene was integrated downstream of the 18S ribosomal promoter region of Leishmania donovani. After initial selection, GFP-expressing parasites-both sodium stibogluconate (SAG)-susceptible (2001) and -resistant (2039) isolates-were grown without adding G418. The infectivity of these transfectants to macrophages (J774.1) as well as to hamsters was checked. The ex vivo screening assay was standardized using standard antileishmanial drugs. RESULTS: A constitutive and enhanced expression of GFP in promastigote and amastigote stages was achieved for approximately 12 months without any need for drug pressure. These transfectants were highly infective to macrophage cell lines as well as to hamsters, as observed by fluorescence microscopy and flow cytometry (FACS). GFP-tagged promastigotes as well as intracellular amastigotes were found to be highly susceptible to miltefosine, amphotericin B and pentamidine, in a concentration-dependent manner. SAG was inactive against the GFP-promastigotes, as well as SAG-resistant intracellular amastigotes, correlating well with earlier reports. CONCLUSIONS: The GFP-transfectants were found to be suitable for FACS-based ex vivo screening assays. They were also infective to hamsters up to day 60 post-infection.

Immunization with the DNA-encoding N-terminal domain of proteophosphoglycan of Leishmania donovani generates Th1-type immunoprotective response against experimental visceral leishmaniasis.

Leishmania produce several types of mucin-like glycoproteins called proteophosphoglycans (PPGs) which exist as secretory as well as surface-bound forms in both promastigotes and amastigotes. The structure and function of PPGs have been reported to be species and stage specific as in the case of Leishmania major and Leishmania mexicana; there has been no such information available for Leishmania donovani. We have recently demonstrated that PPG is differentially expressed in sodium stibogluconate-sensitive and -resistant clinical isolates of L. donovani. To further elucidate the structure and function of the ppg gene of L. donovani, a partial sequence of its N-terminal domain of 1.6 kb containing the majority of antigenic determinants, was successfully cloned and expressed in prokaryotic as well as mammalian cells. We further evaluated the DNA-encoding N-terminal domain of the ppg gene as a vaccine in golden hamsters (Mesocricetus auratus) against the L. donovani challenge. The prophylactic efficacy to the tune of approximately 80% was observed in vaccinated hamsters and all of them could survive beyond 6 mo after challenge. The efficacy was supported by a surge in inducible NO synthase, IFN-gamma, TNF-alpha, and IL-12 mRNA levels along with extreme down-regulation of TGF-beta, IL-4, and IL-10. A rise in the level of Leishmania-specific IgG2 was also observed which was indicative of enhanced cellular immune response. The results suggest the N-terminal domain of L. donovani ppg as a potential DNA vaccine against visceral leishmaniasis.