Fig. 5A depicts the quantification of internalised fluorescence-labelled NPs (Sicastar Red: 6 μg/ml, AmOrSil: 300 μg/ml) in H441 for 4 h with further 20 h cultivation in MC and CC (with ISO-HAS-1). Concentrations were chosen to obtain adequate fluorescence intensities in order to compare mono- and cocultures. A significant increase in fluorescence intensity was observed for NP-incubated H441 in MC for both NPs (Fig. 5A: Sicastar Red: 1.5 ± 0.5-fold of uc and AmOrSil: 2.7 ± 0.3-fold of uc). For H441 in CC, however, an uptake via fluorescence

intensity measurement could not be detected. Based on the visual examination of the microscopic image selleck products (Fig. 5B), the uptake of both NP types in H441 in CC appeared extremely low compared to

the MC. In Fig. 5C, an elevation of the NP-concentration and exposure time revealed an increased uptake of Sicastar Red (60 μg/ml, Trametinib clinical trial 48 h) in H441 in CC. However, an increased uptake of AmOrSil (300 μg/ml, 48 h) could not be verified. The same exposure times and staining procedures as described above (see Fig. 2) were carried out with H441 grown in CC with ISO-HAS-1 to determine if differences in nanoparticle uptake or trafficking behaviour from H441 under different culture conditions compared to the MC occurred. Although the monoculture of H441 showed fluorescent signals inside the cells after only 4 h of incubation, this time period yielded no uptake in H441 in CC with both NP types as detectable by fluorescence microscopy (data not shown). Similar to the findings in the MC, no clear uptake in early endosomes (clathrin heavy chain, caveolin-1 and other markers) was detected in the CC at all time points chosen (4 h and 4 h followed by 20 h cultivation in fresh medium without NPs).

Accumulation of Sicastar Red in flotillin-1- and -2-bearing vesicles occurred after 20 h following the 4 h incubation period (Fig. 6) similar to that observed in MC. AmOrSil however, did not show any colocalisation with flotillin-1 and 2 (data not shown). Fig. 7 (left column) shows exposure of ISO-HAS-1 in MC to NPs as it was applied for the colocalisation studies (Sicastar Red 6 μg/ml and AmOrSil: 300 μg/ml, 4 h with 20 h cultivation in serum-containing medium without NPs. A detectable uptake could be verified with direct exposure to NPs for Dipeptidyl peptidase the MC. To evaluate the transport of NPs across the NP-exposed epithelial layer of the CC, the endothelial layer (ISO-HAS-1) on the lower surface was examined for NPs. For this purpose, NPs (Sicastar Red: 60 μg/ml, AmorSil: 300 μg/ml) were continuously applied on the apical side (on the epithelial monolayer of H441) for 48 h. As a control ISO-HAS-1 was seeded on the lower surface of the transwell filter membrane and cultured for 10 days with subsequent indirect (apical) NP-application without H441 on the top (Fig. 7, middle column). A cellular uptake of both NPs could be detected in the ISO-HAS-1 transwell-monoculture.

The crystals were harvested by centrifugation and then evaporated at 37 °C. CaOX crystals were used at a final concentration of 0.8 mg/ml, buffered with Tris 0.05 mol/L and NaCl 0.15 mol/L at pH 6.5. Experiments were conducted at 37 °C in the absence or presence of the plant extract after stopping the stirring. The percentage aggregation inhibition rate (Ir) was then calculated by comparing the turbidity in the presence of the extract with that obtained in the control using following formula30: Ir=(1−Turbiditysample/Turbiditycontrol)×100Ir=(1−Turbiditysample/Turbiditycontrol)×100 Fig. 1 showed CaOx crystallization without the addition of extract (control) while Fig. 2 showed CaOx

crystallization in the presence of extract in the concentration Obeticholic Acid solubility dmso of 100, 200, 300, 400 and 500 μg/ml respectively. The % inhibition of turbidity (aggregation) in the presence of herb extracts was lower than in the control, showing that crystals were less aggregated. The inhibited aggregation associated with the extract increased with concentration. This inhibition was greatest with aqueous extract of root when compared to petroleum ether, chloroform and methanol extracts of leaf and stem (Fig. 3, Fig. 4, Fig. 5, Fig. 6, Fig. 7 and Fig. 8).

Kidney stone function is a complex process that results from a succession of several physico-chemical events including supersaturation, nucleation, growth, aggregation MAPK Inhibitor Library purchase and retention within renal tubules.31 Thus if supersaturation or later steps in crystallization

can be prevented, then lithiasis should be avoided. Indeed, several measures are usually taken to reduce supersaturation, e.g. increasing fluid intake and medical therapy. In India, as in many less developed areas, phytotherapy is a common method of primary health care because pharmaceutical products are expensive and the ‘folk’ pharmacopoeia provides apparently effective remedies for many diseases. These results could be considered positives because the herb extracts inhibits crystallization and prevents stone formation. The main findings of the present study were that extracts from plants inhibited the crystallization of CaOx in solution, there were less and smaller particles with increasing concentrations Calpain of extract as shown in various microphotographs i.e. Figs. 1 and 2. Fig. 1 showed maximum number and largest size of crystals as it was without plant extracts while Fig. 2 showed comparatively less number and smaller size of crystals. The increasing concentration of plant extracts (100, 200, 300, 400 and 500 μg/ml) had inhibited the CaOx crystal growth (Fig. 2). These results were also supported by the Fig. 3, Fig. 4, Fig. 5, Fig. 6, Fig. 7 and Fig. 8. The extract of plant causes fewer numbers of crystals in solution, thereby reduced supersaturation and the size of the particles.

1) [5], [12] and [28]. Assessment of vaccine-induced immune responses can be

achieved through a range of T cell, B cell, and innate immunity assays. Many of the same assays and reagents used to develop preventive vaccines can be applied to therapeutic vaccine research. However, there is no consensus on assays that would allow for trial comparisons, and on methods to address biological variability in baseline viral load and other responses in HIV positive individuals. One promising and relatively new approach is the measurement of the ability of HIV-specific CD8 T cells to kill infected CD4 T cell targets, which is just beginning to be evaluated in the context of vaccine trials [29] and [30]. Given the focus on curative interventions, a primary outcome measure in most modern therapeutic vaccines studies is the size of the “latent Dinaciclib in vitro reservoir”, perhaps best defined as the residual virus that remains in the setting of apparently effective combination ART, and is able to give rise to recrudescent viral replication and progressive disease

after ART is stopped. At least part of this “reservoir” is composed of virus in latently infected cells, rather than actively replicating virus. Although viral outgrowth assays used to quantify the replication-competent reservoir are viewed as the gold standard, there is no current standard, high-throughput check details measure of the reservoir. Measures of plasma HIV RNA, cell-associated HIV RNA (unspliced, multiply spliced) and cell-associated DNA (integrated, unintegrated, total) are being developed, but these are unlikely to fully resolve the difficulties of distinguishing replication-competent latent proviruses from defective ones [31]. Measurement of the HIV reservoir both in vitro and in vivo has emerged as an important potential biomarker that will require additional development and optimization [32] and [33]. Drs. Nicole Frahm, Felipe Garcia, Jeff Jacobson, John Eldridge, Jean Boyer and George Pavlakis

discussed the lessons that can be learned from past therapeutic vaccine studies in humans (Fig. 2). Therapeutic vaccine candidates recently tested have utilized a variety of platforms and approaches including DNA, viral vectors (alone and with DNA) science [34], [35] and [36] dendritic cells (DC) [37] and [38] and peptides [27], [39] and [40], using a variety of antigens together in some case with adjuvants and immune modulators. A few clinical trials of therapeutic vaccines to date have induced a transitory reduction in viral load in the context of treatment interruption. Some of these trials have shown modest delays in time to viral load rebound, prolongation of time until ART needs to be resumed, and/or sustained reductions of viral load (typically less than 0.5 log10 copies RNA/mL) [12].

It is a temperate genus and grows in the warm temperate regions. About 23 species occur in India. H. candolleanum (Wight et arn), Gamble, an endemic species of Western Ghats, is a large perennial herb with tuberous roots commonly found in the hills and mountains of Peninsular India at higher altitudes. The plant is used in folk and tribal medicine for various purposes.

The kani tribes administer decoction of the whole plant internally for nervous disorders inflammatory conditions. The decoction of the root of this plant is used by the tribals to treat inflammatory condition, as an antiarthritic and nerve tonic. 1 A number of furanocoumarins and two monoterpenoids were reported from the fruits and roots of H. candolleanum 2 Ulixertinib and chemical

composition of essential oil was reported from the rhizomes of H. candolleanum. 3 The essential oil composition of various members of this genus have also been reported, Heracleum persicum, 4 and 5Heracleum dissectum Ledeb, 6Heracleum sphondylium, 7Heracleum crenatifolium Boiss. 8 and 9 Plants belonging to the genus Heracleum are aromatic and are excellent sources of essential oils. Here we report the chemical composition of the oils from the seeds of H. candolleanum. H. candolleanum (Wight et Arn) were collected from Ambalapara, Aralam wild life sanctuary. It was identified by Dr. Udayan. P. S. (Department of botany, Sree Krishna College, Guruvayoor). The herbarium is deposited at Sree Krishna College, first Guruvayoor and Karpagam University, Coimbatore. Voucher No: 0123 Quisinostat on 25. 03.2009. 1 kg of seeds of H. candolleanum was hydrodistilled for 4 h in a modified Clevenger type apparatus to yield 0.4% of essential oil. The essential oil so obtained was stored in a sealed glass tubes with screw lid cover under refrigeration at 4 °C. The essential oil of H. candolleanum was subjected to GC–MS analysis on an Agilent system consisting of a model 6890N gas chromatograph, a model 5975 inert mass selective detector (EIMS, electron energy, 70 eV, scan range 50–1000 amu, and scan rate 2 scans/s), and an Agilent Chem Station data system. The GC column was an DB-5 ms, fused silica capillary with a (5% phenyl)-methyl poly siloxane stationary phase,

film thickness of 0.25 μm, a length of 30 m, and an internal diameter of 0.25 mm. The carrier gas was helium with a column head pressure of 7.07 psi and flow rate of 1.0 mL/min. Inlet temperature was 230 °C and MSD detector temperature was 230 °C. The GC oven temperature program was used as follows: 70 °C @ 5 °C/min, final temperature 120 s ramp @ 10 °C/min, final temperature 280 for 20 min. The sample was dissolved in 10 mL of acetone:toluene (1:1) mixture. 1 μL injections using a split less injection technique was used. Identification of oil components was achieved based on their retention indices, and by comparison of their mass spectral fragmentation patterns with those reported in the literature and stored on the MS library [NIST database (G1036A, revision D.01.

For labour induction, cervical ripening (even with an unfavourable cervix), increases the chance of vaginal delivery [384] and [385]. With severe preeclampsia, this will take more time and be less successful compared with normotensive pregnancy [386] and [387]. Neither IUGR nor oligohydramnios are contraindications

to induction [388]. Rates of vaginal delivery after induction are 6.7–10% at 24–28 weeks (suggesting advisability of Caesarean with viable fetuses), 47.5% at 28–32 weeks, 68.8% at 32–34 weeks, and 30% with birthweights <1500 g [385], [388], [389], [390] and [391]. Vaginal delivery likelihood is reduced (but still exceeds 50%) when there is increased umbilical artery resistance [392] and [393]. The following predict Caesarean delivery: absent or reversed

umbilical artery CDK inhibitor end-diastolic flow, abnormal BPP, and abnormal sequential changes in Doppler studies of the fetal circulation [394], [395], [396] and [397]. Preeclampsia is associated with thrombocytopoenia and coagulopathy, and active management of the third stage [398], avoiding ergometrine (ergonovine maleate), should be performed to avoid postpartum haemorrhage [399], [400], [401], [402], [403] and [404]. 1. The anaesthesiologist should be informed when a woman with preeclampsia is admitted to the delivery suite (II-3B; Low/Strong). 5. Intravenous and oral fluid intake Anti-diabetic Compound Library solubility dmso should be minimized in women with preeclampsia, to avoid pulmonary oedema (II-2B; Low/Strong). 9. Arterial line insertion may be used for continuous arterial BP monitoring when BP control is difficult or there is severe bleeding (II-3B; Very low/Strong). 12. Upon admission to delivery suite, women with preeclampsia should have a platelet count done (II-1A; however Low/Strong).

Communication between caregivers is essential [2]. Early consultation (by telephone if necessary) with anaesthesia should occur, at the latest with delivery suite admission of a woman with preeclampsia. Anaesthesiologists may co-manage hypertension, maternal end-organ dysfunction, and use of medications with anaesthesia/analgesia implications. Early placement of an epidural catheter is advantageous to: (i) attenuate labour pain-induced increases in cardiac output and BP [405], [406] and [407], and in the event that either (ii) thrombocytopoenia develops or (iii) Caesarean delivery is required. Neither epidural nor combined spinal-epidural, analgesia harms the fetus [405], [408] and [409] or increases Caesarean delivery in severe preeclampsia [410] and [411]. If neuraxial analgesia and/or anaesthesia is contraindicated, intravenous opioid analgesia is a reasonable alternative; but neonatal depression may result and require naloxone [412]. For Caesarean delivery, spinal is preferred over epidural anaesthesia (unless already placed) because of its more rapid onset and smaller calibre needle [413].

These classes of drugs interfere with different points in the viral life cycle, so the combination works synergistically.15 Though these combination therapies have increased survival and quality of life enormously, there are also problems associated with these

such as compliance, resistance, many interactions and serious side effects. Reverse transcriptase inhibitors act to inhibit the enzyme reverse find more transcriptase, thus, inhibiting the transcription of viral RNA into DNA. Reverse transcriptase inhibitors are both nucleoside and nucleotide reverse transcriptase inhibitors, and the non-nucleoside reverse transcriptase inhibitors. Patil et al, isolated, from the Malaysian tree Calophyllum inophyllum and also from the giant African snail Achatina fulica which feeds on its leaves, coumarin derivatives designated as inophyllums. Two of the compounds inhibited HIV-1 RT with IC50 PD-1/PD-L1 inhibitor 2 values of 38 and 130 nm respectively

and were active against HIV-1. 16 HIV-1 reverse transcriptase uses nucleotides to reverse transcribe the RNA of the virus into proviral DNA so that this proviral DNA can be inserted into the DNA of the host cell. In the cell, the nucleoside RT inhibitors are then phosphorylated into nucleotides, which are then used by reverse transcriptase to convert RNA into DNA. When reverse transcriptase uses these faulty building blocks, the development of the DNA is terminated and cellular enzymes can destroy the virus particles. Cross resistance between the Linifanib (ABT-869) NRTIs is possible.17 NRTIs, especially Zerit, Videx and Retrovir, are associated with lactic acidosis and hepatic steatosis.18 Nucleoside reverse transcriptase inhibitors can cause hyperlactemia by disrupting the function of the mitochondria, known as mitochondrial toxicity. NRTI’s can also cause hepatic steatosis. However, NRTIs are capable of causing a wide variety of long-term side effects, including myelotoxicity, lactic acidosis, polyneuropathy and pancreatitis. Long-term side effects

are theorized to be related to mitochondrial toxicity (Brinkman et al, 1998). Fast-replicating cells may also be inhibited by NRTIs leading to blood disorders like anemia and neutropenia. Macrocytic anemia and myopathy may occur with Zidovudine and oral ulcers with Zalcitabine and Didanoside. Abacavir can cause severe hypersensitivity reactions and is a contraindication for further treatment. Long-term side effects of the NRTIs are lipoatrophy.18 Nucleoside and nucleotide analogs have become the cornerstone of HAART (Highly Active Antiretroviral Therapy). Unfortunately, these drugs have shown to inhibit cellular polymerases, most notably mitochondrial DNA polymerase gamma. Studies of the NRTIs in enzyme assays and cell cultures demonstrate the following hierarchy of mitochondrial DNA polymerase gamma inhibition: Zalcitabine > Didanosine > Stavudine > Lamivudine > Zidovudine > Abacavir.

Regulatory authorities have recognized the importance of stimulating T cell responses to influenza

and have encouraged the exploration of T cell assays for evaluating vaccine efficacy in general [27] and [28] and, in particular, influenza vaccines including those aimed to protect against avian influenza [29] and [30]. However, standardized and reproducible assays of influenza-specific T cell responses that are too needed to make significant progress in the development of improved influenza vaccines have yet to be validated [29]. Herein, we report the validation of standardized assays of T cell responses that are likely to correlate with protection against influenza [13], [14] and [31]. The assays are based on the detection of effector molecules produced by peripheral Onalespib clinical trial blood mononuclear cells (PBMC) after ex vivo stimulation with live influenza virus. By using multiplex technology, multiple cytokines including IL-2, IL-4, IL-5, IL-10, IL-12, IL-13, IL-17, GM-CSF, IFN-γ, and TNF-α, could efficiently be detected in one sample of PBMC culture supernatant. In addition, a detection assay for granzyme B activity, an essential screening assay effector molecule in the cytotoxic response of CD8+ T cells against virus-infected target cells [32], was validated in lysates of these virus-stimulated PBMC. The validation process was preceded by rigid standardization

of the assays and on-site training of the laboratory technicians following standard operating procedures (SOP) [33]. This work comprised determination of specificity, accuracy,

linearity, range, detection limit, intermediate precision, and robustness by three European and one Canadian laboratory. The validation results showed that these assays of the T cell response to influenza were reproducible and could measure the levels of granzyme B and cytokines in an accurate and specific manner. Human PBMC were isolated from buffy coats of healthy individuals by Lymphoprep (Axis Shield, Oslo, Norway) density centrifugation at 950 × g for 20 min. The PBMC were washed several times with PBS until the supernatant was clear. Subsequently, the PBMC were frozen in multiple aliquots in 90% FCS (Hyclone, Logan, Utah)/10% DMSO (Sigma–Aldrich, St. Louis, USA) and stored at −135 °C. Buffy coats were retrieved in accordance with the human experimental guidelines of Sanquin Blood Bank North West Region (project number S03.0015-X). Influenza below H3N2 A/Wisconsin/67/2005 was produced by infecting MDCK cells. As negative control (mock) medium of uninfected MDCK cells was used. The participating laboratories in alphabetical order, not in order in results, were: 1. National Centre for Epidemiology (NCE), Budapest, Hungary Frozen PBMC were thawed in AIM V medium and rested by incubation for 4 h at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. Pilot experiments showed that this resting period is essential to obtain responses similar to responses with fresh cells (data not shown). Subsequently, PBMC (1.

Both studies examining physical activity interventions adopted different approaches: an environment-focused community awareness campaign promoting physical activity in the local community (Cochrane and Davey, 2008+); and two interventions tested together using a fitness

assessment to tailor an exercise plan and an exercise consultation focused on behaviour change principles, both with vouchers for local facilities (Lowther et al., 2002++). Overall, physical activity interventions showed mixed effectiveness (Supplementary Table 6). One study demonstrated a positive effect on health and mixed effectiveness was found on physical activity behaviour, with one study finding a positive effect and another finding a mixed effect. No studies identified a negative impact on any outcome. One multi-component intervention incorporated PFT�� clinical trial a combination of behaviour change, SCH 900776 molecular weight and educational, empowerment and medical approaches to lifestyle change (Baxter

et al., 1997+) and the other involved providing access to an Internet portal aimed at helping people with heart disease to lead a healthier lifestyle (Lindsay et al., 2008+). Evidence of mixed effectiveness was found on consumption of high fat foods, with one study reporting a positive effect on consumption of low-fat milk but no effect on consumption of low-fat spread, and one study reporting no significant impact ( Supplementary Table 6). Evidence suggested no significant impact on physical activity, weight control, physiological measurements, psychosocial variables and other eating habits. Neither study identified a negative impact on any outcome. We examined the characteristics of studies that were and were not successful across a range of outcomes (sample size, Cell press study design, intervention, duration of intervention

and duration of longest follow-up point). The only difference found was in studies assessing consumption of high fat foods, where the positive effect (for similar interventions) was associated with a shorter follow-up time ( McKellar et al., 2007+). One study that did not find evidence of a positive effect on any outcome was the only study to assess access to a health promotion portal ( Lindsay et al., 2008+). Barriers to and facilitators of lifestyle change identified in included qualitative studies were grouped into several categories, each with one or more themes attached (Supplementary Table 7). Having sufficient available resources was raised as being important in implementing dietary and physical activity interventions ( Bremner et al., 2006+; Dobson et al., 2000+; Kennedy et al., 1998+). Specific barriers included a lack of funding, time and labour for running interventions and a lack of available facilities for preparing, storing and transporting food. Continuous funding from a large award was identified as a facilitator, as was developing a focused action plan to target the funding and labour effectively.

The HPV vaccination programme represents an ideal opportunity to convey the benefit of prevention programmes and reinforcement of this message is needed. Uptake of HPV vaccination was positively correlated with uptake of cervical screening, and cytology results indicate that vaccination has a protective effect against an abnormal result. Women from more socially deprived areas engage less with cervical cancer prevention healthcare services.

New strategies to enhance uptake of screening services need to be directed at young women with a focus on areas classified as socially deprived. SP and SH conceived of the study. HB, SB and MAR collected the data for the study. HB, SH and Pazopanib order SP contributed to the analyses of the study and all authors contributed to the interpretation

of results and the writing of this paper and have approved the final draft. All authors declare no conflicts of interest that could have influenced this work. This study was funded by Cancer Research UK and sponsored by Cardiff University. The research was also supported by The Centre for the Improvement of Population Health through E-records Research (CIPHER). CIPHER is one of four UK e-health Informatics Research Centres funded by a joint investment from: Arthritis Research UK, the British Heart Foundation, Cancer Research UK, the Chief Scientist Office (Scottish Government Health Directorates), the Economic and Social Research Council, the Engineering and Physical Sciences Research Council, the Medical Research Council, the National Institute for Health Research, the National Institute for Social Care and Health Research (Welsh Government) selleck chemicals and the Wellcome Trust (Grant reference: MR/K006525/1). “
“Foot-and-mouth disease (FMD) vaccines are used on an enormous scale across the globe, with over 2 billion doses thought to be used every

year [1]. Despite this, little is done to assess their performance in the field. Vaccine effectiveness, defined as the reduction in risk in vaccinated individuals compared to similarly exposed unvaccinated individuals under field conditions [2], provides a direct measure of vaccine protection within a vaccination programme. FMD in Anatolian Turkey (Fig. 1) poses a threat to the EU which ever is disease free [3]. During 2009–11 (inclusive) approximately twenty-million doses of polyvalent FMD vaccine were used a year for biannual mass vaccination of Turkey’s cattle population [4]. In Turkey, inactivated, oil adjuvanted FMD vaccines with a specified protective effect of >3PD50 (PD50 = 50% protective dose) are administered intra-muscularly. In 2011 Turkey experienced an incursion of the FMD Asia-1 serotype. Although serotypes A and O are endemic this serotype had not been present since 2002 [5]. Vaccine matching tests suggested that the vaccine used at the time (Asia-1 Shamir) would not protect against the new field strain (FMD Asia-1 Sindh-08) [6].

This suggests that the vaccine could offer significant protection in varying geographical settings and over time. This finding also supports

the view that there are multiple determinants that provide a lead to protective immunity. We noted an imbalance of cases associated with G9P[4] but could not identify any biological basis for this imbalance and conclude that this was due to chance alone. The efficacy of the licensed rotavirus vaccines is higher in developed than in developing countries [19], [20], [21], [22] and [23]. Although the efficacy PD0332991 cell line of 116E in the first 2 years of life is modest as it is for other licensed vaccines, the impact on preventing deaths related to severe RVGE is likely to be high in India and other developing countries because of the higher disease burden [2] and [4]. It is for this

reason that the World Health Organization has recommended inclusion of rotavirus vaccine into national immunization programs. BIBF 1120 mouse Finally, the development of 116E is a unique example of team work and global collaboration and represents a novel approach to development of affordable health technologies of particular interest to developing countries [24]. Efforts are underway to understand the reasons underlying the relatively modest efficacy of all live rotavirus vaccines in low middle income countries. NB, JB and MKB prepared the manuscript and all authors reviewed and approved. TRC, AB, JJ, NG, AK, GK, SSR, SJ, JM, AA, HS, VA for design of protocol, trial implementation strategy and conduct. NB, KA and ST contributed to the design of protocol, trial implementation strategy, oversight of trial conduct and data analyses. JB, MKB, GT, RG, why HBG, GC, TSR contributed to the trial design and interpretation of data and laboratory guidance. KM, GVJAH, SP for product development.

MP, RK contributed to data analyses. SV for analyses of specimens. All authors have approved the final manuscript. KM, GVJAH and SP are employees of Bharat Biotech International Limited. Other authors have no conflict of interest. This trial was funded by the Department of Biotechnology, and Biotechnology Industry Research Assistance Council, Government of India, New Delhi, India; by a grant from the Bill & Melinda Gates Foundation (number 52714) to PATH, USA; by the Research Council of Norway, UK Department for International Development; by National Institutes of Health, Bethesda, USA; and by Bharat Biotech International, Hyderabad, India.