Strengthening fruit and vegetable supply-chain policies and programmes in India

India currently has one of the highest numbers of malnourished children in the world – 8% stunted, 43% underweight, and 20% overweight and obese. This distressing public health scenario is further exacerbated by a high prevalence of multiple micronutrient deficiencies among these children – such as iron deficiency anaemia and Vitamin A deficiency. Evidence shows linkages between early life malnourishment (either underweight or overweight-obesity) and predisposition to developing chronic diseases in adult life. Consuming 400g/day of fresh fruits and vegetables can help prevent micronutrient deficiencies while promoting overall growth and development. However, national averages indicate that children do not consume even 40% of the daily recommended amounts. Public Health Foundation of India (PHFI) undertook a study titled ‘Leveraging fruit and vegetable supply policies to tackle the dual burden of malnutrition in India’ supported by the Leveraging Agriculture for Nutrition in South Asia (LANSA) consortium at the M S Swaminathan Research Foundation (MSSRF). The study, discussed in this Brief aimed to analyse the policy environment related with fruit and vegetable (FV) supply in India to identify opportunities for policy to increase access to, and thus intakes of FV, especially among children.Department for International Development (DFID)UKAI

Commit, hide and escape: the story of Plasmodium gametocytes

Malaria is the major cause of mortality and morbidity in tropical countries. The causative agent, Plasmodium sp., has a complex life cycle and is armed with various mechanisms which ensure its continuous transmission. Gametocytes represent the sexual stage of the parasite and are indispensable for the transmission of the parasite from the human host to the mosquito. Despite its vital role in the parasite's success, it is the least understood stage in the parasite's life cycle. The presence of gametocytes in asymptomatic populations and induction of gametocytogenesis by most antimalarial drugs warrants further investigation into its biology. With a renewed focus on malaria elimination and advent of modern technology available to biologists today, the field of gametocyte biology has developed swiftly, providing crucial insights into the molecular mechanisms driving sexual commitment. This review will summarise key current findings in the field of gametocyte biology and address the associated challenges faced in malaria detection, control and elimination

A disrupted transsulphuration pathway results in accumulation of redox metabolites and induction of gametocytogenesis in malaria

Intra-erythrocytic growth of malaria parasite is known to induce redox stress. In addition to haem degradation which generates reactive oxygen species (ROS), the parasite is also thought to efflux redox active homocysteine. To understand the basis underlying accumulation of homocysteine, we have examined the transsulphuration (TS) pathway in the parasite, which is known to convert homocysteine to cysteine in higher eukaryotes. Our bioinformatic analysis revealed absence of key enzymes in the biosynthesis of cysteine namely cystathionine-beta-synthase and cystathionine-gamma-lyase in the parasite. Using mass spectrometry, we confirmed the absence of cystathionine, which is formed by enzymatic conversion of homocysteine thereby confirming truncation of TS pathway. We also quantitated levels of glutathione and homocysteine in infected erythrocytes and its spent medium. Our results showed increase in levels of these metabolites intracellularly and in culture supernatants. Our results provide a mechanistic basis for the long-known occurrence of hyperhomocysteinemia in malaria. Most importantly we find that homocysteine induces the transcription factor implicated in gametocytogenesis namely AP2-G and consequently triggers sexual stage conversion. We confirmed this observation both in vitro using Plasmodium falciparum cultures, and in vivo in the mouse model of malaria. Our study implicates homocysteine as a potential physiological trigger of gametocytogenesis

DataSheet_4_Identification and characterization of extracellular vesicles from red cells infected with Babesia divergens and Babesia microti.docx

Babesiosis is a zoonosis and an important blood-borne human parasitic infection that has gained attention because of its growing infection rate in humans by transfer from animal reservoirs. Babesia represents a potential threat to the blood supply because asymptomatic infections in man are common, and blood from such donors can cause severe disease in certain recipients. Extracellular vesicles (EVs) are vesicles released by cells that contain a complex mixture of proteins, lipids, glycans, and genetic information that have been shown to play important roles in disease pathogenesis and susceptibility, as well as cell–cell communication and immune responses. In this article, we report on the identification and characterization of EVs released from red blood cells (RBCs) infected by two major human Babesia species—Babesia divergens from in vitro culture and those from an in vivo B. microti mouse infection. Using nanoparticle tracking analysis, we show that there is a range of vesicle sizes from 30 to 1,000 nm, emanating from the Babesia-infected RBC. The study of these EVs in the context of hemoparasite infection is complicated by the fact that both the parasite and the host RBC make and release vesicles into the extracellular environment. However, the EV frequency is 2- to 10-fold higher in Babesia-infected RBCs than uninfected RBCs, depending on levels of parasitemia. Using parasite-specific markers, we were able to show that ~50%–60% of all EVs contained parasite-specific markers on their surface and thus may represent the specific proportion of EVs released by infected RBCs within the EV population. Western blot analysis on purified EVs from both in vivo and in vitro infections revealed several parasite proteins that were targets of the host immune response. In addition, microRNA analysis showed that infected RBC EVs have different microRNA signature from uninfected RBC EVs, indicating a potential role as disease biomarkers. Finally, EVs were internalized by other RBCs in culture, implicating a potential role for these vesicles in cellular communication. Overall, our study points to the multiple functional implications of EVs in Babesia–host interactions and support the potential that EVs have as agents in disease pathogenesis.</p

DataSheet_2_Identification and characterization of extracellular vesicles from red cells infected with Babesia divergens and Babesia microti.xlsx

Babesiosis is a zoonosis and an important blood-borne human parasitic infection that has gained attention because of its growing infection rate in humans by transfer from animal reservoirs. Babesia represents a potential threat to the blood supply because asymptomatic infections in man are common, and blood from such donors can cause severe disease in certain recipients. Extracellular vesicles (EVs) are vesicles released by cells that contain a complex mixture of proteins, lipids, glycans, and genetic information that have been shown to play important roles in disease pathogenesis and susceptibility, as well as cell–cell communication and immune responses. In this article, we report on the identification and characterization of EVs released from red blood cells (RBCs) infected by two major human Babesia species—Babesia divergens from in vitro culture and those from an in vivo B. microti mouse infection. Using nanoparticle tracking analysis, we show that there is a range of vesicle sizes from 30 to 1,000 nm, emanating from the Babesia-infected RBC. The study of these EVs in the context of hemoparasite infection is complicated by the fact that both the parasite and the host RBC make and release vesicles into the extracellular environment. However, the EV frequency is 2- to 10-fold higher in Babesia-infected RBCs than uninfected RBCs, depending on levels of parasitemia. Using parasite-specific markers, we were able to show that ~50%–60% of all EVs contained parasite-specific markers on their surface and thus may represent the specific proportion of EVs released by infected RBCs within the EV population. Western blot analysis on purified EVs from both in vivo and in vitro infections revealed several parasite proteins that were targets of the host immune response. In addition, microRNA analysis showed that infected RBC EVs have different microRNA signature from uninfected RBC EVs, indicating a potential role as disease biomarkers. Finally, EVs were internalized by other RBCs in culture, implicating a potential role for these vesicles in cellular communication. Overall, our study points to the multiple functional implications of EVs in Babesia–host interactions and support the potential that EVs have as agents in disease pathogenesis.</p

DataSheet_1_Identification and characterization of extracellular vesicles from red cells infected with Babesia divergens and Babesia microti.xlsx

Babesiosis is a zoonosis and an important blood-borne human parasitic infection that has gained attention because of its growing infection rate in humans by transfer from animal reservoirs. Babesia represents a potential threat to the blood supply because asymptomatic infections in man are common, and blood from such donors can cause severe disease in certain recipients. Extracellular vesicles (EVs) are vesicles released by cells that contain a complex mixture of proteins, lipids, glycans, and genetic information that have been shown to play important roles in disease pathogenesis and susceptibility, as well as cell–cell communication and immune responses. In this article, we report on the identification and characterization of EVs released from red blood cells (RBCs) infected by two major human Babesia species—Babesia divergens from in vitro culture and those from an in vivo B. microti mouse infection. Using nanoparticle tracking analysis, we show that there is a range of vesicle sizes from 30 to 1,000 nm, emanating from the Babesia-infected RBC. The study of these EVs in the context of hemoparasite infection is complicated by the fact that both the parasite and the host RBC make and release vesicles into the extracellular environment. However, the EV frequency is 2- to 10-fold higher in Babesia-infected RBCs than uninfected RBCs, depending on levels of parasitemia. Using parasite-specific markers, we were able to show that ~50%–60% of all EVs contained parasite-specific markers on their surface and thus may represent the specific proportion of EVs released by infected RBCs within the EV population. Western blot analysis on purified EVs from both in vivo and in vitro infections revealed several parasite proteins that were targets of the host immune response. In addition, microRNA analysis showed that infected RBC EVs have different microRNA signature from uninfected RBC EVs, indicating a potential role as disease biomarkers. Finally, EVs were internalized by other RBCs in culture, implicating a potential role for these vesicles in cellular communication. Overall, our study points to the multiple functional implications of EVs in Babesia–host interactions and support the potential that EVs have as agents in disease pathogenesis.</p

DataSheet_3_Identification and characterization of extracellular vesicles from red cells infected with Babesia divergens and Babesia microti.docx

Babesiosis is a zoonosis and an important blood-borne human parasitic infection that has gained attention because of its growing infection rate in humans by transfer from animal reservoirs. Babesia represents a potential threat to the blood supply because asymptomatic infections in man are common, and blood from such donors can cause severe disease in certain recipients. Extracellular vesicles (EVs) are vesicles released by cells that contain a complex mixture of proteins, lipids, glycans, and genetic information that have been shown to play important roles in disease pathogenesis and susceptibility, as well as cell–cell communication and immune responses. In this article, we report on the identification and characterization of EVs released from red blood cells (RBCs) infected by two major human Babesia species—Babesia divergens from in vitro culture and those from an in vivo B. microti mouse infection. Using nanoparticle tracking analysis, we show that there is a range of vesicle sizes from 30 to 1,000 nm, emanating from the Babesia-infected RBC. The study of these EVs in the context of hemoparasite infection is complicated by the fact that both the parasite and the host RBC make and release vesicles into the extracellular environment. However, the EV frequency is 2- to 10-fold higher in Babesia-infected RBCs than uninfected RBCs, depending on levels of parasitemia. Using parasite-specific markers, we were able to show that ~50%–60% of all EVs contained parasite-specific markers on their surface and thus may represent the specific proportion of EVs released by infected RBCs within the EV population. Western blot analysis on purified EVs from both in vivo and in vitro infections revealed several parasite proteins that were targets of the host immune response. In addition, microRNA analysis showed that infected RBC EVs have different microRNA signature from uninfected RBC EVs, indicating a potential role as disease biomarkers. Finally, EVs were internalized by other RBCs in culture, implicating a potential role for these vesicles in cellular communication. Overall, our study points to the multiple functional implications of EVs in Babesia–host interactions and support the potential that EVs have as agents in disease pathogenesis.</p

A secreted Heat shock protein 90 of <i>Trichomonas vaginalis</i>

<div><p><i>Trichomonas vaginalis</i> is a causative agent of Trichomoniasis, a leading non-viral sexually transmitted disease worldwide. In the current study, we show Heat shock protein 90 is essential for its growth. Upon genomic analysis of the parasite, it was found to possess seven ORFs which could potentially encode Hsp90 isoforms. We identified a cytosolic Hsp90 homolog, four homologs which can align to truncated cytosolic Hsp90 gene products along with two Grp94 homologs (ER isoform of Hsp90). However, both Grp94 orthologs lacked an ER retention motif. In cancer cells, it is very well established that Hsp90 is secreted and regulates key clients involved in metastases, migration, and invasion. Since <i>Trichomonas</i> Grp94 lacks ER retention motif, we examined the possibility of its secretion. By using cell biology and biochemical approaches we show that the Grp94 isoform of Hsp90 is secreted by the parasite by the classical ER-Golgi pathway. This is the first report of a genome encoded secreted Hsp90 in a clinically important parasitic protozoan.</p></div

TV910 is secreted via a BFA-sensitive secretory pathway.

<p>(A) Immunoblot shows inhibition of TV910 secretion into medium upon BFA treatment. Ponceau profile is shown as loading control. (B) The graph shows quantitation of signal. (C) Autoradiograph of IP for secreted TV910 at 0 and 3 H of the chase after 2 H pulse of S³⁵ labeling with and without BFA treatment. Upon BFA treatment no signal for secreted TV910 was observed at 3 H. (D) Autoradiograph of total labeled proteins of lysate shows a time-dependent decrease from 0 to 3 H. Upon BFA treatment higher signal was observed in cells due to inhibition of signal. (E) Autoradiograph of total labeled secreted proteins show a time-dependent increase in signal and upon BFA treatment a clear decrease was seen in signal due to inhibition of secretion.</p

TV910 is secreted by <i>Trichomonas</i>.

<p><b>(</b>A) Immunoblot shows TV910 in the cellular lysate and spent medium consistent with the secretion of TV910. No signal for alpha-tubulin was observed in spent media confirming cellular integrity. (B) Immunoblot for lysate and spent medium of <i>Giardia</i> trophozoites shows a prominent signal in the lysate and no signal in spent medium. (C) Coomassie profile of total secretome of <i>Trichomonas vaginalis</i>. (D) MS/MS spectra of the peptide ‘VTEDPRGNTLGR’ of TV910 detected in the tryptic digest corresponding to 75–100 kDa region. (E) TV910 sequence showing the peptides identified by MS/MS analysis. Peptides in green were identified with a confidence of 95% or more. (F) TV910 is secreted in a time-dependent manner. (a) Autoradiograph of IP of TV910 in the spent medium for different time points following the 2 H pulse of labeling. Protein A lane does not show any signal. A time-dependent increase is seen in the TV910 signal. (b) Autoradiograph of total labeled proteins of lysate shows a time-dependent decrease. (c) Autoradiograph of total labeled secreted proteins shows a time-dependent increase in signal. (d) Autoradiograph of IP for GlHsp90 after pulse-chase shows signal only in the lysate and no signal in spent media.</p