Assessment of aflatoxin exposure, growth faltering and the gut microbiome among children in rural Guatemala: protocol for an observational prospective cohort and bioreactor simulations.

INTRODUCTION: Aflatoxin B1 (AFB1) is a carcinogen produced by Aspergillus flavus and Aspergillus parasiticus which grow on maize. Given the high prevalence of child stunting (ie, impaired growth) and other nutritional disorders in low-income and middle-income countries, where maize is consumed, the role of aflatoxin exposure may be significant. Observational reports have demonstrated associations between aflatoxin exposure and impaired child growth; however, most have been cross-sectional and have not assessed seasonal variations in aflatoxin, food preparation and dynamic changes in growth. Biological mechanistic data on how aflatoxin may exert an impact on child growth is missing. This study incorporates a prospective cohort of children from rural Guatemala to assess (1) temporal associations between aflatoxin exposure and child growth and (2) possible mediation of the gut microbiome among aflatoxin exposure, inflammation and child growth. METHODS AND ANALYSIS: We will prospectively evaluate aflatoxin exposure and height-for-age difference trajectories for 18 months in a cohort of 185 children aged 6-9 months at enrolment. We will assess aflatoxin exposure levels and biomarkers of gut and systemic inflammation. We will examine the faecal microbiome of each child and identify key species and metabolic pathways for differing AFB1 exposure levels and child growth trajectories. In parallel, we will use bioreactors, inoculated with faeces, to investigate the response of the gut microbiome to varying levels of AFB1 exposure. We will monitor key microbial metabolites and AFB1 biotransformation products to study nutrient metabolism and the impact of the gut microbiome on aflatoxin detoxification/metabolism. Finally, we will use path analysis to summarise the effect of aflatoxin exposure and the gut microbiome on child growth. ETHICS AND DISSEMINATION: Ethics approval was obtained from Arizona State University Institutional Review Board (IRB; STUDY00016799) and Wuqu' Kawoq/Maya Health Alliance IRB (WK-2022-003). Findings will be disseminated in scientific presentations and peer-reviewed publications.

Prevalence of Hypertension, Diabetes, and Other Cardiovascular Disease Risk Factors in Two Indigenous Municipalities in Rural Guatemala: A Population-Representative Survey.

Background: Nearly 50% of Guatemalans are Indigenous Maya, yet few studies have examined the prevalence of modifiable cardiovascular disease (CVD) risk factors in Indigenous Maya populations. Therefore, we sought to estimate the prevalence of modifiable CVD risk factors in two Indigenous Maya areas in Guatemala. Methods: We conducted, between June 2018 and October 2019, a population-representative survey of adults aged 18 years and older in two rural Indigenous Maya municipalities in Guatemala. Our primary outcomes were five modifiable CVD risk factors: diabetes, hypertension, obesity, smoking, and alcohol use. We estimated the crude and age-standardized prevalence of each outcome. We also constructed multivariable logistic regression models to assess prevalence over covariates including age, sex, education level, ethnicity, and poverty. Sampling weights adjusted for nonresponse, and appropriate survey commands were used in all analyses. Results: The crude prevalence of diabetes was 12.5% (95% confidence Interval [CI] 9.6% to 16.1%), hypertension 20.3% (95% CI 17.1% to 23.9%), obesity 23.7% (95% CI 19.4% to 28.6%), smoking 10.7% (95% CI 7.8% to 14.5%), and high alcohol use 0.9% (95% CI 0.5% to 1.6%). Age-standardized prevalence of each outcome was similar to the crude prevalence. The prevalence of multiple CVD risk factors increased between the age groups 18-29 years and 50-59 years before decreasing among older age groups. Men had twenty-fold higher smoking prevalence than women (20.5% vs. 1.2%, respectively) and women had nearly double the age-adjusted prevalence of obesity as men (30.1% vs. 17.0%, respectively). Conclusion: There is a substantial prevalence of modifiable CVD risk factors in rural, Indigenous populations in Guatemala, in particular hypertension, diabetes, obesity (among women), and smoking (among men). These findings can help catalyze policy and clinical investments to improve the prevention, management, and control of CVD risk factors in these historically marginalized communities.

Loss to Follow-Up and the Care Cascade for Cervical Cancer Care in Rural Guatemala: A Cross-Sectional Study.

PURPOSE: More than 80% of cervical cancer cases and deaths occur in low- and middle-income countries. Here, we analyze a large geographically extensive cross-sectional data set from the Western rural highlands of Guatemala. Our objective is to better characterize weak points in care along the cervical cancer care continuum and investigate sociodemographic and clinical correlates of loss to follow-up. METHODS: We conducted a retrospective review of electronic health records data from July 21, 2015, through December 10, 2020 for a cytology-based screening and cervical cancer treatment program. We used a care cascade analysis to characterize the progression of individuals through screening, confirmatory testing, and treatment. We examined demographic and clinical factors correlated with screening and loss to follow-up using multivariate logistic regression. RESULTS: A total of 8,872 individuals were included in the analysis. Five thousand nine hundred thirteen cervical cancer screenings were conducted. 4.1% of all screening tests were abnormal, including 0.61% cervical intraepithelial neoplasia or overt cervical cancer. Care cascade analysis showed that 67% of eligible women accepted screening. Of those requiring confirmatory testing or treatment, 73% completed recommended follow-up. In adjusted multivariable analysis, prior history of sexual transmitted infection, prior experience with cervical cancer screening, older age, and current contraceptive use were associated with accepting screening. Age and contraceptive use were also associated with retention in care after a positive first screen. CONCLUSION: In a large rural Guatemalan retrospective cohort, a care continuum analysis showed that both declining the opportunity to receive cervical cancer screening as well as declining confirmatory testing after a first positive screen were both important weak points along the care continuum. These data support the need for comprehensive and culturally appropriate initiatives to improve screening uptake and retention in care.

Mobile Technologies and Cervical Cancer Screening in Low- and Middle-Income Countries: A Systematic Review.

PURPOSE: Cervical cancer screening is not well implemented in many low- and middle-income countries (LMICs). Mobile health (mHealth) refers to utilization of mobile technologies in health promotion and disease management. We aimed to qualitatively synthesize published articles reporting the impact of mHealth on cervical cancer screening-related health behaviors. METHODS: Three reviewers independently reviewed articles with the following criteria: the exposure or intervention of interest was mHealth, including messages or educational information sent via mobile telephone or e-mail; the comparison was people not using mHealth technology to receive screening-related information, and studies comparing multiple different mHealth interventional strategies were also eligible; the primary outcome was cervical cancer screening uptake, and secondary outcomes included awareness, intention, and knowledge of screening; appropriate research designs included randomized controlled trials and quasi-experimental or observational research; and the study was conducted in an LMIC. RESULTS: Of the 8 selected studies, 5 treated mobile telephone or message reminders as the exposure or intervention, and 3 compared the effects of different messages on screening uptake. The outcomes were diverse, including screening uptake (n = 4); health beliefs regarding the Papanicolaou (Pap) test (n = 1); knowledge of, attitude toward, and adherence to colpocytologic examination (n = 1); interest in receiving messages about Pap test results or appointment (n = 1); and return for Pap test reports (n = 1). CONCLUSION: Overall, our systematic review suggests that mobile technologies, particularly telephone reminders or messages, lead to increased Pap test uptake; additional work is needed to unequivocally verify whether mhealth interventions can improve knowledge regarding cervical cancer. Our study will inform mHealth-based interventions for cervical cancer screening promotion in LMICs.

Impact of school and work status on diet and physical activity in rural Guatemalan adolescent girls: a qualitative study.

In low- and middle-income countries, gender norms and access to energy-dense, nutrient-poor foods are well-studied determinants of food and physical activity choices for adolescent girls. However, most work has been done in urban and school settings. In many settings, a large proportion of the adolescent population is rural or not enrolled in school. We conducted in-depth interviews with 20 adolescent girls (ages 15-19) in Tecpán, Guatemala, a rural, largely indigenous Maya community. Interviews were coded and themes analyzed for insights into eating habits, food choices, and physical activity. Twelve participants were enrolled in school and eight were employed and not in school. Girls enrolled in school had more disrupted eating schedules and consumed more energy-dense, nutrient-poor foods. Girls not enrolled in school had fewer opportunities for physical activity and were more sedentary. To our knowledge, this study is the first in a low- and middle-income country to consider diet and physical activity of adolescent girls enrolled and not enrolled in school. Key implications include (1) the need to reduce exposure to nutrient-poor foods and promote healthy eating schedules in schools, and (2) the need to create community-based opportunities for the activity for girls no longer enrolled in school.

Barriers to Cervical Cancer Screening and the Cervical Cancer Care Continuum in Rural Guatemala: A Mixed-Method Analysis.

Purpose Cervical cancer is an important cause of mortality in low- and middle-income countries. Although screening technologies continue to improve, systems of care remain fragmented. It is important to better understand factors that affect use of screening services and loss to follow-up along the care continuum. Methods We conducted a mixed-methods study of a cytology-based screening program in rural Guatemala. A retrospective electronic chart review was performed on data from all patients from 2013 to 2014. We analyzed progression through care and calculated loss-to-follow-up rates. We also analyzed the prior experiences of patients with cervical cancer screening on the basis of self-reported historical data available in the chart review. Structured interviews with a subset of individuals to explore social supports and barriers to screening and engagement in care were conducted at the time of screening. Results The analysis included 515 women (median age, 36 years). Cytologic screening showed concern for neoplastic changes in 0.83%; half resulted in biopsy-proven cervical intraepithelial neoplasia. An additional 9.9% showed severe inflammation. The rate of loss to follow-up was 11.3%. All losses to follow-up occurred for severe inflammation, not for cervical intraepithelial neoplasia. Historical data showed that 73% of the cohort had previously been screened and had high levels of loss to follow-up (57.4%). Qualitative interviews revealed factors that promoted loss to follow-up; these included cost, lack of social supports, transportation, distrust in public facilities, long turn-around times, and failure to return test results or offer follow-up treatments. Conclusions Taken together, these quantitative and qualitative results highlight the need for cervical cancer screening programs in Guatemala to improve uptake of screening services by eligible women and to improve follow-up after a first abnormal screen.

Screening for chronic kidney disease in a community-based diabetes cohort in rural Guatemala: a cross-sectional study.

OBJECTIVE: Screening is a key strategy to address the rising burden of chronic kidney disease (CKD) in low-income and middle-income countries. However, there are few reports regarding the implementation of screening programmes in resource-limited settings. The objectives of this study are to (1) to share programmatic experiences implementing CKD screening in a rural, resource-limited setting and (2) to assess the burden of renal disease in a community-based diabetes programme in rural Guatemala. DESIGN: Cross-sectional assessment of glomerular filtration rate (GFR) and urine albumin. SETTING: Central Highlands of Guatemala. PARTICIPANTS: We enrolled 144 adults with type 2 diabetes in a community-based CKD screening activity carried out by the sponsoring institution. OUTCOME MEASURES: Prevalence of renal disease and risk of CKD progression using Kidney Disease: Improving Global Outcomes definitions and classifications. RESULTS: We found that 57% of the sample met GFR and/or albuminuria criteria suggestive of CKD. Over half of the sample had moderate or greater increased risk for CKD progression, including nearly 20% who were classified as high or very high risk. Hypertension was common in the sample (42%), and glycaemic control was suboptimal (mean haemoglobin A1c 9.4%±2.5% at programme enrolment and 8.6%±2.3% at time of CKD screening). CONCLUSIONS: The high burden of renal disease in our patient sample suggests an imperative to better understand the burden and risk factors of CKD in Guatemala. The implementation details we share reveal the tension between evidence-based CKD screening versus screening that can feasibly be delivered in resource-limited global settings.

Obstetric care navigation: a new approach to promote respectful maternity care and overcome barriers to safe motherhood.

BACKGROUND: Disrespectful and abusive maternity care is a common and pervasive problem that disproportionately impacts marginalized women. By making mothers less likely to agree to facility-based delivery, it contributes to the unacceptably high rates of maternal mortality in low- and middle-income countries. Few programmatic approaches have been proposed to address disrespectful and abusive maternity care. OBSTETRIC CARE NAVIGATION: Care navigation was pioneered by the field of oncology to improve health outcomes of vulnerable populations and promote patient autonomy by providing linkages across a fragmented care continuum. Here we describe the novel application of the care navigation model to emergency obstetric referrals to hospitals for complicated home births in rural Guatemala. Care navigators offer women accompaniment and labor support intended to improve the care experience-for both patients and providers-and to decrease opposition to hospital-level obstetric care. Specific roles include deflecting mistreatment from hospital staff, improving provider communication through language and cultural interpretation, advocating for patients' right to informed consent, and protecting patients' dignity during the birthing process. Care navigators are specifically chosen and trained to gain the trust and respect of patients, traditional midwives, and biomedical providers. We describe an ongoing obstetric care navigator pilot program employing rapid-cycle quality improvement methods to quickly identify implementation successes and failures. This approach empowers frontline health workers to problem solve in real time and ensures the program is highly adaptable to local needs. CONCLUSION: Care navigation is a promising strategy to overcome the "humanistic barrier" to hospital delivery by mitigating disrespectful and abusive care. It offers a demand-side approach to undignified obstetric care that empowers the communities most impacted by the problem to lead the response. Results from an ongoing pilot program of obstetric care navigation will provide valuable feedback from patients on the impact of this approach and implementation lessons to facilitate replication in other settings.

Delays in diagnosis and treatment of extrapulmonary tuberculosis in Guatemala.

A 23-year-old indigenous Guatemalan man presented in 2016 to our clinic in Sololá, Guatemala, with 10 months of recurrent neck swelling, fevers, night sweats and weight loss. Previously, he had sought care in three different medical settings, including a private physician-run clinic, a tertiary private cancer treatment centre and, finally, a rural government health post. With assistance from our institution's accompaniment staff, the patient was admitted to a public tertiary care hospital for work-up. Rifampin-susceptible tuberculosis was diagnosed, and appropriate treatment was begun. The case illustrates how low tuberculosis recognition among community health workers and health system segmentation creates obstacles to appropriate care, especially for patients with limited means. As a result, significant diagnostic and treatment delays can occur, increasing the public health burden of tuberculosis.

Use of propranolol in a remote region of rural Guatemala to treat a large facial infantile haemangioma.

We present a female infant with a right-sided facial and neck haemangioma, from a remote, resource-poor community in rural Guatemala. She received first-line treatment, propranolol, with marked reduction in tumour size and erythema. Treatment was stopped after 35 weeks due to recurrent diarrhoea and sustained weight loss. Propranolol can be used to safely treat infants with haemangiomas in remote, rural communities if there is adequate follow-up, education and communication. Periocular haemangiomas should be treated promptly to avoid visual impairment. Infants with large facial haemangiomas should be screened for Posterior fossa anomalies, Hemangioma, Arterial anomalies, Cardiac anomalies, and Eye anomalies (PHACE) syndrome, and specialists should be involved. The case also highlights the difficulty of providing treatment for a complex illness when basic health needs, such as food security and water sanitation, are limited.

Myxoedema in a patient with achondroplasia in rural area of Guatemala.

A 43-year-old indigenous Guatemalan woman with achondroplasia presented to our clinic with chronic fatigue and generalised oedema. She had limited contact with the formal healthcare system. However, 1 year prior, she had sought medical evaluation from a private physician. Her symptoms had been attributed to a combination of heart failure and physical disability due to the musculoskeletal complications of her achondroplasia. She was lost to follow-up due to inability to pay for further testing or treatment. On initial laboratory evaluation in our clinic, she was found to have a thyrotropin level greater than assay. With initiation of oral levothyroxine supplementation, her dyspnoea and oedema completely resolved. The case illustrates how indigenous patients in rural Guatemala experience many barriers to accessing high-quality medical care. As a result, presentations of common illnesses are often very advanced and definitive diagnoses and treatments are frequently delayed.

Major challenges to scale up of visual inspection-based cervical cancer prevention programs: the experience of Guatemalan NGOs.

BACKGROUND: Like many other low- and middle-income countries, Guatemala has adopted visual inspection with acetic acid (VIA) as a low-resource alternative to the Pap smear for cervical cancer screening. Nongovernmental organizations (NGOs) introduced VIA to Guatemala in 2004, and a growing number of NGOs, working both independently and in collaboration with the Guatemalan Ministry of Health, employ VIA in cervical cancer prevention programs today. While much research describes VIA efficacy and feasibility in Latin America, little is known about NGO involvement with VIA programming or experiences with VIA outside the context of clinical trials and pilot projects in the region. METHODS: To explore challenges faced by NGOs implementing VIA programs in Guatemala, we conducted semi-structured interviews with 36 NGO staff members involved with 20 VIA programs as direct service providers, program administrators, and training course instructors. Additionally, we collected data through observation at 30 NGO-sponsored cervical cancer screening campaigns, 8 cervical cancer prevention conferences, and 1 week-long NGO-sponsored VIA training course. RESULTS: Frequently highlighted challenges included staff turnover, concerns over training quality, a need for opportunities for continued supervision, and problems with cryotherapy referrals when immediate treatment for VIA-positive women was unavailable. CONCLUSIONS: Reducing staff turnover, budgeting to train replacement providers, standardizing training curricula, and offering continued supervision are key strategies to improve VIA service quality and program sustainability. Alternative training methods, such as on-the-job mentoring and course prerequisites of online learning, could help increase training time available for clinical supervision. Efforts should be made to ensure that VIA testing is coupled with immediate cryotherapy, that providers trained in VIA are also trained in cryotherapy, and that cryotherapy supplies and equipment are maintained. Where this is not possible and only VIA screening is available, referral systems must be strengthened.

Adaptor protein-3 (AP-3) complex mediates the biogenesis of acidocalcisomes and is essential for growth and virulence of Trypanosoma brucei.

Acidocalcisomes are acidic calcium and polyphosphate storage organelles found in a diverse range of organisms. Here we present evidence that the biogenesis of acidocalcisomes in Trypanosoma brucei is linked to the expression of adaptor protein-3 (AP-3) complex. Localization studies in cell lines expressing ß3 and δ subunits of AP-3 fused to epitope tags revealed their partial co-localization with the vacuolar proton pyrophosphatase, a marker of acidocalcisomes, with the Golgi marker Golgi reassembly and stacking protein, and with antibodies against the small GTPase Rab11. Ablation of the ß3 subunit by RNA interference (RNAi) resulted in disappearance of acidocalcisomes from both procyclic and bloodstream form trypanosomes, as revealed by immmunofluorescence and electron microscopy assays, with no alterations in trafficking of different markers to lysosomes. Knockdown of the ß3 subunit resulted in lower acidic calcium, pyrophosphate, and polyphosphate content as well as defects in growth in culture, resistance to osmotic stress, and virulence in mice. Similar results were obtained by knocking down the expression of the δ subunit of AP-3. These results indicate that AP-3 is essential for the biogenesis of acidocalcisomes and for growth and virulence of T. brucei.

Calcium uptake and proton transport by acidocalcisomes of Toxoplasma gondii.

Acidocalcisomes are acidic calcium stores found in diverse organisms, being conserved from bacteria to humans. They possess an acidic matrix that contains several cations bound to phosphates, which are mainly present in the form of short and long polyphosphate chains. Their matrix is acidified through the action of proton pumps such as a vacuolar proton ATPase and a vacuolar proton pyrophosphatase. Calcium uptake occurs through a Ca(2+)/H(+) countertransporting ATPase located in the membrane of the organelle. Acidocalcisomes have been identified in a variety of microorganisms, including Apicomplexan parasites such as Plasmodium and Eimeria species, and in Toxoplasma gondii. We report the purification and characterization of an acidocalcisome fraction from T. gondii tachyzoites after subcellular fractionation and further discontinuous iodixanol gradient purification. Proton and calcium transport activities in the fraction were characterized by fluorescence microscopy and spectrophotometric methods using acridine orange and arsenazo III, respectively. This work will facilitate the understanding of the function of acidocalcisomes in Apicomplexan parasites, as we can now isolate highly purified fractions that could be used for proteomic analysis to find proteins that may clarify the biogenesis of these organelles.

A contractile vacuole complex is involved in osmoregulation in Trypanosoma cruzi.

Acidocalcisomes are dense, acidic organelles with a high concentration of phosphorus present as pyrophosphate and polyphosphate complexed with calcium and other cations. Acidocalcisomes have been linked to the contractile vacuole complex in Chlamydomonas reinhardtii, Dictyostelium discoideum, and Trypanosoma cruzi. A microtubule- and cyclic AMP-mediated fusion of acidocalcisomes to the contractile vacuole complex in T. cruzi results in translocation of aquaporin and the resulting water movement which, in addition to swelling of acidocalcisomes, is responsible for the volume reversal not accounted for by efflux of osmolytes. Polyphosphate hydrolysis occurs during hyposmotic stress, probably increasing the osmotic pressure of the contractile vacuole and facilitating water movement.

Overexpression of a Zn2+-sensitive soluble exopolyphosphatase from Trypanosoma cruzi depletes polyphosphate and affects osmoregulation.

We report the cloning, expression, purification, and characterization of the Trypanosoma cruzi exopolyphosphatase (TcPPX). The product of this gene (TcPPX), has 383 amino acids and a molecular mass of 43.1 kDa. TcPPX differs from most exopolyphosphatases in its preference for short-chain polyphosphate (poly P). Heterologous expression of TcPPX in Escherichia coli produced a functional enzyme that had a neutral optimum pH and was dramatically inhibited by low concentrations of Zn2+, high concentrations of basic amino acids (lysine and arginine), and heparin. TcPPX is a processive enzyme and does not hydrolyze ATP, pyrophosphate, or p-nitrophenyl phosphate, although it hydrolyzes guanosine 5'-tetraphosphate very efficiently. Overexpression of TcPPX resulted in a dramatic decrease in total short-chain poly P and partial decrease in long-chain poly P. This was accompanied by a delayed regulatory volume decrease after hyposmotic stress. These results support the role of poly P in T. cruzi osmoregulation.

Ablation of a small transmembrane protein of Trypanosoma brucei (TbVTC1) involved in the synthesis of polyphosphate alters acidocalcisome biogenesis and function, and leads to a cytokinesis defect.

Inorganic poly P (polyphosphate) is an abundant component of acidocalcisomes of Trypanosoma brucei. In the present study we report the presence of a protein homologous with the yeast Vtc1p (vacuolar transporter chaperone 1) in T. brucei that is essential for poly P synthesis, acidocalcisome biogenesis and cytokinesis. Localization studies in a cell line expressing a TbVTC1 fused to GFP (green fluorescent protein) revealed its co-localization with the V-H+-PPase (vacuolar H+-pyrophosphatase), a marker for acidocalcisomes. Western blot analysis of acidocalcisome fractions and immunogold electron microscopy using polyclonal antibodies against a fragment of TbVTC1 confirmed the acidocalcisome localization. Ablation of TbVTC1 expression by RNA interference caused an abnormal morphology of acidocalcisomes, indicating that their biogenesis was disturbed, with a decreased pyrophosphate-driven H+ uptake and Ca2+ content, a significant decrease in the amount of poly P and a deficient response to hyposmotic stress. Ablation of TbVTC1 expression for longer periods produced marked gross morphological alterations compatible with a defect in cytokinesis, followed by cell death. Overexpression of the TbVTC1 gene caused mild alterations in growth rate, but had no perceptible effect on acidocalcisome morphology. We propose that the PP(i)-driven H+ pumping deficiency induced by ablation of TbVTC1 leads to alterations in the protonmotive force of acidocalcisomes, which results in deficient fusion or budding of the organelles, decreased H+ and Ca2+ content, and decreased synthesis of poly P. A decrease in the poly P content would lead to osmotic sensitivity and defects in cytokinesis.

A solanesyl-diphosphate synthase localizes in glycosomes of Trypanosoma cruzi.

We report the cloning of a Trypanosoma cruzi gene encoding a solanesyl-diphosphate synthase, TcSPPS. The amino acid sequence (molecular mass approximately 39 kDa) is homologous to polyprenyl-diphosphate synthases from different organisms, showing the seven conserved motifs and the typical hydrophobic profile. TcSPPS preferred geranylgeranyl diphosphate as the allylic substrate. The final product, as determined by TLC, had nine isoprene units. This suggests that the parasite synthesizes mainly ubiquinone-9 (UQ-9), as described for Trypanosoma brucei and Leishmania major. In fact, that was the length of the ubiquinone extracted from epimastigotes, as determined by high-performance liquid chromatography. Expression of TcSPPS was able to complement an Escherichia coli ispB mutant. A punctuated pattern in the cytoplasm of the parasite was detected by immunofluorescence analysis with a specific polyclonal antibody against TcSPPS. An overlapping fluorescence pattern was observed using an antibody directed against the glycosomal marker pyruvate phosphate dikinase, suggesting that this step of the isoprenoid biosynthetic pathway is located in the glycosomes. Co-localization in glycosomes was confirmed by immunogold electron microscopy and subcellular fractionation. Because UQ has a central role in energy production and in reoxidation of reduction equivalents, TcSPPS is promising as a new chemotherapeutic target.

Ammonium production during hypo-osmotic stress leads to alkalinization of acidocalcisomes and cytosolic acidification in Trypanosoma cruzi.

Osmotic swelling of Trypanosoma cruzi epimastigotes resulted in alkalinization of acidocalcisomes, as revealed by changes in acridine orange fluorescence of intact cells. Concomitant with these changes, intracellular ammonium levels increased while extracellular ammonium levels decreased significantly. Hypo-osmotic stress also resulted in cytosolic acidification. The observed changes in intracellular pH (pH(i)) were independent of extracellular calcium, and other ions concentration. Taken together, these results are consistent with a stimulation of ammonium production upon hypo-osmotic stress and its accumulation in acidocalcisomes resulting in their alkalinization, which might be responsible for polyphosphate hydrolysis and osmotic changes in the organelles.