A Leishmania amazonensis ZIP family iron transporter is essential for parasite replication within macrophage phagolysosomes

Infection of mammalian hosts with Leishmania amazonensis depends on the remarkable ability of these parasites to replicate within macrophage phagolysosomes. A critical adaptation for survival in this harsh environment is an efficient mechanism for gaining access to iron. In this study, we identify and characterize LIT1, a novel L. amazonensis membrane protein with extensive similarity to IRT1, a ZIP family ferrous iron transporter from Arabidopsis thaliana. The ability of LIT1 to promote iron transport was demonstrated after expression in yeast and in L. amazonensis LIT1-null amastigotes. Endogenous LIT1 was only detectable in amastigotes replicating intracellularly, and its intracellular expression was accelerated under conditions predicted to result in iron deprivation. Although L. amazonensis lacking LIT1 grew normally in axenic culture and had no defects differentiating into infective forms, replication within macrophages was abolished. Consistent with an essential role for LIT1 in intracellular growth as amastigotes, Δlit1 parasites were avirulent. After inoculation into highly susceptible mice, no lesions were detected, even after extensive periods of time. Despite the absence of pathology, viable Δlit1 parasites were recovered from the original sites of inoculation, indicating that L. amazonensis can persist in vivo independently of the ability to grow in macrophages. Our findings highlight the essential role played by intracellular iron acquisition in Leishmania virulence and identify this pathway as a promising target for therapeutic intervention

Leishmania sexual reproductive strategies as resolved through computational methods designed for aneuploid genomes

A cryptic sexual reproductive cycle i

Position Statement Executive Summary: Guidelines and Recommendations for Laboratory Analysis in the Diagnosis and Management of Diabetes Mellitus

Background: Multiple laboratory tests are used in the diagnosis and management of patients with diabetes mellitus. The quality of the scientific evidence supporting the use of these assays varies substantially. Approach: An expert committee compiled evidence-based recommendations for the use of laboratory analysis in patients with diabetes. A new system was developed to grade the overall quality of the evidence and the strength of the recommendations. A draft of the guidelines was posted on the Internet, and the document was modified in response to comments. The guidelines were reviewed by the joint Evidence-Based Laboratory Medicine Committee of the AACC and the National Academy of Clinical Biochemistry and were accepted after revisions by the Professional Practice Committee and subsequent approval by the Executive Committee of the American Diabetes Association. Content: In addition to the long-standing criteria based on measurement of venous plasma glucose, diabetes can be diagnosed by demonstrating increased hemoglobin A1c (HbA1c) concentrations in the blood. Monitoring of glycemic control is performed by the patients measuring their own plasma or blood glucose with meters and by laboratory analysis of HbA1c. The potential roles of noninvasive glucose monitoring, genetic testing, and measurement of autoantibodies, urine albumin, insulin, proinsulin, C-peptide, and other analytes are addressed. Summary: The guidelines provide specific recommendations based on published data or derived from expert consensus. Several analytes are found to have minimal clinical value at the present time, and measurement of them is not recommended

Gene expression in Leishmania is regulated predominantly by gene dosage

ABSTRACT Leishmania tropica, a unicellular eukaryotic parasite present in North and East Africa, the Middle East, and the Indian subcontinent, has been linked to large outbreaks of cutaneous leishmaniasis in displaced populations in Iraq, Jordan, and Syria. Here, we report the genome sequence of this pathogen and 7,863 identified protein-coding genes, and we show that the majority of clinical isolates possess high levels of allelic diversity, genetic admixture, heterozygosity, and extensive aneuploidy. By utilizing paired genome-wide high-throughput DNA sequencing (DNA-seq) with RNA-seq, we found that gene dosage, at the level of individual genes or chromosomal “somy” (a general term covering disomy, trisomy, tetrasomy, etc.), accounted for greater than 85% of total gene expression variation in genes with a 2-fold or greater change in expression. High gene copy number variation (CNV) among membrane-bound transporters, a class of proteins previously implicated in drug resistance, was found for the most highly differentially expressed genes. Our results suggest that gene dosage is an adaptive trait that confers phenotypic plasticity among natural Leishmania populations by rapid down- or upregulation of transporter proteins to limit the effects of environmental stresses, such as drug selection. IMPORTANCE Leishmania is a genus of unicellular eukaryotic parasites that is responsible for a spectrum of human diseases that range from cutaneous leishmaniasis (CL) and mucocutaneous leishmaniasis (MCL) to life-threatening visceral leishmaniasis (VL). Developmental and strain-specific gene expression is largely thought to be due to mRNA message stability or posttranscriptional regulatory networks for this species, whose genome is organized into polycistronic gene clusters in the absence of promoter-mediated regulation of transcription initiation of nuclear genes. Genetic hybridization has been demonstrated to yield dramatic structural genomic variation, but whether such changes in gene dosage impact gene expression has not been formally investigated. Here we show that the predominant mechanism determining transcript abundance differences (>85%) in Leishmania tropica is that of gene dosage at the level of individual genes or chromosomal somy

Deficiency in β1,3-Galactosyltransferase of a Leishmania major Lipophosphoglycan Mutant Adversely Influences the Leishmania-Sand Fly Interaction

To study the function of side chain oligosaccharides of the cell-surface lipophosphoglycan (LPG), mutagenized Leishmania major defective in side chain biosynthesis were negatively selected by agglutination with the monoclonal antibody WIC79.3, which recognizes the galactose-containing side chains of L. major LPG. One such mutant, called Spock, lacked the ability to bind significantly to midguts of the natural L. major vector, Phlebotomus papatasi, and to maintain infection in the sand fly after excretion of the digested bloodmeal. Biochemical characterization of Spock LPG revealed its structural similarity to the LPG of Leishmania donovani, a species whose inability to bind to and maintain infections in P. papatasi midguts has been strongly correlated with the expression of a surface LPG lacking galactose-terminated oligosaccharide side chains. An in vitro galactosyltransferase assay using wild-type or Spock membranes was used to determine that the defect in Spock LPG biosynthesis is a result of defective beta1,3-galactosyltransferase activity as opposed to a modification of LPG, which would prevent it from serving as a competent substrate for galactose addition. The results of these experiments show that Spock lacks the beta1, 3-galactosyltransferase for side chain addition and that the LPG side chains are required for L. major to bind to and to produce transmissible infection in P. papatasi

Nonalcoholic and Alcoholic Beverage Intakes by Adults across 5 Upper-Middle- and High-Income Countries.

BACKGROUND: Despite considerable public health interest in sugary drink consumption, there has been little comparison of intake across countries. OBJECTIVES: This study aimed to compare the consumption frequency and amounts of commonly consumed beverages among adults in 5 upper-middle- and high-income countries, and examine differences in consumption between population subgroups. METHODS: Adults aged 18-65 y completed online surveys in December 2017 in Australia (n = 3264), Canada (n = 2745), Mexico (n = 3152), the United Kingdom (n = 3221), and the USA (n = 4015) as part of the International Food Policy Study. The frequency of consuming beverages from 22 categories in the past 7 d was estimated using the Beverage Frequency Questionnaire. Regression models were used to examine differences in the likelihood of any consumption and in the amounts consumed of sugar-sweetened beverages (SSBs), sugary drinks (SSBs and 100% juice), diet, and alcoholic beverages between countries and across sociodemographic subgroups. RESULTS: The prevalence of reported SSB consumption in the past 7 d ranged from 47% (United Kingdom) to 81% (Mexico), and that of sugary drinks ranged from 62% (United Kingdom) to 87% (Mexico). Rates of consumption of diet drinks ranged from 26% (Mexico) to 37% (United Kingdom), whereas alcoholic drink consumption rates ranged from 45% (USA) to 52% (Canada). Respondents in Mexico were more likely to consume SSBs and sugary drinks, and in greater amounts, than those in other countries. Respondents in the United Kingdom were more likely to consume diet drinks than those in Australia, Canada, and Mexico, and greater amounts of diet drinks were consumed in the United Kingdom and the USA. Across countries, younger respondents and males were more likely to consume greater amounts of SSBs and sugary drinks. CONCLUSIONS: Most adult respondents across all countries consumed SSBs and sugary drinks, with greater consumption in Mexico and the USA. Consumption varied greatly across countries, but patterns of association among subpopulations were relatively similar.The first two waves of the International Food Policy Study were funded by a population health intervention research operating grant from the Canadian Institutes of Health Research (CIHR). Additional support was provided by a CIHR – Public Health Agency of Canada (PHAC) Applied Public Health Chair held by David Hammond. JA receives funding from the Centre for Diet and Activity Research (CEDAR), a UKCRC Public Health Research Centre of Excellence. Funding from the British Heart Foundation, Cancer Research UK, Economic and Social Research Council, Medical Research Council, the National Institute for Health Research, and the Wellcome Trust, under the auspices of the UK Clinical Research Collaboration, is gratefully acknowledged. GS is supported by a Heart Foundation Future Leader Fellowship (102035) from the National Heart Foundation of Australia. He is also a researcher within National Health and Medical Research Council (NHMRC) Centres for Research Excellence entitled Reducing Salt Intake Using Food Policy Interventions (APP1117300) and a Centre of Research Excellence in Food Retail Environments for Health (RE-FRESH) (APP1152968) (Australia). He has also received other funding from the NHMRC, Australian Research Council (ARC) and the World Health Organization (WHO). Data described in the manuscript, code book, and analytic code will be made available upon request pending application and approval by DH