Leishmania infantum (JPCM5) Transcriptome, Gene Models and Resources for an Active Curation of Gene Annotations.

Leishmania infantum is one of the causative agents of visceral leishmaniases, the most severe form of leishmaniasis. An improved assembly for the L. infantum genome was published five years ago, yet delineation of its transcriptome remained to be accomplished. In this work, the transcriptome annotation was attained by a combination of both short and long RNA-seq reads. The good agreement between the results derived from both methodologies confirmed that transcript assembly based on Illumina RNA-seq and further delimitation according to the positions of spliced leader (SAS) and poly-A (PAS) addition sites is an adequate strategy to annotate the transcriptomes of Leishmania, a procedure previously used for transcriptome annotation in other Leishmania species and related trypanosomatids. These analyses also confirmed that the Leishmania transcripts boundaries are relatively slippery, showing extensive heterogeneity at the 5'- and 3'-ends. However, the use of RNA-seq reads derived from the PacBio technology (referred to as Iso-Seq) allowed the authors to uncover some complex transcription patterns occurring at particular loci that would be unnoticed by the use of short RNA-seq reads alone. Thus, Iso-Seq analysis provided evidence that transcript processing at particular loci would be more dynamic than expected. Another noticeable finding was the observation of a case of allelic heterozygosity based on the existence of chimeric Iso-Seq reads that might be generated by an event of intrachromosomal recombination. In addition, we are providing the L. infantum gene models, including both UTRs and CDS regions, that would be helpful for undertaking whole-genome expression studies. Moreover, we have built the foundations of a communal database for the active curation of both gene/transcript models and functional annotations for genes and proteins.

Gene Annotation and Transcriptome Delineation on a De Novo Genome Assembly for the Reference Leishmania major Friedlin Strain.

Leishmania major is the main causative agent of cutaneous leishmaniasis in humans. The Friedlin strain of this species (LmjF) was chosen when a multi-laboratory consortium undertook the objective of deciphering the first genome sequence for a parasite of the genus Leishmania. The objective was successfully attained in 2005, and this represented a milestone for Leishmania molecular biology studies around the world. Although the LmjF genome sequence was done following a shotgun strategy and using classical Sanger sequencing, the results were excellent, and this genome assembly served as the reference for subsequent genome assemblies in other Leishmania species. Here, we present a new assembly for the genome of this strain (named LMJFC for clarity), generated by the combination of two high throughput sequencing platforms, Illumina short-read sequencing and PacBio Single Molecular Real-Time (SMRT) sequencing, which provides long-read sequences. Apart from resolving uncertain nucleotide positions, several genomic regions were reorganized and a more precise composition of tandemly repeated gene loci was attained. Additionally, the genome annotation was improved by adding 542 genes and more accurate coding-sequences defined for around two hundred genes, based on the transcriptome delimitation also carried out in this work. As a result, we are providing gene models (including untranslated regions and introns) for 11,238 genes. Genomic information ultimately determines the biology of every organism; therefore, our understanding of molecular mechanisms will depend on the availability of precise genome sequences and accurate gene annotations. In this regard, this work is providing an improved genome sequence and updated transcriptome annotations for the reference L. major Friedlin strain.

The Experimental Proteome of Leishmania infantum Promastigote and Its Usefulness for Improving Gene Annotations.

Leishmania infantum causes visceral leishmaniasis (kala-azar), the most severe form of leishmaniasis, which is lethal if untreated. A few years ago, the re-sequencing and de novo assembling of the L. infantum (JPCM5 strain) genome was accomplished, and now we aimed to describe and characterize the experimental proteome of this species. In this work, we performed a proteomic analysis from axenic cultured promastigotes and carried out a detailed comparison with other Leishmania experimental proteomes published to date. We identified 2352 proteins based on a search of mass spectrometry data against a database built from the six-frame translated genome sequence of L. infantum. We detected many proteins belonging to organelles such as glycosomes, mitochondria, or flagellum, as well as many metabolic enzymes and many putative RNA binding proteins and molecular chaperones. Moreover, we listed some proteins presenting post-translational modifications, such as phosphorylations, acetylations, and methylations. On the other hand, the identification of peptides mapping to genomic regions previously annotated as non-coding allowed for the correction of annotations, leading to the N-terminal extension of protein sequences and the uncovering of eight novel protein-coding genes. The alliance of proteomics, genomics, and transcriptomics has resulted in a powerful combination for improving the annotation of the L. infantum reference genome.

Leishmania Mitochondrial Genomes: Maxicircle Structure and Heterogeneity of Minicircles.

The mitochondrial DNA (mtDNA), which is present in almost all eukaryotic organisms, is a useful marker for phylogenetic studies due to its relative high conservation and its inheritance manner. In Leishmania and other trypanosomatids, the mtDNA (also referred to as kinetoplast DNA or kDNA) is composed of thousands of minicircles and a few maxicircles, catenated together into a complex network. Maxicircles are functionally similar to other eukaryotic mtDNAs, whereas minicircles are involved in RNA editing of some maxicircle-encoded transcripts. Next-generation sequencing (NGS) is increasingly used for assembling nuclear genomes and, currently, a large number of genomic sequences are available. However, most of the time, the mitochondrial genome is ignored in the genome assembly processes. The aim of this study was to develop a pipeline to assemble Leishmania minicircles and maxicircle DNA molecules, exploiting the raw data generated in the NGS projects. As a result, the maxicircle molecules and the plethora of minicircle classes for Leishmania major, Leishmania infantum and Leishmania braziliensis have been characterized. We have observed that whereas the heterogeneity of minicircle sequences existing in a single cell hampers their use for Leishmania typing and classification, maxicircles emerge as an extremely robust genetic marker for taxonomic studies within the clade of kinetoplastids.

Complete assembly of the Leishmania donovani (HU3 strain) genome and transcriptome annotation.

Leishmania donovani is a unicellular parasite that causes visceral leishmaniasis, a fatal disease in humans. In this study, a complete assembly of the genome of L. donovani is provided. Apart from being the first published genome of this strain (HU3), this constitutes the best assembly for an L. donovani genome attained to date. The use of a combination of sequencing platforms enabled to assemble, without any sequence gap, the 36 chromosomes for this species. Additionally, based on this assembly and using RNA-seq reads derived from poly-A + RNA, the transcriptome for this species, not yet available, was delineated. Alternative SL addition sites and heterogeneity in the poly-A addition sites were commonly observed for most of the genes. After a complete annotation of the transcriptome, 2,410 novel transcripts were defined. Additionally, the relative expression for all transcripts present in the promastigote stage was determined. Events of cis-splicing have been documented to occur during the maturation of the transcripts derived from genes LDHU3_07.0430 and LDHU3_29.3990. The complete genome assembly and the availability of the gene models (including annotation of untranslated regions) are important pieces to understand how differential gene expression occurs in this pathogen, and to decipher phenotypic peculiarities like tissue tropism, clinical disease, and drug susceptibility.

Complete and de novo assembly of the Leishmania braziliensis (M2904) genome

Leishmania braziliensis is the etiological agent of American mucosal leishmaniasis, one of the most severe clinical forms of leishmaniasis. Here, we report the assembly of the L. braziliensis (M2904) genome into 35 continuous chromosomes. Also, the annotation of 8395 genes is provided. The public availability of this information will contribute to a better knowledge of this pathogen and help in the search for vaccines and novel drug targets aimed to control the disease caused by this Leishmania species.

Complete and de novo assembly of the Leishmania braziliensis (M2904) genome.

Leishmania braziliensis is the etiological agent of American mucosal leishmaniasis, one of the most severe clinical forms of leishmaniasis. Here, we report the assembly of the L. braziliensis (M2904) genome into 35 continuous chromosomes. Also, the annotation of 8395 genes is provided. The public availability of this information will contribute to a better knowledge of this pathogen and help in the search for vaccines and novel drug targets aimed to control the disease caused by this Leishmania species.

Genomic and transcriptomic alterations in Leishmania donovani lines experimentally resistant to antileishmanial drugs.

Leishmaniasis is a serious medical issue in many countries around the World, but it remains largely neglected in terms of research investment for developing new control and treatment measures. No vaccines exist for human use, and the chemotherapeutic agents currently used are scanty. Furthermore, for some drugs, resistance and treatment failure are increasing to alarming levels. The aim of this work was to identify genomic and trancriptomic alterations associated with experimental resistance against the common drugs used against VL: trivalent antimony (SbIII, S line), amphotericin B (AmB, A line), miltefosine (MIL, M line) and paromomycin (PMM, P line). A total of 1006 differentially expressed transcripts were identified in the S line, 379 in the A line, 146 in the M line, and 129 in the P line. Also, changes in ploidy of chromosomes and amplification/deletion of particular regions were observed in the resistant lines regarding the parental one. A series of genes were identified as possible drivers of the resistance phenotype and were validated in both promastigotes and amastigotes from Leishmania donovani, Leishmania infantum and Leishmania major species. Remarkably, a deletion of the gene LinJ.36.2510 (coding for 24-sterol methyltransferase, SMT) was found to be associated with AmB-resistance in the A line. In the P line, a dramatic overexpression of the transcripts LinJ.27.T1940 and LinJ.27.T1950 that results from a massive amplification of the collinear genes was suggested as one of the mechanisms of PMM resistance. This conclusion was reinforced after transfection experiments in which significant PMM-resistance was generated in WT parasites over-expressing either gene LinJ.27.1940 (coding for a D-lactate dehydrogenase-like protein, D-LDH) or gene LinJ.27.1950 (coding for an aminotransferase of branched-chain amino acids, BCAT). This work allowed to identify new drivers, like SMT, the deletion of which being associated with resistance to AmB, and the tandem D-LDH-BCAT, the amplification of which being related to PMM resistance.

Stability of unstimulated and stimulated whole saliva flow rates in children.

OBJECTIVE: To analyze the stability of the unstimulated saliva flow rate (USFR) and the stimulated saliva flow rate (SSFR) in children followed from age 7 to 12 years old. METHODS: Longitudinal study. Whole saliva samples were collected from school children (50 girls and 50 boys). Forty-four girls and 32 boys remained in this cohort for 6 years (dropout rate 24%). Variables that could influence USFR or SSFR patterns were analyzed in a repeated-measures manova. RESULTS: Over a 6-year follow-up, the children's USFR ranged from 0.41 to 0.46 mL/min in the initial and final observation, respectively, and showed no significant differences (P = 0.4455) during the follow-up. The children consistently belonged to one of three distinct SSFR groups (P < 0.0001). A repeated-measures manova model showed that USFR and SSFR were not associated with sex, body mass index, or indoor temperature during sampling. CONCLUSION: These results indicate that USFR and SSFR patterns were stable from 7 to 12 years old. This finding may be useful in oral health prevention or treatment and in the search for biomarkers in saliva for screening or diagnostic purposes.

HPV oral lesions in HIV-infected patients: the impact of long-term HAART.

BACKGROUND: Since the introduction of highly active antiretroviral therapy (HAART), an increase in the frequency of human papillomavirus-associated oral lesions (HPV-OL) has been observed. Thus, the aim of this study was to determine the prevalence and factors associated with HPV-OL in Mexican HIV-infected patients, as well as its genotyping, in the HAART era. METHODS: In a cross-sectional study developed at an HIV/AIDS referral center in Mexico City, HIV-infected patients were consecutively included from 2004 to 2011. An oral exam was performed; lymphocyte CD4(+) count, HIV-viral load, CDC-stage, and HAART use were recorded. HPV-OL samples were taken for routine histopathological analysis (H-E) and HPV-DNA amplification/sequencing. Logistic regression models were performed and the interactions tested using the STATA software. RESULTS: Among 787 HIV patients, 55 (6.9%) showed HPV-OL. HPV-OLs were independently associated with age (≥40 years) and with a longer time of HAART use (≥12 months). The most frequent lesion was squamous cell papilloma in 22 (40%) cases, followed by multifocal epithelial hyperplasia in 15 (27.3%) cases. Labial mucosa was the most common site involved (56.4%). Of the sequences obtained, 65.4% corresponded to low risk and 11.5% to high risk. Mixed high- and low-risk infection were identified in 7.7% of the cases. CONCLUSIONS: Human papillomavirus-associated oral lesions were associated with older age and longer HAART use. All lesions were benign in nature and most of the HPV sequences corresponded to low-risk types. The rise of HPV-OLs in HIV patients on HAART may be related with the longer life expectancy of individuals with an impaired immune system rather than a direct effect of HAART.

Prospective evaluation of oral mucositis in acute leukemia patients receiving chemotherapy.

PURPOSE: Knowledge of oral mucositis (OM) in patients with acute leukemia (AL) and chemotherapy (CT) has remained limited. Thus, a prospective, longitudinal study was undertaken to characterize clinical features, associated risk factors, and behavior of OM in a cohort of AL patients starting CT. METHODS: Prospective and longitudinal study. A cohort of patients, older than 15 years of age with AL, scheduled to receive CT, was followed from March 2006 to October 2007. At baseline and three times per week, for 21 days, patients had an oral examination performed using the Oral Mucositis Assessment Scale (OMAS); also, oral pain and difficulty to swallow were recorded using a visual analog scale. Weekly, salivary flow measurements (Schirmer's test modified version) were done. RESULTS: A cohort of 29 AL patients was followed for a median time of 21 (range, 14-53) days; 12 (41.4%) developed OM, with a mean OMAS score of 0.181 (SD +/- 0.56) and a mean peak OMAS score of 1.8 (SD +/- 0.56). The OM onset mean time was 9.8 (range, 2-20, SD +/- 6.09) days, with a mean duration of 7 (range, 3-14, SD +/- 4.15) days. OM was significantly correlated with salivary flow [rs = 0.420 (P = 0.0051)], oral pain [rs = 0.47 (P < 0.0001)], ability to swallow [rs = 0.36 (P = 0.0001)], and type of food intake [rs = 0.38 (P < 0.0001)]. CONCLUSIONS: OM is a frequent and early side effect of CT closely correlated with oral pain, difficulty to swallow, and impairment in food intake.

Prevención de la caries: recomendaciones actualizadas y estatus del conocimiento directamente / Caries prevention: updated recommendations and status of the knowledge directly applied to the Mexican scope

Objetivo: Establecer el impacto potencial de los procedimientos clínicos y de salud pública en cuanto a caries, en términos de lineamientos explícitos de evaluación y en el contexto de la investigación científica/profesional directamente relevante a México. Métodos: Revisión estructurada de la literatura sobre temas de prevención y salud de caries en México (1990-2004); se cotejaron los hallazgos con esquemas actualizados de evaluación de la calidad de la evidencia científica y recomendaciones asociadas (Canadian Task Force on Preventive Health Care). Resultados: Los esquemas de evaluación arrojaron una clasificación cualitativa de la evidencia científica/profesional y de recomendaciones directamente relevantes a intervenciones preventivas de caries. Ciento diecinueve artículos publicados entre 1990 y 2004 (inclusive) cumplieron con criterios de solidez metodológica y fueron agrupados en dos áreas temáticas y cinco niveles de calidad de evidencia. Grosso modo, esta literatura no cubre con la diversidad suficiente ni a profundidad la multiplicidad de temas y retos pertinentes a la salud dental en México. Conclusiones: Este enfoque, permitió identificar la necesidad de continuar documentando ciertos problemas de salud epidemiológicamente importantes. Es fundamental establecer acciones y métodos claramente definidos para extender el conocimiento actualmente disponible, así como operacionalizar esta información en las actividades clínicas y de salud pública

Línea basal de factores de riesgo a caries en escolares / Baseline risk factors for caries in elementary school aged children

Resumen Introducción. El objetivo de este reporte es presentar las asociaciones iniciales de los factores de riesgo inmutable (género y edad) y los factores de riesgo biológico en la línea basal de un modelo diagnóstico de predicción de riesgo a caries. Material y métodos. Se registró en 110 niños entre 6 y 7 años que asisten a 2 escuelas públicas de la Ciudad de México, los siguientes indicadores: clínicos o dentales (morfología, índices de caries y lesiones activas); salivales (flujo salival estimulado y prueba de Snyder); y bacteriológicos (cuentas de Lactobacillus y Streptococcus mutans en saliva y placa dentobacteriana). Resultados. La experiencia de caries se asoció con los siguientes indicadores: morfología, prueba de Snyder y 3 de los indicadores bacteriológicos (ANOVA). Los puntos de corte de riesgo más altos en esta muestra fueron: conteo de S. mutans en saliva (0.96), prueba de Snyder (0.61) y experiencia de caries (0.58). Se usaron 3 modelos de ANOVA con y sin ponderación de los factores de riesgo inmutables, identificando que los indicadores: morfología, lesiones de caries activa y conteo de Lactobacillus salivales son los factores que se asocian de manera significativa con la experiencia de caries en los 3 modelos. Conclusión. El modelo que mejor explica la caries es el que pondera el género R² 62%.

Introduction. The objective of this report is to present the initial associations between age and gender and the biological risk factors at the baseline of a diagnostic model to predict the risk for caries. Material and methods. The following indicators were registered in 110 elementary school children between 6-7 years of age, who attended to 2 public schools in Mexico City: dental (morphology, caries indexes and active caries lesions); salivary (stimulated flow rate and Snyder test); and bacteriological (Streptococcus mutans and Lactobacillus counts in plaque and saliva). Results. Caries experience was associated with dental morphology, Snyder test and with 3 of the bacterial indicators (ANOVA). When the biological risk factors were analyzed by the cut off point for risk, the following variables were identified: salivary S. mutans counts (0.96), Snyder test (0.61) and history of caries (0.58) as highest. Three models of ANOVA were used with/without weighted immutable risk factors, identifying that the indicators: morphology, active lesions and salivary Lactobacillus counts as the factors significantly associated with the caries experience in the 3 models. Conclusion. The model that best explains the risk for caries is the 1 that weighted for gender R² 62%.

[Association of oral lesions with HIV serological status]. / Asociación de lesiones bucales con el estado serológico para el VIH.

OBJECTIVE: To estimate the prevalence of oral lesions and its association with HIV serological status. MATERIAL AND METHODS: A cross-sectional descriptive study was conducted between 1998 and 1999 in Mexico City, among 512 subjects attending two information centers of Centro Nacional para la Prevención del VIH/SIDA e ITS (CONASIDA, National Center for Prevention of HIV/AIDS and Sexually Transmitted Infections) for HIV serologic testing. The oral examination was performed without knowledge of the HIV status. Statistical analysis was conducted using Student's t test, Fisher's exact test and the chi 2 test; odds ratios and 95% CI were also calculated. RESULTS: A total of 512 individuals were examined, 68 of whom were HIV-positive. HIV-related oral lesions (OL), were evident in 65% (44/68) of the HIV-positive individuals; 95% of them consisted in oral candidosis (OC) and hairy leukoplakia (HL). OC and HL were strongly associated with seropositivity to HIV. CONCLUSIONS: OC and HL were the oral lesions most strongly associated to HIV seropositivity. The English version of this paper is available at: http://www.insp.mx/salud/index.html.

Asociación de lesiones bucales con el estado serológico para el VIH / Association of oral lesions with HIV serological status

OBJECTIVE: To estimate the prevalence of oral lesions and its association with HIV serological status. MATERIAL AND METHODS: A cross-sectional descriptive study was conducted between 1998 and 1999 in Mexico City, among 512 subjects attending two information centers of Centro Nacional para la Prevención del VIH/SIDA e ITS (CONASIDA, National Center for Prevention of HIV/AIDS and Sexually Transmitted Infections) for HIV serologic testing. The oral examination was performed without knowledge of the HIV status. Statistical analysis was conducted using Student's t test, Fisher's exact test and the chi 2 test; odds ratios and 95 CI were also calculated. RESULTS: A total of 512 individuals were examined, 68 of whom were HIV-positive. HIV-related oral lesions (OL), were evident in 65 (44/68) of the HIV-positive individuals; 95 of them consisted in oral candidosis (OC) and hairy leukoplakia (HL). OC and HL were strongly associated with seropositivity to HIV. CONCLUSIONS: OC and HL were the oral lesions most strongly associated to HIV seropositivity. The English version of this paper is available at: http://www.insp.mx/salud/index.html.

Estudio antropométrico en un grupo de hombres y mujeres de la tercera edad en la Ciudad de México / Anthropometric study in a group of elderly men and women from Mexico city

Estimar valores antropométricos en un grupo de hombres y mujeres de la tercera edad en la Ciudad de México. Material y métodos. Se realizó un estudio de corte transversal en 508 sujetos mayores de 60 años, registrados como jubilados y pensionados del Instituto Mexicano del Seguro Social o asistentes al Instituto Nacional de la Senectud, durante el segundo semestre de 1995. Se utilizaron protocolos de estandarización para registrar sus caractarísticas antropométricas. Se realizaron pruebas t de Student para detectar diferencias en los valores promedio entre hombres y mujeres, en general, como en cada subgrupo de edad conformado; también el análisis de correlación de Pearson del índice de masa corporal (IMC) con las variables antropométricas; se tomó un nivel de significancia de p<0.05. Resultados. Doscientos treinta individuos de la población de estudio correspondieron al sexo masculino y 278 al femenino, la edad promedio fue de 66.9 y 67.3 años, respectivamente. El promedio de peso en los hombres fue de 70.7 kg, con una desviación estándar (DE) de 9.9, la estatura de 164 cm (DE 6.5) y el IMC de 26.4 (DE 3.7). La distribución del IMC mostró que 50.9 por ciento de los hombres y 54 por ciento de las mujeres evaluados se ubicaron en el rango de 25.0 a 29.9; se obtuvo un coeficiente de correlación superior a r 0.70 (p<0.001) entre el IMC con las circunferencias de cintura y cadera. Conclusiones. Con base en el IMC, aproximadamente tres cuartas partes de la población de estudio presentó sobrepeso u obesidad. Es posible que la evaluación tanto de sobrepeso como de obesidad en el anciano requiera de un ajuste en los valores de normalidad de este índice

The aim of the present study was to estimate basic anthropometric measurements in a group of elderly men and women from Mexico City. Material and methods. A cross-sectional study was carried out among senior citizens registered in the National Institute of the Elderly and National Institute of Social security in Mexico City. Standardized protocols were used to measure the anthropometric characteristics of the study group. The analysis included Student t tests to detect differences in average values between men and women in general and in each age subgroup formed. In addition, Pearson correlation analysis of the body mass index (BMI) with anthropometric variables was performed; p<0.05 was taken as the level of significance. Results. A total of 508 people aged 60 or older participated in the study; 230 were males and 278 were females. The average age was 66.9-years-old in the male group and 67.3-years-old in the female group. Among men the average weight was 70.7 kg, standard deviation (SD 9.9), height was 164 cm (SD 6.5) and BMI was 26.4 (SD 3.7). Among women the average weight was 60.8 kg (SD 9.9), height was 150 cm (SD 5.9) and BMI was 27.1 (SD 4.0). The distribution of the BMI showed that 50.9% of men and 54% of women were between 25.0 and 29.9. A correlation coefficient over r 0.70 (