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ABSTRACT We present the case of a 37-year-old woman who underwent bilateral penetrating keratoplasty for congenital hereditary endothelial dystrophy at the age of 10 years. Over the subsequent 27 years, the patient's vision slowly deteriorated. Our examination revealed decompensation of the right corneal graft. We addressed this with regraft surgery. We then learned that the patient had been suffering from progressive hearing loss since adolescence. Tonal audiometry revealed hearing per ceptive deafness of 25 dB, which was more prominent in the left ear. Because the patterns of progressive sensorineural hearing loss and congenital hereditary endothelial dystrophy have both been linked to the same gene, slc4a11, we tested our patient for mutations in this gene. The test was positive for a heterozygous slc4a11 gene fifth exon mutation on chromosome 20p13-p12, which causes a frameshift. A combined clinical and genetic evaluation confirmed a diagnosis of Harboyan syndrome. After the genetic diagnosis of the disease, she was evaluated for the need for a hearing aid due to her hearing loss. The patient was also informed about genetic counseling.

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We searched for the prevalence of actionable somatic mutations in exon 2 of the KRAS gene in western Mexican patients with CRC. Tumor tissue DNA samples from 150 patients with sporadic CRC recruited at the Civil Hospital of Guadalajara were analyzed. Mutations in exon 2 of the KRAS gene were identified using Sanger sequencing, and the data were analyzed considering clinical-pathological characteristics. Variants in codon 12 (rs121913529 G>A, G>C, and G>T) and codon 13 (rs112445441 G>A) were detected in 26 patients (with a prevalence of 17%). No significant associations were found between these variants and clinical-pathological characteristics (p > 0.05). Furthermore, a comprehensive search was carried out in PubMed/NCBI and Google for the prevalence of KRAS exon 2 mutations in Latin American populations. The 17 studies included 12,604 CRC patients, with an overall prevalence of 30% (95% CI = 0.26-0.35), although the prevalence ranged from 13 to 43% across the different data sources. Determining the variation and frequency of KRAS alleles in CRC patients will enhance their potential to receive targeted treatments and contribute to the understanding of the genomic profile of CRC.

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Protein folding and evolution are intimately linked phenomena. Here, we revisit the concept of exons as potential protein folding modules across a set of 38 abundant and conserved protein families. Taking advantage of genomic exon-intron organization and extensive protein sequence data, we explore exon boundary conservation and assess the foldon-like behavior of exons using energy landscape theoretic measurements. We found deviations in the exon size distribution from exponential decay indicating selection in evolution. We show that when taken together there is a pronounced tendency to independent foldability for segments corresponding to the more conserved exons, supporting the idea of exon-foldon correspondence. While 45% of the families follow this general trend when analyzed individually, there are some families for which other stronger functional determinants, such as preserving frustrated active sites, may be acting. We further develop a systematic partitioning of protein domains using exon boundary hotspots, showing that minimal common exons correspond with uninterrupted alpha and/or beta elements for the majority of the families but not for all of them.

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En el cáncer de pulmón de células no pequeñas (CPCNP) con mutación clásica de EGFR, los inhibidores de la tirosina quinasa (TKI) de EGFR produce mejores resultados que la quimioterapia basada en platino. Sin embargo, la eficacia terapéutica es bastante diferente en pacientes con mutaciones de inserción del exón 20 del EGFR (ex20ins) versus mutaciones comunes. Los pacientes con mutaciones ex20ins son insensibles a los EGFR-TKI y tienen mal pronóstico. Es importante conocer las características demográficas y clínicas en este grupo de pacientes y la prevalencia en nuestra región. Metodología: Revisión retrospectiva, única instituciónal, serie de casos de pacientes con cáncer de pulmón de células no pequeñas con mutaciones de inserción del exón 20 desde 2017-2023. Los pacientes habían recibido terapia de primera línea para enfermedad avanzada y tuvieron estudios de imágenes para evaluar la respuesta. Se registraron los datos demográficos, las características y tratamiento de cada paciente. La respuesta al tratamiento se evaluó utilizando los criterios RECIST v1.1 y la supervivencia global se calculó mediante el método de Kaplan Meier. Resultados: Entre los 15 pacientes identificados con cáncer de pulmón de células no pequeñas con mutaciones de inserción del exón 20 en nuestra institución, la incidencia para la mutación fue del 1.5%. La edad promedio fue de 60 años, el 46,7% eran mujeres, 14 pacientes hispanos y 1 paciente asiático, solo 3 pacientes tenían antecedentes de tabaquismo. El 40% de los pacientes tuvo una escala funcional según el Grupo Cooperativo de Oncología Oriental (ECOG) de 2. El subtipo histológico fue adenocarcinoma en todos los casos. De los 13 (86.7%) pacientes que recibieron tratamiento de primera línea, se les realizaron exploraciones evaluables para determinar la respuesta, 11 progresaron, 1 paciente obtuvo enfermedad estable y otro tuvo respuesta parcial. La mediana de supervivencia global (SG) fue de 5 meses. Conclusiones: Los pacientes con mutaciones de inserción del exón 20 tienen resistencia a los inhibidores de tirosina quinasa, lo cual le confiere un peor pronóstico. Es vital conocer en nuestra región la incidencia de la mutación y las características de los pacientes para ofrecer un diagnóstico y tratamiento oportuno. Nuestros resultados proporcionan un contexto importante para el desarrollo de nuevas terapias que puedan aprobarse en primera línea de tratamiento y no en líneas subsecuentes. (provisto por Infomedic International)

In non-small cell lung cancer (NSCLC) with classical EGFR mutation, EGFR tyrosine kinase inhibitors (TKIs) produce better results than platinum-based chemotherapy. However, therapeutic efficacy is quite different in patients with EGFR exon 20 insertion mutations (ex20ins) versus common mutations. Patients with ex20ins mutations are insensitive to EGFR-TKIs and have poor prognosis. It is important to know the demographic and clinical characteristics in this group of patients and the prevalence in our region. Methodology: retrospective, single institution, case series review of patients with non-small cell lung cancer with exon 20 insertion mutations from 2017-2023. Patients had received first-line therapy for advanced disease and had imaging studies to assess response. Demographics, characteristics, and treatment of each patient were recorded. Treatment response was assessed using RECIST v1.1 criteria and overall survival was calculated using the Kaplan Meier method. Results: Among the 15 patients identified with non-small cell lung cancer with exon 20 insertion mutations at our institution, the incidence for the mutation was 1.5%. The mean age was 60 years, 46.7% were women, and the incidence of the mutation was 1.5%. The average age was 60 years, 46.7% were women, 14 patients were Hispanic and 1 patient was Asian, only 3 patients had a history of smoking. Forty percent of the patients had an Eastern Cooperative Oncology Group (ECOG) functional score of 2. The histologic subtype was adenocarcinoma in all cases. Of the 13 (86.7%) patients who received first-line treatment had evaluable scans to determine response, 11 progressed, 1 patient had stable disease, and 1 patient had a partial response. The median overall survival (OS) was 5 months. Conclusions: Patients with exon 20 insertion mutations have resistance to tyrosine kinase inhibitors, which confers a worse prognosis. It is vital to know in our region the incidence of the mutation and patient characteristics to provide timely diagnosis and treatment. Our results provide an important context for the development of new therapies that can be approved in the first line of treatment and not in subsequent lines. (provided by Infomedic International)

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Precision medicine has helped identify several tumor molecular aberrations to be treated with targeted therapies. These therapies showed substantial improvement in efficacy without excessive toxicity in patients with specific oncogenic drivers with advanced cancers. In metastatic lung cancers, the implementation of broad platforms for molecular tumor sequencing has helped oncology providers identify oncogenic drivers linked with better outcomes when treated upfront with targeted therapies. Mesenchymal-epithelial transition factor (MET) alterations are present in up to 60% of non-small cell lung cancer and are associated with a poor prognosis. Capmatinib and tepotinib are currently the only two approved targeted therapies by the U.S. Food and Drug Administration (FDA) for patients with MET exon 14 skipping mutation. Several agents are being developed to tackle an unmet need in patients with MET alterations. Some of these agents are being used in combination with EGFR targeted therapy to mitigate resistance to EGFR inhibitor. These agents are poised to provide new hope for these patients.

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The Multidrug Resistance protein (ABCB1, MDR1) is involved in the transport of xenobiotics and antiretroviral drugs. Some variants of the ABCB1 gene are of clinical importance; among them, exon 12 (c.1236C>T, rs1128503), 21 (c.2677G>T/A, rs2032582), and 26 (c.3435C>T, rs1045642) have a high incidence in Caucasians. Several protocols have been used for genotyping the exon 21 variants, such as allele-specific PCR-RFLP using adapted primer to generate a digestion site for several enzymes and automatic sequencing to detect the SNVs, TaqMan Allele Discrimination assay and High-Resolution Melter analysis (HRMA). The aim was to describe a new approach to genotype the three variants c.2677G>T/A for the exon 21 doing only one PCR with the corresponding primers and the digestion of the PCR product with two restriction enzymes: BrsI to identify A allele and BseYI to differentiate between G or T. An improvement of this methodology was also described. The proposal technique here described is demonstrated to be very efficient, easy, fast, reproducible, and cost-effective.

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Familial hypercholesterolemia (FH) is a monogenic disease characterized by high plasma low-density lipoprotein cholesterol (LDL-c) levels and increased risk of premature atherosclerotic cardiovascular disease. Mutations in FH-related genes account for 40% of FH cases worldwide. In this study, we aimed to assess the pathogenic variants in FH-related genes in the Brazilian FH cohort FHBGEP using exon-targeted gene sequencing (ETGS) strategy. FH patients (n = 210) were enrolled at five clinical sites and peripheral blood samples were obtained for laboratory testing and genomic DNA extraction. ETGS was performed using MiSeq platform (Illumina). To identify deleterious variants in LDLR, APOB, PCSK9, and LDLRAP1, the long-reads were subjected to Burrows-Wheeler Aligner (BWA) for alignment and mapping, followed by variant calling using Genome Analysis Toolkit (GATK) and ANNOVAR for variant annotation. The variants were further filtered using in-house custom scripts and classified according to the American College Medical Genetics and Genomics (ACMG) guidelines. A total of 174 variants were identified including 85 missense, 3 stop-gain, 9 splice-site, 6 InDel, and 71 in regulatory regions (3'UTR and 5'UTR). Fifty-two patients (24.7%) had 30 known pathogenic or likely pathogenic variants in FH-related genes according to the American College Medical and Genetics and Genomics guidelines. Fifty-three known variants were classified as benign, or likely benign and 87 known variants have shown uncertain significance. Four novel variants were discovered and classified as such due to their absence in existing databases. In conclusion, ETGS and in silico prediction studies are useful tools for screening deleterious variants and identification of novel variants in FH-related genes, they also contribute to the molecular diagnosis in the FHBGEP cohort.

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ERBB2 exon 20 insertions may impact the clinical management of lung cancer patients. However, the frequency of ERBB2 exon 20 insertions in lung cancer patients in Brazil is scarce. Here, we analyzed 722 Brazilian non-small cell lung cancer (NSCLC) patients from Barretos Cancer Hospital that were indicated to require routine lung cancer molecular testing. ERBB2 exon 20 insertions were evaluated by a targeted panel using next-generation sequencing (NGS). Clinicopathological and molecular data were collected from patient medical records. Among the 722 NSCLC patients, 85.2% had lung adenocarcinomas, 53.9% were male, 66.8% were quitter or current smokers, and 63.2% were diagnosed at an advanced stage of the disease. We identified 0.8% (6/722) of patients who harbored the insertion p.(Tyr772_Ala775dup) at exon 20 of the ERBB2 gene. All ERBB2 mutated patients were diagnosed with lung adenocarcinoma, were never smokers, and wild-type for EGFR, KRAS, and ALK hotspot alterations. Less than 1% of Brazilian NSCLC patients harbor ERBB2 exon 20 insertions, yet they could benefit in future from the new drugs in development.

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PURPOSE: The identification of subpopulations harboring druggable targets has become a major step forward in the subclassification of solid tumors into small groups suitable for specific therapies. BRAF fusions represent a paradigm of uncommon and targetable oncogenic events and have been widely correlated to the development of specific malignancies. However, they are only present in a limited frequency across most common tumor types. At this regard, we performed a genomic screening aimed to identifying rare variants associated to advanced prostate cancer development. METHODS: Tumoral tissue genomic screening of 41 patients developing advanced prostate cancer was performed at our center as part of the GETHI XX study. The project, sponsored by the Spanish Collaborative Group in Rare Cancers (GETHI), aims to analyze the molecular background of rare tumors and to discover unfrequent molecular variants in common tumors. RESULTS: Here we present the clinical outcome and an in-deep molecular analysis performed in a case harboring a SND1-BRAF fusion gene. The identification of such rearrangement in a patient refractory to standard therapies led to the administration of trametinib (MEK inhibitor). Despite unsensitive to standard therapies, the patient achieved a dramatic response to trametinib. A comprehensive study of the tumor demonstrated this event to be a trunk alteration with higher expression of MEK in areas of tumor invasion. CONCLUSIONS: Our study describes the patient-driven discovery of the first BRAF fusion-driven prostate cancer effectively treated with trametinib. Consequently, MAPK pathway activation could define a new subtype of prostate cancer susceptible to a tailored management.

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Several oncogenic mechanisms have been identified for MET, including MET amplification, fusions, mutations in the tyrosine kinase domain and exon 14 skipping alterations. MET exon 14 mutations are found in about 3-5% of non-small-cell lung cancers. Dysregulation of the MET receptor leads to cell proliferation and survival by activation of the PI3K-AKT-TOR and RAS-RAF-MET-ERK canonical pathways. Targeting the MET tyrosine kinase domain in the setting of MET exon 14 mutations using effective MET tyrosine kinase inhibitors is a current targeted therapy option for patients with metastatic lung cancer. In this Review, we focus on the management of patients with MET exon 14 skipping alterations by addressing the biology of the MET receptor and exon 14 skipping mutations, current treatment strategies, and sequential treatment options based on resistance mechanisms to MET inhibitors in patients with non-small-cell lung cancer.

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OBJECTIVE: To evaluate the genomic and immune characteristics of non-small-cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) exon 20 insertion (ex20ins) mutations from a retrospective dataset with molecular spectrum, tumor mutational burden (TMB), and programmed death-ligand 1 (PD-L1) expression, as well as to evaluate the efficacy of immune checkpoint inhibitors (ICIs). METHODS: A total of 283 patients with EGFR ex20ins mutations who were diagnosed with NSCLC at our hospital from August 2013 to September 2020 were enrolled in this single-center retrospective study. RESULTS: Among the 283 patients with EGFR ex20ins mutations, 182 patients received next-generation sequencing (NGS) test, and 51 different subtypes of insertion variants were recorded. The most common mutations were A767_V769dup (21.4%), S768_D770dup (19.2%) and A763_Y764insFQEA (7.1%). The most common co-occurring mutations were EGFR amplification (37.9%), TP53 mutation (35.0%) and PIK3CA mutation (8.7%). PD-L1 status was available for 141 patients, and 75.9% (107/141) of these samples showed negative PD-L1 expression. In the 36 cases with TMB tested by NGS, the median TMB was 4.6 mutations/Mb. Then 12 patients received ICIs monotherapy or combination therapy. No severe adverse events were observed. CONCLUSION: Low PD-L1 expression and TMB were observed in NSCLC patients harboring EGFR ex20ins mutations. Further investigations are needed to confirm the therapeutic sensitivity of ICIs in this subgroup of EGFR mutations.

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BACKGROUND: Duchenne and Becker muscular dystrophies (DMD/BMD) are the most common human dystrophinopathies with recessive X-linked inheritance. Dystrophin gene deletions and duplications are the most common mutations, followed by point mutations. The aim of this study is to characterize the mutational profile of the dystrophin gene in Colombian patients with DMD/BMD. MATERIAL AND METHODS: Mutational profiling was determined in 69 affected patients using Sanger sequencing, next-generation sequencing (NGS) and/or multiplex ligation dependent-probes amplification (MLPA). Genetic variants were classified according to molecular consequence and new variants were determined through database and literature analysis. RESULTS: Mutational profile in affected patients revealed that large deletions/duplications analyzed by MLPA accounted for 72.5% of all genetic variations. By using Sanger sequencing or NGS, we identified point mutations in 15.9% and small deletions in 11.6% of the patients. New mutations were found, most of them were point mutations or small deletions (10.1%). CONCLUSION: Our results described the genetic profile of the dystrophin gene in Colombian patients with DMD and contribute to efforts to identify molecular variants in Latin American populations. For our population, 18.8% of cases could be treated with FDA or MDA approved molecular therapies based on specific mutations. These data contribute to the establishment of appropriate genetic counseling and potential treatment.

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INTRODUCTION: Photoaging is the process by which ultraviolet rays gradually induce clinical and histological changes in the skin through the production and organization of biological molecules, such as elastin, which is critical to skin strength and elasticity. After exposure to radiation, elastin may undergo alternative mRNA splicing, resulting in modified proteins that contribute to the formation of aging characteristics, such as solar elastosis. The present work aimed to study two different forms of elastin under these conditions: normal elastin and elastin that had been altered in exon 26A. METHODS: These different forms of elastin were characterized for gene expression by quantitative real-time polymerase chain reaction (qPCR) and for protein expression by immunohistochemistry of ex vivo skins (from photoexposed and non-photoexposed areas) and in vitro reconstituted skin. In addition, up- and downstream molecules in the elastin signaling cascade were evaluated. RESULTS: As a result, a significant increase in the gene expression of elastin 26A was observed in both ex vivo photoexposed skin tissues and the in vitro photoexposed reconstituted skins. Additionally, significant increases in the gene expression levels of matrix metalloproteinase-12 (MMP12) and lysyl oxidase (LOX) were observed in the ex vivo skin model. The evaluation of protein expression levels of some photoaging markers on the reconstituted skin revealed increased tropoelastin and fibrillin-1 expression after photoexposure. CONCLUSION: This work contributes to a better understanding of the biological mechanisms involved in photoaging, making it possible to obtain new strategies for the development of dermocosmetic active ingredients to prevent and treat skin aging.

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The structure of eukaryotic genes is generally a combination of exons interrupted by intragenic non-coding DNA regions (introns) removed by RNA splicing to generate the mature mRNA. A fraction of genes, however, comprise a single coding exon with introns in their untranslated regions or are intronless genes (IGs), lacking introns entirely. The latter code for essential proteins involved in development, growth, and cell proliferation and their expression has been proposed to be highly specialized for neuro-specific functions and linked to cancer, neuropathies, and developmental disorders. The abundant presence of introns in eukaryotic genomes is pivotal for the precise control of gene expression. Notwithstanding, IGs exempting splicing events entail a higher transcriptional fidelity, making them even more valuable for regulatory roles. This work aimed to infer the functional role and evolutionary history of IGs centered on the mouse genome. IGs consist of a subgroup of genes with one exon including coding genes, non-coding genes, and pseudogenes, which conform approximately 6% of a total of 21,527 genes. To understand their prevalence, biological relevance, and evolution, we identified and studied 1,116 IG functional proteins validating their differential expression in transcriptomic data of embryonic mouse telencephalon. Our results showed that overall expression levels of IGs are lower than those of MEGs. However, strongly up-regulated IGs include transcription factors (TFs) such as the class 3 of POU (HMG Box), Neurog1, Olig1, and BHLHe22, BHLHe23, among other essential genes including the ß-cluster of protocadherins. Most striking was the finding that IG-encoded BHLH TFs fit the criteria to be classified as microproteins. Finally, predicted protein orthologs in other six genomes confirmed high conservation of IGs associated with regulating neural processes and with chromatin organization and epigenetic regulation in Vertebrata. Moreover, this study highlights that IGs are essential modulators of regulatory processes, such as the Wnt signaling pathway and biological processes as pivotal as sensory organ developing at a transcriptional and post-translational level. Overall, our results suggest that IG proteins have specialized, prevalent, and unique biological roles and that functional divergence between IGs and MEGs is likely to be the result of specific evolutionary constraints.

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Dystrophinopathies cover a spectrum of rare progressive X-linked muscle diseases, arising from DMD mutations. They are among the most common pediatric muscular dystrophies, being Duchenne muscular dystrophy (DMD) the most severe form. Despite the fact that there is still no cure for these serious diseases, unprecedented advances are being made for the development of therapies for DMD. Some of which are already conditionally approved: exon skipping and premature stop codon read-through. The present work aimed to characterize the mutational spectrum of DMD in an Argentinian cohort, to identify candidates for available pharmacogenetic treatments and finally, to conduct a comparative analysis of the Latin American (LA) frequencies of mutations amenable for available DMD therapies. We studied 400 patients with clinical diagnosis of dystrophinopathy, implementing a diagnostic molecular algorithm including: MLPA/PCR/Sanger/Exome and bioinformatics. We also performed a meta-analysis of LA's metrics for DMD available therapies. The employed algorithm resulted effective for the achievement of differential diagnosis, reaching a detection rate of 97%. Because of this, corticosteroid treatment was correctly indicated and validated in 371 patients with genetic confirmation of dystrophinopathy. Also, 20 were eligible for exon skipping of exon 51, 21 for exon 53, 12 for exon 45 and another 70 for premature stop codon read-through therapy. We determined that 87.5% of DMD patients will restore the reading frame with the skipping of only one exon. Regarding nonsense variants, UGA turned out to be the most frequent premature stop codon observed (47%). According to the meta-analysis, only four LA countries (Argentina, Brazil, Colombia and Mexico) provide the complete molecular algorithm for dystrophinopathies. We observed different relations among the available targets for exon skipping in the analyzed populations, but a more even proportion of nonsense variants (∼40%). In conclusion, this manuscript describes the theragnosis carried out in Argentinian dystrophinopathy patients. The implemented molecular algorithm proved to be efficient for the achievement of differential diagnosis, which plays a crucial role in patient management, determination of the standard of care and genetic counseling. Finally, this work contributes with the international efforts to characterize the frequencies and variants in LA, pillars of drug development and theragnosis.

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Populations along steep environmental gradients are subject to differentiating selection that can result in local adaptation, despite countervailing gene flow, and genetic drift. In montane systems, where species are often restricted to narrow ranges of elevation, it is unclear whether the selection is strong enough to influence functional differentiation of subpopulations differing by a few hundred meters in elevation. We used targeted capture of 12 501 exons from across the genome, including 271 genes previously implicated in altitude adaptation, to test for adaptation to local elevations for 2 highland hummingbird species, Coeligena violifer (n = 62) and Colibri coruscans (n = 101). For each species, we described population genetic structure across the complex geography of the Peruvian Andes and, while accounting for this structure, we tested whether elevational allele frequency clines in single nucleotide polymorphisms (SNPs) showed evidence for local adaptation to elevation. Although the 2 species exhibited contrasting population genetic structures, we found signatures of clinal genetic variation with shifts in elevation in both. The genes with SNP-elevation associations included candidate genes previously discovered for high-elevation adaptation as well as others not previously identified, with cellular functions related to hypoxia response, energy metabolism, and immune function, among others. Despite the homogenizing effects of gene flow and genetic drift, natural selection on parts of the genome evidently optimizes elevation-specific cellular function even within elevation range-restricted montane populations. Consequently, our results suggest local adaptation occurring in narrow elevation bands in tropical mountains, such as the Andes, may effectively make them "taller" biogeographic barriers.

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We report the case of a 75-year-old female patient with a big tumour in the lower rectum with intestinal obstruction and lower gastrointestinal bleeding history who underwent a tumour biopsy under laparotomy and end colostomy at another hospital in Peru. She came to our institution for clinical evaluation with a pathology result of a rectal gastrointestinal stromal tumour. An extra elevator abdominoperineal resection was performed with tumour-free margins. The histology confirmed a high-grade (G2) rectal gastrointestinal stromal tumour with a mitotic index of 27/50. DOC-1 (+) and CD117 (+) in immunohistochemistry. Genomic DNA was extracted from the paraffin-fixed tumour sample, and c.1504_1509dupGCCTAT (p.Ala502_Tyr503dup) mutation was detected in exon 9 of the KIT gene. Imatinib 400 mg per day for 3 years was indicated as adjuvant treatment. Currently, she has a disease-free survival of 12 months.

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BACKGROUND: Trypanosoma cruzi, the causative agent of Chagas disease, and T. rangeli are kinetoplastid parasites endemic to Latin America. Although closely related to T. cruzi and capable of infecting humans, T. rangeli is non-pathogenic. Both parasite species are transmitted by triatomine bugs, and the presence of T. rangeli constitutes a confounding factor in the study of Chagas disease prevalence and transmission dynamics. Trypanosoma cruzi possesses high molecular heterogeneity: seven discrete typing units (DTUs) are currently recognized. In Ecuador, T. cruzi TcI and T. rangeli KP1(-) predominate, while other genetic lineages are seldom reported. METHODS: Infection by T. cruzi and/or T. rangeli in different developmental stages of triatomine bugs from two communities of southern Ecuador was evaluated via polymerase chain reaction product size polymorphism of kinetoplast minicircle sequences and the non-transcribed spacer region of the mini-exon gene (n = 48). Forty-three mini-exon amplicons were also deep sequenced to analyze single-nucleotide polymorphisms within single and mixed infections. Mini-exon products from ten monoclonal reference strains were included as controls. RESULTS: Trypanosoma cruzi genetic richness and diversity was not significantly greater in adult vectors than in nymphal stages III and V. In contrast, instar V individuals showed significantly higher T. rangeli richness when compared with other developmental stages. Among infected triatomines, deep sequencing revealed one T. rangeli infection (3%), 8 T. cruzi infections (23.5%) and 25 T. cruzi + T. rangeli co-infections (73.5%), suggesting that T. rangeli prevalence has been largely underestimated in the region. Furthermore, deep sequencing detected TcIV sequences in nine samples; this DTU had not previously been reported in Loja Province. CONCLUSIONS: Our data indicate that deep sequencing allows for better parasite identification/typing than amplicon size analysis alone for mixed infections containing both T. cruzi and T. rangeli, or when multiple T. cruzi DTUs are present. Additionally, our analysis showed extensive overlap among the parasite populations present in the two studied localities (c.28 km apart), suggesting active parasite dispersal over the study area. Our results highlight the value of amplicon sequencing methodologies to clarify the population dynamics of kinetoplastid parasites in endemic regions and inform control campaigns in southern Ecuador.

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Micro Exon Gene (MEG) proteins are thought to play major roles in the infection and survival of parasitic Schistosoma mansoni worms in host organisms. Here, the physical chemical properties of two small MEG proteins found in the genome of S. mansoni, named MEG-24 and MEG-27, were examined by a combination of biophysical techniques such as differential scanning calorimetry, tensiometry, circular dichroism, fluorescence, and electron spin resonance spectroscopies. The proteins are surface active and structurally arranged as cationic amphipathic α-helices that can associate with lipid membranes and cause their disruption. Upon adsorption to lipid membranes, MEG-27 strongly affects the fluidity of erythrocyte ghost membranes, whereas MEG-24 forms pores in erythrocytes without modifying the ghost membrane fluidity. Whole-mount in situ hybridization experiments indicates that MEG-27 and MEG-24 transcripts are located in the parasite esophagus and subtegumental cells, respectively, suggesting a relevant role of these proteins in the host-parasite interface. Taken together, these characteristics lead us to propose that these MEG proteins may interact with host cell membranes and potentially modulate the immune process using a similar mechanism as that described for α-helical membrane-active peptides.

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