Congenital heart disease (CHD) can be complicated by pulmonary arterial hypertension (PAH). Cardiopulmonary bypass (CPB) for corrective surgery may cause endothelial dysfunction, involving ...endothelin-1 (ET-1), circulating endothelial cells (CECs), and endothelial progenitor cells (EPCs). These markers can gauge disease severity, but their levels in children’s peripheral blood still lack consensus for prognostic value. The aim of our study was to investigate changes in ET-1, cytokines, and the absolute numbers (Ɲ) of CECs and EPCs in children 24 h before and 48 h after CPB surgery to identify high-risk patients of complications. A cohort of 56 children was included: 41 cases with CHD-PAH (22 with high pulmonary flow and 19 with low pulmonary flow) and 15 control cases. We observed that Ɲ-CECs increased in both CHD groups and that Ɲ-EPCs decreased in the immediate post-surgical period, and there was a strong negative correlation between ET-1 and CEC before surgery, along with significant changes in ET-1, IL8, IL6, and CEC levels. Our findings support the understanding of endothelial cell precursors’ role in endogenous repair and contribute to knowledge about endothelial dysfunction in CHD.
Next-Generation Sequencing (NGS) is widely used to investigate genomic variation. In several studies, the genetic variation of Mycobacterium tuberculosis has been analyzed in sputum samples without ...previous culture, using target enrichment methodologies for NGS. Alignments obtained by different programs generally map the sequences under default parameters, and from these results, it is assumed that only Mycobacterium reads will be obtained. However, variants of interest microorganism in clinical samples can be confused with a vast collection of reads from other bacteria, viruses, and human DNA. Currently, there are no standardized pipelines, and the cleaning success is never verified since there is a lack of rigorous controls to identify and remove reads from other sputum-microorganisms genetically similar to M. tuberculosis. Therefore, we designed a bioinformatic pipeline to process NGS data from sputum samples, including several filters and quality control points to identify and eliminate non-M. tuberculosis reads to obtain a reliable genetic variant report. Our proposal uses the SURPI software as a taxonomic classifier to filter input sequences and perform a mapping that provides the highest percentage of Mycobacterium reads, minimizing the reads from other microorganisms. We then use the filtered sequences to perform variant calling with the GATK software, ensuring the mapping quality, realignment, recalibration, hard-filtering, and post-filter to increase the reliability of the reported variants. Using default mapping parameters, we identified reads of contaminant bacteria, such as Streptococcus, Rhotia, Actinomyces, and Veillonella. Our final mapping strategy allowed a sequence identity of 97.8% between the input reads and the whole M. tuberculosis reference genome H37Rv using a genomic edit distance of three, thus removing 98.8% of the off-target sequences with a Mycobacterium reads loss of 1.7%. Finally, more than 200 unreliable genetic variants were removed during the variant calling, increasing the report's reliability.
CATSPER1 gene encodes a pore-forming and pH-sensing subunit of the CatSper Ca2+- permeable channel, a protein in the flagellum essential for sperm hyperactivation. Previous studies have shown that ...the murine Catsper1 gene promoter is regulated by different Sox proteins. Likewise, it is acknowledged that the human CATSPER1 gene promoter sequence is enriched in potential interaction sites for the sex-determining region Y gene (SRY), which suggest a novel regulatory transcriptional mechanism for CatSper1 channel expression. Therefore, in this work, we sought to determine whether the human CATSPER1 gene expression is regulated by the SRY transcription factor. To this end, a series of deletions and mutations were introduced in the wild- type CATSPER1 gene promoter to eliminate the SRY sites, and the different constructs were tested for their ability to activate transcription in human embryonic kidney and murine spermatogonial germ cell lines (HEK-293 and GC1-spg, respectively) using luciferase assays. In addition, by using a strategy that combines electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation (ChIP) we investigated whether the CATSPER1 gene expression is regulated by the SRY transcription factor both in vitro and in vivo. Our results show that the transcriptional factor SRY specifically binds to different sites in the promoter sequence and has the ability to control CATSPER1 gene transcription.
Tuberculosis (TB) remains one of the most important infectious diseases globally. Establishing a resistance profile from the initial TB diagnosis is a priority. Rapid molecular tests evaluate only ...the most common genetic variants responsible for resistance to certain drugs, and Whole Genome Sequencing (WGS) needs culture prior to next-generation sequencing (NGS), limiting their clinical value. Targeted sequencing (TS) from clinical samples avoids these drawbacks, providing a signature of genetic markers that can be associated with drug resistance and phylogeny. In this study, a proof-of-concept protocol was developed for detecting genomic variants associated with drug resistance and for the phylogenetic classification of Mycobacterium Tuberculosis (Mtb) in sputum samples. Initially, a set of Mtb reference strains from the WHO were sequenced (WGS and TS). The results from the protocol agreed >95% with WHO reported data and phenotypic drug susceptibility testing (pDST). Lineage genetics results were 100% concordant with those derived from WGS. After that, the TS protocol was applied to sputum samples from TB patients to detect resistance to first- and second-line drugs and derive phylogeny. The accuracy was >90% for all evaluated drugs, except Eto/Pto (77.8%), and 100% were phylogenetically classified. The results indicate that the described protocol, which affords the complete drug resistance profile and phylogeny of Mtb from sputum, could be useful in the clinical area, advancing toward more personalized and more effective treatments in the near future.
The COVID-19 pandemic negatively affected the progress in accessing essential Tuberculosis (TB) services and reducing the burden of TB disease, resulting in a decreased detection of new cases and increased deaths. Generating molecular diagnostic tests with faster results without losing reliability is considered a priority. Specifically, developing an antimicrobial resistance profile from the initial stages of TB diagnosis is essential to ensure appropriate treatment. Currently available rapid molecular tests evaluate only the most common genetic variants responsible for resistance to certain drugs, limiting their clinical value. In this work, targeted sequencing on sputum samples from TB patients was used to identify Mycobacterium tuberculosis mutations in genes associated with drug resistance and to derive a phylogeny of the infecting strain. This protocol constitutes a proof-of-concept toward the goal of helping clinicians select a timely and appropriate treatment by providing them with actionable information beyond current molecular approaches.
Background
In Mexico, the incidence of acute myeloid leukemia (AML) has increased in the last few years. Mortality is higher than in developed countries, even though the same chemotherapy protocols ...are used. CCAAT Enhancer Binding Protein Alpha (
CEBPA
) mutations are recurrent in AML, influence prognosis, and help to define treatment strategies.
CEBPA
mutational profiles and their clinical implications have not been evaluated in Mexican pediatric AML patients.
Aim of the Study
To identify the mutational landscape of the
CEBPA
gene in pediatric patients with
de novo
AML and assess its influence on clinical features and overall survival (OS).
Materials and Methods
DNA was extracted from bone marrow aspirates at diagnosis. Targeted massive parallel sequencing of
CEBPA
was performed in 80 patients.
Results
CEBPA
was mutated in 12.5% (10/80) of patients. Frameshifts at the N-terminal region were the most common mutations 57.14% (8/14).
CEBPA
biallelic (
CEBPA
BI
) mutations were identified in five patients. M2 subtype was the most common in
CEBPA
positive patients (
CEBPA
POS
) (
p
= 0.009); 50% of the
CEBPA
POS
patients had a WBC count > 100,000 at diagnosis (
p
= 0.004). OS > 1 year was significantly better in
CEBPA
negative (
CEBPA
NEG
) patients (
p
= 0.0001).
CEBPA
POS
patients (either bi- or monoallelic) had a significantly lower OS (
p
= 0.002). Concurrent mutations in
FLT3
,
CSF3R
, and
WT1
genes were found in
CEBPA
POS
individuals. Their contribution to poor OS cannot be ruled out.
Conclusion
CEBPA mutational profiles in Mexican pediatric AML patients and their clinical implications were evaluated for the first time. The frequency of
CEBPA
POS
was in the range reported for pediatric AML (4.5–15%).
CEBPA
mutations showed a negative impact on OS as opposed to the results of other studies.
We report the effect of the Sesquiterpene Lactones Ambrosin, Incomptine B and Glaucolide E against seven strains of Trypanosoma cruzi, the etiological agent of Chagas Disease. These compounds were ...isolated from Parthenium hysterophorus, Decachaeta incompta, and Vernonia liatroides, respectively. We evaluated by flow cytometry the viability of epimastigotes. Ambrosin was the most effective, then Incomptine B, and Glaucolide E (IC50 = 67.1, 123.7, and 215.1 μM, respectively). These compounds were more potent than the drugs Benznidazole (IC50 > 400 μM) and Nifurtimox (IC50 = 199.7 to >400 μM). Toxicity to mammalian Vero and Jurkat cells was also determined in vitro. All the compounds had a poor selective index (0.003–1.859). Toxicoinformatics is useful to forecast in silico toxicological and pharmacokinetic properties. Ambrosin and Incomptine B may not possess mutagenic, tumorigenic, or reproductive effects. Glaucolide E could possess a low mutagenic and high tumorigenic effects, and probably target the Amine Oxidase A, Prostaglandin and G/H Synthase I. Interestingly, Ambrosin, Incomptine B and Glaucolide E, comply with Lipinsky Rule of Five, indicating a suitable pharmacokinetic profile. Ambrosin and Incomptine B possess high trypanocidal activity, and pharmaceutical properties suitable for development; however, their safety profile should be optimized by structural modifications.
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•Sesquiterpene Lactones Ambrosin, Incomptine B and Glaucolide E have trypanocidal activity.•Ambrosin and Incomptine B possess pharmaceutical properties suitable for development.•The safety profile of Ambrosin and Incomptine B should be optimized before their use as novel trypanocidal drugs.
INTRODUCTIONThe main cause of cervical cancer is an infection of keratinocytes in the basal layer of the stratified epithelium of the cervix by human papillomavirus (HPV). Other than in cervical ...samples, HPV DNA has been found in serum and other fluids but its origin is unclear. Extracellular vesicles (EV) could be a conveyance of viral DNA given their emerging role in cellular communication. The content of EV derived from cervical cells has not been properly explored and it is not known whether or not they contain HPV DNA. METHODSWe evaluated the DNA content of exosomes purified from cultures of HeLa cells by Next Generation Sequencing (NGS) and confirmed its presence by PCR. The presence of HPV DNA was also evaluated by PCR and NGS in EV from HPV-positive cervical samples without apparent lesion or with LSIL. RESULTSWe detected the integrated form of viral-DNA in exosomes from HeLa cells by NGS and confirmed its presence by PCR. The search for HPV sequences in EV obtained from cervical exudate samples without apparent lesion or with LSIL, where we expected to find the viral genome as an episome, indicated that HPV DNA, including the E6 and E7 oncogenes, is present in these EV. CONCLUSIONHPV DNA, including the viral oncogenes E6/E7, is found in exosomes regardless of the integration status of the virus in the infected cell.
•Catsper is a Ca2+permeable channel required for sperm hyperactivation.•Catsper play a central role in mammalian fertilization.•The transcriptional mechanisms that regulate Catsper1 expression are ...ill defined.•We identify and characterize regulatory elements in the murine Catsper1 promoter.•Transcription factor Sox5 and Sox9 increase Catsper1 promoter activity.
Catsper is a Ca2+permeable channel required for sperm hyperactivation. In spite of its central role in male fertility, the transcriptional mechanisms that regulate Catsper1 expression are ill defined. In this work, we describe the identification and characterization of important regulatory elements in the murine Catsper1 gene proximal promoter. Four transcription start sites and three functional Sox-binding sites were identified in the Catsper1 promoter. Interestingly, transcription factors Sox5 and Sox9 caused a significant increase in transactivation of the Catsper1 promoter in heterologous systems, and chromatin immunoprecipitation assays showed that both transcription factors interact with the Catsper1 promoter in vivo. These results provide new insights into the molecular mechanisms that control Catsper channel expression.
Recurrent genetic alterations contributing to leukemogenesis have been identified in pediatric B-cell Acute Lymphoblastic Leukemia (B-ALL), and some are useful for refining classification, prognosis, ...and treatment selection.
is a complex biomarker associated with a poor prognosis. It is characterized by
deletion coexisting with
,
, or PAR1 region deletions. The mutational spectrum and clinical impact of these alterations have scarcely been explored in Mexican pediatric patients with B-ALL. Here, we report the frequency of the
profile and the mutational spectrum of
, and
genes and evaluate their impact on overall survival (OS) in a group of patients with B-ALL.
A total of 206 pediatric patients with
B-ALL were included. DNA was obtained from bone marrow samples at diagnosis before treatment initiation. A custom-designed next-generation sequencing panel was used for mutational analysis. Kaplan-Meier analysis was used for OS estimation.
We identified the
profile in 21.8% of patients, which was higher than that previously reported in other studies. A significantly older age (
), a trend toward high-risk stratification (
), and a decrease in 5-year Overall Survival (OS) (
) were observed, although heterogeneous treatment protocols in our cohort would have impacted OS. A mutation frequency higher than that reported was found for
(35.9%) and
(35.9%) but lower for
(26.6%).
group was older at diagnosis (
), and most of them were classified as high-risk (73.8%,
), while patients with
had a higher leukocyte count (
) and a tendency toward a higher percentage of blasts (98.6%, >50% blasts,
) than the non-mutated patients. A decrease in OS was found in
and
patients, but the significance was lost after
was removed.
Our findings demonstrated that Mexican patients with B-ALL have a higher prevalence of genetic markers associated with poor outcomes. Incorporating genomic methodologies into the diagnostic process, a significant unmet need in low- and mid-income countries, will allow a comprehensive identification of relevant alterations, improving disease classification, treatment selection, and the general outcome.
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