The oral bacterium, Aggregatibacter actinomycetemcomitans, which is associated with localized aggressive periodontitis, as well as systemic infections including endocarditis, produces numerous ...virulence factors, including a repeats-in-toxin (RTX) protein called leukotoxin (LtxA), which kills human immune cells. The strains of A. actinomycetemcomitans most closely associated with disease have been shown to produce the most LtxA, suggesting that LtxA plays a significant role in the virulence of this organism. LtxA, like many of the RTX toxins, can be divided into four functional domains: an N-terminal hydrophobic domain, which contains a significant fraction of hydrophobic residues and has been proposed to play a role in the membrane interaction of the toxin; the central domain, which contains two lysine residues that are the sites of post-translational acylation; the repeat domain that is characteristic of the RTX toxins, and a C-terminal domain thought to be involved in secretion. In its initial interaction with the host cell, LtxA must bind to both cholesterol and an integrin receptor, lymphocyte function-associated antigen-1 (LFA-1). While both interactions are essential for toxicity, the domains of LtxA involved remain unknown. We therefore undertook a series of experiments, including tryptophan quenching and trypsin digestion, to characterize the structure of LtxA upon interaction with membranes of various lipid compositions. Our results demonstrate that LtxA adopts a U-shaped conformation in the membrane, with the N- and C-terminal domains residing outside of the membrane.
Objective
To evaluate survival outcomes of fetuses with right sided congenital diaphragmatic hernia (CDH) treated in Latin American centres and to assess the utility of left lung area to predict ...neonatal survival.
Methods
A retrospective cohort including isolated right sided CDH cases managed expectantly during pregnancy in six tertiary centers from five Latin American countries. The utility of the observed/expected lung‐to‐head ratio (O/E‐LHR) in predicting neonatal survival was assessed, and the best cut‐off to predict prognosis was automatically selected by decision tree analysis.
Results
A total of 99 right sided CDH cases were recruited, 58 isolated fetuses were selected at a median gestational age of 26.2 weeks, showing an overall survival rate of 26.2%. A linear trend was observed between survival and the O/E‐LHR, showing that at higher O/E‐LHR, the greater probability of survival (r = 0.56, p < 0.001). O/E‐LHR discriminates two groups with different survival outcomes: fetuses with an O/E‐LHR ≥65% showed a significantly higher survival rate than those with an O/E‐LHR <65% (81.8% vs. 15.6%, p < 0.01).
Conclusions
Overall survival rate in right sided CDH is lower in Latin American countries. The severity category of pulmonary hypoplasia should be classified according to lung area and the survival rate in such population.
Key points
What is already known about this topic?
Isolated left congenital diaphragmatic hernia (CDH) is associated with high perinatal mortality secondary to lung hypoplasia and pulmonary hypertension. The risk of neonatal death is higher in prenatally diagnosed right sided CDH than left CDH. The only parameter to classify the degree of lung hypoplasia and to predict the risk of neonatal survival in right‐sided CDH is evaluation of the observed/expected lung‐to‐head circumference ratio (O/E‐LHR). In North American and European countries, those fetuses with O/E‐LHR <45% are considered severe cases since this group shows overall survival rate of less than 17%.
What does this study add?
This report describes neonatal survival according to O/E‐LHR in fetuses with right congenital diaphragmatic hernia (CDH) managed expectantly during pregnancy in Latin American countries and demonstrates that overall survival rate in these countries is lower than European and North American countries, and that severe pulmonary hypoplasia in such centers should be defined by an O/E‐LHR below 65%, since this subgroup shows an overall survival rate of about 15%.
ERK5, the last MAP kinase family member discovered, is activated by the upstream kinase MEK5 in response to growth factors and stress stimulation. MEK5-ERK5 pathway has been associated to different ...cellular processes, playing a crucial role in cell proliferation in normal and cancer cells by mechanisms that are both dependent and independent of its kinase activity. Thus, nuclear ERK5 activates transcription factors by either direct phosphorylation or acting as co-activator thanks to a unique transcriptional activation TAD domain located at its C-terminal tail. Consequently, ERK5 has been proposed as an interesting target to tackle different cancers, and either inhibitors of ERK5 activity or silencing the protein have shown antiproliferative activity in cancer cells and to block tumor growth in animal models. Here, we review the different mechanisms involved in ERK5 nuclear translocation and their consequences. Inactive ERK5 resides in the cytosol, forming a complex with Hsp90-Cdc37 superchaperone. In a canonical mechanism, MEK5-dependent activation results in ERK5 C-terminal autophosphorylation, Hsp90 dissociation, and nuclear translocation. This mechanism integrates signals such as growth factors and stresses that activate the MEK5-ERK5 pathway. Importantly, two other mechanisms, MEK5-independent, have been recently described. These mechanisms allow nuclear shuttling of kinase-inactive forms of ERK5. Although lacking kinase activity, these forms activate transcription by interacting with transcription factors through the TAD domain. Both mechanisms also require Hsp90 dissociation previous to nuclear translocation. One mechanism involves phosphorylation of the C-terminal tail of ERK5 by kinases that are activated during mitosis, such as Cyclin-dependent kinase-1. The second mechanism involves overexpression of chaperone Cdc37, an oncogene that is overexpressed in cancers such as prostate adenocarcinoma, where it collaborates with ERK5 to promote cell proliferation. Although some ERK5 kinase inhibitors have shown antiproliferative activity it is likely that those tumors expressing kinase-inactive nuclear ERK5 will not respond to these inhibitors.
Brain specific kinases (BRSKs) are serine/threonine kinases, preferentially expressed in the brain after Embryonic Day 12. Although BRSKs are crucial neuronal development factors and regulation of ...their enzymatic activity has been widely explored, little is known of their transcriptional regulation. In this work, we show that Neuronal Growth Factor (NGF) increased the expression of Brsk1 in PC12 cells. Furthermore, during neuronal differentiation, Brsk1 mRNA increased through a MAPK-dependent Sp1 activation. To gain further insight into this regulation, we analyzed the transcriptional activity of the Brsk1 promoter in PC12 cells treated with NGF. Initially, we defined the minimal promoter region (−342 to +125 bp) responsive to NGF treatment. This region had multiple Sp1 binding sites, one of which was within a CpG island. In vitro binding assays showed that NGF-induced differentiation increased Sp1 binding to this site and that DNA methylation inhibited Sp1 binding. In vitro methylation of the Brsk1 promoter reduced its transcriptional activity and impaired the NGF effect. To evaluate the participation of DNA methyltransferases in Brsk1 gene regulation, the 5′Aza-dC inhibitor was used. 5′Aza-dC acted synergistically with NGF to promote Brsk1 promoter activity. Accordingly, DNMT3B overexpression abolished the response of the Brsk1 promoter to NGF. Surprisingly, we found Dnmt3b to be a direct target of NGF regulation, via the MAPK pathway. In conclusion, our results provide evidence of a novel mechanism of Brsk1 transcriptional regulation changing the promoter's methylation status, which was incited by the NGF-induced neuronal differentiation process.
•NGF increased the mRNA expression of Brsk1 in a PC12 cell model through a MAPK-dependent Sp1 activation.•NGF promoted the change in the methylation status of the brsk1 promoter.•NGF downregulates Dnmt3b expression through MAPK pathway activation.
Leukotoxin (LtxA), from oral pathogen
is a secreted membrane-damaging protein. LtxA is internalized by β2 integrin LFA-1 (CD11a/CD18)-expressing leukocytes and ultimately causes cell death; however, ...toxin localization in the host cell is poorly understood and these studies fill this void. We investigated LtxA trafficking using multi-fluor confocal imaging, flow cytometry and Rab5a knockdown in human T lymphocyte Jurkat cells. Planar lipid bilayers were used to characterize LtxA pore-forming activity at different pHs. Our results demonstrate that the LtxA/LFA-1 complex gains access to the cytosol of Jurkat cells without evidence of plasma membrane damage, utilizing dynamin-dependent and presumably clathrin-independent mechanisms. Upon internalization, LtxA follows the LFA-1 endocytic trafficking pathways, as identified by co-localization experiments with endosomal and lysosomal markers (Rab5, Rab11A, Rab7, and Lamp1) and CD11a. Knockdown of Rab5a resulted in the loss of susceptibility of Jurkat cells to LtxA cytotoxicity, suggesting that late events of LtxA endocytic trafficking are required for toxicity. Toxin trafficking via the degradative endocytic pathway may culminate in the delivery of the protein to lysosomes or its accumulation in Rab11A-dependent recycling endosomes. The ability of LtxA to form pores at acidic pH may result in permeabilization of the endosomal and lysosomal membranes.
Chronic kidney disease affects a large part of the population with hypertension, diabetes mellitus as well as those over 50 years of age. Research reported that male sex and other comorbidities such ...as obesity and anemia are more frequent in Chronic kidney disease, as well as uncontrolled diabetes mellitus or hypertension.
To determine the risk factors associated with the development of chronic kidney disease in adults with arterial hypertension.
Retrospective cohort study of 455 patients with hypertension treated in a primary health care hospital. Medical records and laboratory information were reviewed for the diagnosis of chronic kidney disease and its staging. Patients aged 40 years and older, of both sexes and evaluated between the years 2015 -2017 were included. Logistic regression analysis allowed the identification of risk factors associated with the development of chronic kidney disease.
63.7% were female and 36.3% male. The average age for 2015 was 69.79 ± 9.03, more than half of participants had diabetes mellitus and controlled hypertension and the predominant nephroprotection was with Losartan (53%) that year. Male sex (OR 1.68, CI 1.03-2,76), age: 60 years or older (OR 6.38, CI 2.65-15,37) and anemia (OR 1.71, CI 1.03-2,85), were risk factors for the development of chronic kidney disease (p < 0.05), whereas nephroprotection (OR 0.39, CI 0.18-0,88) and controlled diabetes mellitus (OR: 0.18, CI 0.07-0,47) were shown to be protective factors (p < 0.05). The prevalence of chronic kidney disease between 2015 and 2017 was 19% and 45%, respectively, with predominance of category G2. The comparison group is the same cohort analyzed in each year under study.
Male sex, age over 60 years, and anemia are risk factors for chronic kidney disease. Nephroprotection, controlled diabetes mellitus, and patient follow-up are factors that prevent its development.
To describe the clinical results of patients admitted and managed as cases of placenta accreta spectrum (PAS) at a Central American public hospital and the influence of the prenatal diagnosis on the ...condition.
A retrospective analysis of PAS patients treated at Hospital Bertha Calderón Roque, in Managua, Nicaragua, between June 2017 and September 2021. The diagnostic criteria used were those of the International Federation of Gynecology and Obstetrics (Fédération Internationale de Gynécologie et d'Obstétrique, FIGO, in French). The population was divided into patients with a prenatal ultrasonographic diagnosis of PAS (group 1) and those whose the diagnosis of PAS was established at the time of the caesarean section (group 2).
During the search, we found 103 cases with a histological and/or clinical diagnosis of PAS; groups 1 and 2 were composed of 51 and 52 patients respectively. Regarding the clinical results of both groups, the patients in group 1 presented a lower frequency of transfusions (56.9% versus 96.1% in group 2), use of a lower number of red blood cell units (RBCUs) among those undergoing transfusions (median: 1; interquartile range: IQR: 0-4 versus median: 3; IQR: 2-4 in group 2), and lower frequency of 4 or more RBCU transfusions (29.4% versus 46.1% in group 2). Group 1 also exhibited a non-significant trend toward a lower volume of blood loss (1,000 mL IQR: 750-2,000 mL versus 1,500 mL IQR: 1,200-1,800 mL in group 2), and lower requirement of pelvic packing (1.9% versus 7.7% in group 2).
Establishing a prenatal diagnosis of PAS is related to a lower frequency of transfusions. We observed a high frequency of prenatal diagnostic failures of PAS. It is a priority to improve prenatal detection of this disease.
Peptides are very diverse molecules that can participate in a wide variety of biological processes. In this way, peptides are attractive for doping, since these molecules can activate or trigger ...biological processes that can improve the sports performance of athletes. Peptide molecules are found in the official World Anti-Doping Agency lists, mainly in sections S2, S4, and S5. In most cases, these molecules have a very short half-life in the body and/or are identical to natural molecules in the body, making it difficult to analyze them as performance-enhancing drugs. This article reviews the role of peptides in doping, with special emphasis on the peptides used as reference materials, the pretreatment of samples in biological matrices, the instrumentation, and the validation of analytical methodologies for the analysis of peptides used in doping. The growing need to characterize and quantify these molecules, especially in complex biological matrices, has generated the need to search for robust strategies that allow for obtaining sensitive and conclusive results. In this sense, strategies such as solid phase peptide synthesis (SPPS), seeking to obtain specific peptides, metabolites, or isotopically labeled analogs, is a key tool for adequate quantification of different peptide molecules in biological matrices. This, together with the use of optimal methodologies for sample pretreatment (e.g., SPE or protein precipitation), and for subsequent analysis by high-resolution techniques (mainly hyphenated LC-HRMS techniques), have become the preferred instrumentation to meet the analytical challenge involved in the analysis of peptides in complex matrices.
PDF
