Prolactin Protects the Structural Integrity of Human Fetal Membranes by Downregulating Inflammation-induced Secretion of Matrix Metalloproteinases.

Prolactin (PRL) is a pleiotropic hormone with a key role in pregnancy. In fetal membranes, PRL can regulate the secretion of pro-inflammatory factors, which induces the activation of matrix metalloproteinases (MMPs). The increase and activation of MMPs deregulate the turnover of the extracellular matrix in the fetal membranes, altering its structure and function, causing premature rupture of the membranes and preterm labor. In this work, we evaluate the effect of PRL upon the secretion of MMP-1, MMP-2, MMP-9, MMP-13, and the tissue inhibitors of metalloproteinases (TIMPs) in human fetal membranes after lipopolysaccharide (LPS) challenge. Nine fetal membranes from healthy non-laboring cesarean deliveries at term were cultured in a 2-independent chamber system and pre-treated with 250, 500, 1000 or 4000 ng/ml of PRL for 24 h, then choriodecidual region was stimulated with 500 ng/ml of LPS plus fresh PRL for 24 h. The MMPs and TIMPs secretion were quantified by ELISA, additionally MMP-2 and MMP-9 gelatinolytic activity was measured by zymography. LPS induced the MMP-9 and MMP-1 secretion, but no MMP-2 or MMP-13 in comparison with basal levels. PRL co-treatment decreased the MMP-2, MMP-9 and MMP-1 secretion induced by LPS. The active forms were present in the tissue extract, showing a response consistent with the secretion profile. TIMP-1 and TIMP-2 secretion was decreased after LPS treatment and the PRL co-treatment reverts this effect. The present results support that PRL may favor the balance between these factors involved in the structural maintenance of fetal membranes in an inflammatory event.

Case Series Study of Melioidosis, Colombia.

We report 7 cases of melioidosis in Colombia and comparision of 4 commercial systems for identifying Burkholderia pseudomallei. Phoenix systems were not a definitive method for identifying B. pseudomallei. For accurate identification, we recommend including this bacterium in the library databases of matrix-assisted laser desorption/ionization mass spectrometry systems in Latin America.

[Molecular identification of glucose-6-phosphate dehydrogenase (G6PD) detected in neonatal screening]. / Identificación molecular de la glucosa-6-fosfato deshidrogenasa (G6PD) detectada en el tamiz neonatal.

AIMS: To present the strategy of identifying the molecular variants of G6PD detected in neonatal screening (NS). MATERIAL AND METHODS: We present a series of incident cases of newborns positive for G6PD deficiency detected in NS. From nuclear DNA with the methodology of real-time PCR we sought molecular G6PD variants: G202A, A376G, T968C and C563T. RESULTS: Of a total of 21,619 neonates, 41 cases were reactive in NS for G6PD (189.6/100,000 RN screened rate), 34 cases confirmed the molecular variant of G6PD (157.3/100,000 RN screened rate). The most frequent allele combination G202A/A376G (G6PD ratio and median activity, 0.460 and 1.72 ± 0.35 U/g Hb, respectively), followed by G202A (0.170 and 1.74 ± 0.27 U/g Hb) and A376G/T968C (ratio 0.150 and 1.10 ± 0.44 U/g Hb). The T968C allelic variant showed lower enzyme activity than the rest (1.1 ± 0.4; p = 0.02). Two women were detected with G6PD deficiency with G202A/A376G and G202A variant. CONCLUSIONS: African alleles were prevalently detected in neonatal screening. This strategy allows the identification of molecular variants involved in 80% of cases.

Quantifying the reduction in potential health risks by determining the sensitivity of poliovirus type 1 chat strain and rotavirus SA-11 to electron beam irradiation of iceberg lettuce and spinach.

Fresh produce, such as lettuce and spinach, serves as a route of food-borne illnesses. The U.S. FDA has approved the use of ionizing irradiation up to 4 kGy as a pathogen kill step for fresh-cut lettuce and spinach. The focus of this study was to determine the inactivation of poliovirus and rotavirus on lettuce and spinach when exposed to various doses of high-energy electron beam (E-beam) irradiation and to calculate the theoretical reduction in infection risks that can be achieved under different contamination scenarios and E-beam dose applications. The D(10) value (dose required to reduce virus titers by 90%) (standard error) of rotavirus on spinach and lettuce was 1.29 (± 0.64) kGy and 1.03 (± 0.05) kGy, respectively. The D(10) value (standard error) of poliovirus on spinach and lettuce was 2.35 (± 0.20) kGy and 2.32 (± 0.08) kGy, respectively. Risk assessment of data showed that if a serving (∼14 g) of lettuce was contaminated with 10 PFU/g of poliovirus, E-beam irradiation at 3 kGy will reduce the risk of infection from >2 in 10 persons to approximately 6 in 100 persons. Similarly, if a serving size (∼0.8 g) of spinach is contaminated with 10 PFU/g of rotavirus, E-beam irradiation at 3 kGy will reduce infection risks from >3 in 10 persons to approximately 5 in 100 persons. The results highlight the value of employing E-beam irradiation to reduce public health risks but also the critical importance of adhering to good agricultural practices that limit enteric virus contamination at the farm and in packing houses.

Smart pooling: AI-powered COVID-19 informative group testing.

Massive molecular testing for COVID-19 has been pointed out as fundamental to moderate the spread of the pandemic. Pooling methods can enhance testing efficiency, but they are viable only at low incidences of the disease. We propose Smart Pooling, a machine learning method that uses clinical and sociodemographic data from patients to increase the efficiency of informed Dorfman testing for COVID-19 by arranging samples into all-negative pools. To do this, we ran an automated method to train numerous machine learning models on a retrospective dataset from more than 8000 patients tested for SARS-CoV-2 from April to July 2020 in Bogotá, Colombia. We estimated the efficiency gains of using the predictor to support Dorfman testing by simulating the outcome of tests. We also computed the attainable efficiency gains of non-adaptive pooling schemes mathematically. Moreover, we measured the false-negative error rates in detecting the ORF1ab and N genes of the virus in RT-qPCR dilutions. Finally, we presented the efficiency gains of using our proposed pooling scheme on proof-of-concept pooled tests. We believe Smart Pooling will be efficient for optimizing massive testing of SARS-CoV-2.

Effect of Oropharyngeal Administration of Colostrum in Premature Newborns ≤32 Weeks of Gestation on the Immune Response and Neonatal Morbidity: A Double-Blind Randomized Clinical Trial.

Introduction: The mother's colostrum carries immunological components, such as cytokines and immunoglobulins (Igs), derived from the maternal circulation with bacteriostatic properties. Objective: The objective of this study was to evaluate the effect of oropharyngeal administration of colostrum (OPAC) vs. placebo in the first 4 days of life in premature newborns ≤32 weeks of gestation on serum Ig concentration, neonatal morbidity, and total days of hospitalization. Hypothesis: The OPAC increases serum Igs and decreases morbidity and total days of hospitalization. Materials and Methods: A double-blind randomized controlled trial was carried out. Participants were randomly assigned to one of the two groups, namely, group 1: placebo (P) (n = 50) and group 2: colostrum (C) (n = 46). A blood sample was obtained at baseline and 7 and 28 days of life to quantify immunoglobulin G (IgG), immunoglobulin A (IgA), and IgM. Results: The C group showed an increase in serum IgA on day 28 expressed as median and [interquartile range]; C: 25 [12-35] vs. P: 11 [8-18], p < 0.001. There were no significant differences in neonatal morbidity. Newborns in the colostrum group showed the completed enteral feeding earlier (days), C: 13.9 ± 7 vs. P: 17.4 ± 8.4, p < 0.04; they reached the birth weight earlier, C: 10.9 ± 2.8 vs. P: 12.9 ± 4, p < 0.01, and had less days of hospitalization, C: 60.2 ± 33.8 vs. P: 77.2 ± 47.3, p < 0.04. Neonatal mortality was lower in the colostrum group than the placebo group 0% vs. 12%, respectively, without a statistical difference (p = 0.06). Conclusion: In premature newborns ≤32 weeks of gestation, the OPAC within 4 days after birth increases serum IgA concentration at day 28 compared to placebo. Similarly, OPAC decreased the days to complete enteral feeding and reach the birth weight and total days of hospitalization. Clinical Trial Registration: [https://clinicaltrials.gov/ct2/show/NCT03578341], identifier: [NCT03578341].

Multiparameter flow cytometry analysis of leukocyte markers for diagnosis in preterm neonatal sepsis.

PURPOSE: Neonatal sepsis is an important public health concern worldwide due to its immediate lethality and long-term morbidity rates, Clinical evaluation and laboratory analyses are indispensable for diagnosis of neonatal sepsis. However, assessing multiple biomarkers in neonates is difficult due to limited blood availability. The aim is to investigate if the neonatal sepsis in preterm could be identified by multiparameter analysis with flow cytometry. MATERIALS AND METHODS: The expression of activation-related molecules was evaluated by flow cytometry in newborn with or without risk factors for sepsis. RESULTS: Our analysis revealed that several markers could be useful for sepsis diagnosis, such as CD45RA, CD45RO, or CD71 on T cells; HLA-DR on NKT or classic monocytes, and TREM-1 on non-classic monocytes or neutrophils. However, ROC analysis shows that the expression of CD45RO on T lymphocytes is the only useful biomarker for diagnosis of neonatal late-onset sepsis. Also, decision tree analyses showed that CD45RO plus CD27 could help differentiate the preterm septic neonates from those with risk factors. CONCLUSIONS: Our study shows a complementary and practical strategy for biomarker assessment in neonatal sepsis.

Prolactin modifies the in vitro LPS-induced chemotactic capabilities in human fetal membranes at the term of gestation.

PROBLEM: Immune responses of fetal membranes involve the production of chemoattractant mediators causing infiltration of maternal and fetal leukocytes, intrauterine inflammation and potentially the disruption of maternal-fetal tolerance. Prolactin (PRL) has deep immunoregulatory effects in the fetal-maternal interface. We aimed to test the in vitro PRL effect upon chemotactic capacities of human fetal membranes. METHOD OF STUDY: Fetal membranes and umbilical cord blood were collected from healthy non-laboring caesarean deliveries at term. Fetal membranes were cultured in Transwell® frames to mimic the barrier function between choriodecidual and amniotic sides. Tissues were treated with PRL, Lipopolysaccharide (LPS), or both simultaneously. Then, RANTES, MCP-1, MIP-1α, IP-10, and PECAM-1 were quantified in a conditioned medium by choriodecidual or amniotic sides. The chemotaxis of subsets of migrating mononuclear cells from umbilical cord blood was evaluated in a Boyden Chamber in response to the conditioned medium by both sides. RESULTS: Lipopolysaccharide stimulates the production of RANTES, MCP-1, MIP-1α, and PECAM-1 in choriodecidua, while MIP-1α and PECAM-1 only increase in amnion. PRL decrease RANTES, MCP-1, and MIP-1 only in choriodecidua, but PECAM-1 was decreased mainly in amnion. The leukocyte migration was regulated significantly in response to the conditioned medium by the amnion, increase in the conditioned medium after LPS treatment, contrary with, the leukocyte migration decreased in a significant manner in response to conditioned medium after PRL and LPS-PRL co-treatment. Finally, T cells were the most responsive subset of cells. CONCLUSIONS: Prolactin modified in a tissue-specific manner the chemotactic factor and the leukocyte migration differentially in fetal membranes.

Transcriptome analysis of genes controlled by luxS/autoinducer-2 in Salmonella enterica serovar Typhimurium.

The enteric pathogen Salmonella enterica serovar Typhimurium uses autoinducer-2 (AI-2) as a signaling molecule. AI-2 requires the luxS gene for its synthesis. The regulation of global gene expression in Salmonella Typhimurium by luxS/AI-2 is currently not known; therefore, the focus of this study was to elucidate the global gene expression patterns in Salmonella Typhimurium as regulated by luxS/AI-2. The genes controlled by luxS/AI-2 were identified using microarrays with RNA samples from wild-type (WT) Salmonella Typhimurium and its isogenic DeltaluxS mutant, in two growth conditions (presence and absence of glucose) at mid-log and early stationary phases. The results indicate that luxS/AI-2 has very different effects in Salmonella Typhimurium depending on the stage of cell growth and the levels of glucose. Genes with p < or = 0.05 were considered to be significantly expressed differentially between WT and DeltaluxS mutant. In the mid-log phase of growth, AI-2 activity was higher (1500-fold) in the presence of glucose than in its absence (450-fold). There was differential gene expression of 13 genes between the WT and its isogenic DeltaluxS mutant in the presence of glucose and 547 genes in its absence. In early stationary phase, AI-2 activity was higher (650-fold) in the presence of glucose than in its absence (1.5-fold). In the presence of glucose, 16 genes were differentially expressed, and in its absence, 60 genes were differentially expressed. Our microarray study indicates that both luxS and AI-2 could play a vital role in several cellular processes including metabolism, biofilm formation, transcription, translation, transport, and binding proteins, signal transduction, and regulatory functions in addition to previously identified functions. Phenotypic analysis of DeltaluxS mutant confirmed the microarray results and revealed that luxS did not influence growth but played a role in the biofilm formation and motility.

Rapid concentration of Bacillus and Clostridium spores from large volumes of milk, using continuous flow centrifugation.

Deliberate or accidental contamination of foods such as milk, soft drinks, and drinking water with infectious agents or toxins is a major concern to health authorities. There is a critical need to develop technologies that can rapidly and efficiently separate and concentrate biothreat agents from food matrices. A key limitation of current centrifugation and filtration technologies is that they are batch processes with extensive hands-on involvement and processing times. The objective of our studies was to evaluate the continuous flow centrifugation (CFC) technique for the rapid separation and concentration of bacterial spores from large volumes of milk. We determined the effectiveness of the CFC technology for concentrating approximately 10(3) bacterial spores in 3.7 liters (1 gal) of whole milk and skim milk, using Bacillus subtilis, Bacillus atrophaeus, and Clostridium sporogenes spores as surrogates for biothreat agents. The spores in the concentrated samples were enumerated by using standard plating techniques. Three independent experiments were performed at 10,000 rpm and 0.7 liters/min flow rate. The mean B. subtilis spore recoveries were 71.3 and 56.5% in skim and whole milk, respectively, and those for B. atrophaeus were 55 and 59.3% in skim and whole milk, respectively. In contrast, mean C. sporogenes spore recoveries were 88.2 and 78.6% in skim and whole milk, respectively. The successful use of CFC to concentrate these bacterial spores from 3.7 liters of milk in 10 min shows promise for rapidly concentrating other spores from large volumes of milk.

Recovery and functional validation of hidden soil enzymes in metagenomic libraries.

The vast microbial diversity on the planet represents an invaluable source for identifying novel activities with potential industrial and therapeutic application. In this regard, metagenomics has emerged as a group of strategies that have significantly facilitated the analysis of DNA from multiple environments and has expanded the limits of known microbial diversity. However, the functional characterization of enzymes, metabolites, and products encoded by diverse microbial genomes is limited by the inefficient heterologous expression of foreign genes. We have implemented a pipeline that combines NGS and Sanger sequencing as a way to identify fosmids within metagenomic libraries. This strategy facilitated the identification of putative proteins, subcloning of targeted genes and preliminary characterization of selected proteins. Overall, the in silico approach followed by the experimental validation allowed us to efficiently recover the activity of previously hidden enzymes derived from agricultural soil samples. Therefore, the methodology workflow described herein can be applied to recover activities encoded by environmental DNA from multiple sources.

Grapefruit juice and its furocoumarins inhibits autoinducer signaling and biofilm formation in bacteria.

Cell-to-cell communications in bacteria mediated by small diffusible molecules termed as autoinducers (AI) are known to influence gene expression and pathogenicity. Oligopeptides and N-acylhomoserine lactones (AHL) are major AI molecules involved in intra-specific communication in gram-positive and gram-negative bacteria respectively, whereas boronated-diester molecules (AI-2) are involved in inter-specific communication among both gram-positive and gram-negative bacteria. Naturally occurring furocoumarins from grapefruit showed >95% inhibition of AI-1 and AI-2 activities based on the Vibrio harveyi based autoinducer bioassay. Grapefruit juice and furocoumarins also inhibited biofilm formation by Escherichia coli O157:H7, Salmonella typhimurium and Pseudomonas aeruginosa. These results suggest that grape fruit juice and furocoumarins could serve as a source to develop bacterial intervention strategies targeting microbial cell signaling processes.

Identification of ground beef-derived fatty acid inhibitors of autoinducer-2-based cell signaling.

Autoinducer-2 (AI-2) molecules are used by several microorganisms to modulate various processes, including bioluminescence, biofilm formation, and virulence expression. Certain food matrices, including ground beef extracts, possess compounds capable of inhibiting AI-2 activity. In the present study, we identified and characterized these AI-2 inhibitors from ground beef extract using hexane solvent extraction and gas chromatography. Gas chromatographic analysis revealed the presence of several fatty acids such as palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:omega9), and linoleic acid (C18:omega6) that were capable of inhibiting AI-2 activity. These fatty acids were tested (using Vibrio harveyi BB170 and MM32 reporter strains) at different concentrations (1, 5, and 10 mM) to identify differences in the level of AI-2 activity inhibition. AI-2 inhibition ranged from 25 to 90%. A mixture of these fatty acids (prepared at concentrations equivalent to those present in the ground beef extract) produced 52 to 65% inhibition of AI-2 activity. The fatty acid mixture also negatively influenced Escherichia coli K-12 biofilm formation. These results demonstrate that both medium- and long-chain fatty acids in ground beef have the ability to interfere with AI-2-based cell signaling.

Autoinducer AI-2 is involved in regulating a variety of cellular processes in Salmonella Typhimurium.

Salmonella Typhimurium is known to exhibit LuxS/AI-2-mediated cell signaling. We investigated the role of LuxS/AI-2 system on Salmonella Typhimurium protein expression using a proteomics approach based on two-dimensional gel electrophoresis (2DGE)-MALDI-MS. The global protein expression profiles of the wild-type, a luxS mutant, and a luxS mutant strain supplemented with AI-2 were compared. Seven proteins were differentially expressed when comparing the wild-type and luxS mutant strains, whereas 13 proteins were differentially expressed when comparisons were made between luxS mutant strains with and without AI-2 supplementation. The seven proteins that were differentially expressed between the wild-type and the luxS mutant strain were also differentially expressed in the luxS mutant strain supplemented with AI-2. The level of PhoP, a virulence determinant, was higher in the presence of AI-2. Proteins associated with the carbohydrate metabolism (pfkA, gpmI, and talB) and ATP synthesis (Pta gene product) were up-regulated by the presence of AI-2 molecules. These results provide experimental evidence that AI-2 molecules regulate a variety of cellular processes in Salmonella Typhimurium.

Necrotizing fasciitis caused by Apophysomyces variabilis in an immunocompetent patient.

Mucormycosis caused by Apophysomyces variabilis is rarely reported in humans. A case of A. variabilis infection in an immunocompetent men after friction burns in a car accident is described. The infection presented as a rapidly progressive necrotizing infection of the skin and soft tissue, which required extensive surgical debridement and total colonic defunctioning colostomy associated with prolonged antifungal therapy. A. variabilis infection should be considered as a differential diagnosis of rapidly progressive necrotizing skin and soft tissue infections in immunocompetent individuals.

Respiratory tract clinical sample selection for microbiota analysis in patients with pulmonary tuberculosis.

BACKGROUND: Changes in respiratory tract microbiota have been associated with diseases such as tuberculosis, a global public health problem that affects millions of people each year. This pilot study was carried out using sputum, oropharynx, and nasal respiratory tract samples collected from patients with pulmonary tuberculosis and healthy control individuals, in order to compare sample types and their usefulness in assessing changes in bacterial and fungal communities. FINDINGS: Most V1-V2 16S rRNA gene sequences belonged to the phyla Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, and Fusobacteria, with differences in relative abundances and in specific taxa associated with each sample type. Most fungal ITS1 sequences were classified as Ascomycota and Basidiomycota, but abundances differed for the different samples. Bacterial and fungal community structures in oropharynx and sputum samples were similar to one another, as indicated by several beta diversity analyses, and both differed from nasal samples. The only difference between patient and control microbiota was found in oropharynx samples for both bacteria and fungi. Bacterial diversity was greater in sputum samples, while fungal diversity was greater in nasal samples. CONCLUSIONS: Respiratory tract microbial communities were similar in terms of the major phyla identified, yet they varied in terms of relative abundances and diversity indexes. Oropharynx communities varied with respect to health status and resembled those in sputum samples, which are collected from tuberculosis patients only due to the difficulty in obtaining sputum from healthy individuals, suggesting that oropharynx samples can be used to analyze community structure alterations associated with tuberculosis.

Proteomic analysis to identify the role of LuxS/AI-2 mediated protein expression in Escherichia coli O157:H7.

Microorganisms employ autoinducer molecules to modulate various bacterial processes including virulence expression, biofilm development, and bioluminescence. The universal autoinducer molecule AI-2 is hypothesized to mediate cell signaling in Escherichia coli O157:H7. We investigated the role of AI-2 on the E. coli O157:H7 cellular proteins using a two-dimensional (2D) gel electrophoresis-based proteomic approach. The protein expression patterns between two experimental comparisons were studied namely, 1) a wild type E. coli O157:H7 and its isogenic luxS mutant, and 2) the luxS mutant and the luxS mutant supplemented with AI-2 molecules. Eleven proteins were differentially expressed between the wild type and the luxS mutant strain, whereas 18 proteins were differentially expressed in the luxS mutant strain when supplemented with AI-2. The tryptophan repressor binding protein (WrbA), phosphoglycerate mutase (GpmA), and a putative protein YbbN were found to be differentially expressed under both experimental comparisons. The FliC protein which is involved in flagellar synthesis and motility was up-regulated in the wild type strain but was not influenced by the addition of synthetic AI-2 molecules to the luxS mutant suggesting the involvement of signaling molecules other than AI-2 on flagellar synthesis and motility.

Neurodesarrollo en infantes con antecedente de hipotiroidismo congénito / Neurodevelopment in infants with congenital hypothyroidism antecedents

Introducción: Los estudios de seguimiento de infantes con hipotiroidismo congénito con tratamiento temprano, muestran que no existen diferencias con respecto al coeficiente intelectual que se encuentra en la población general. Material y métodos: Estudio de cohorte retrospectivo. Veinticuatro hipotiroideos fueron detectados a través del Programa de Tamiz Neonatal y comparados con un grupo testigo. Los recién nacidos fueron valorados entre los seis meses y los siete años de edad mediante el índice de desarrollo (ID) de Bayley, el coeficiente intelectuales (Cl) de Terman Merril y la prueba Hiskey Nebraska para detectar infantes con hipoacusia. Resultados: Dieciocho infantes hipotiroideos mostraron Cl o ID normal o alto. Uno con ID de Bayley porabajo del promedio, dos con Cl por abajo del promedio y uno con hipoacusia y capacidad de aprendizaje baja. No se encontraron diferencias con respecto al grupo control. El uso de ototóxicos, prematurez e hiperbilirrubinemia, fueron las causas que explicaron los coeficientes bajos entre los hipotiroideos y el grupo control. Conclusiones: El Cl y el ID en el grupo de hipotiroidismo tratado tempranamente no difirió con los resultados del grupo testigo.

Introduction: Follow-up studies of hypothyroid infants who received early treatment evidence the presence of the same IQ vs. the general population. Material and methods: Retrospective cohort study. Twenty-four hypothyroid infants detected in neonatal screening were compared to a control group. The newborns were assessed from six months to seven years based on Bayley's infant development scales (ID), the Terman-Merril IQ (Cl) and the learning aptitude of the heard of hearing with the Hiskey-Nebraska test (CA). Results: Twenty hypothyroid infants showed a normal or high Cl or ID. One of the infants with a ID result below the mean, two with an Cl below the mean, one hearing impaired infant with low learning aptitude. No differences were found vs. the control group. The use of ototoxic medications, prematurity and hyperbilirubinemia explain the low coefficients between the hypothyroid individuals and the control. Conclusions: The Cl and the ID results in the hypothyroid group who received early treatment showed no difference vs. the control group.