Evolution of the regulation of developmental gene expression in blind Mexican cavefish.

Developmental evolution and diversification of morphology can arise through changes in the regulation of gene expression or protein-coding sequence. To unravel mechanisms underlying early developmental evolution in cavefish of the species Astyanax mexicanus, we compared transcriptomes of surface-dwelling and blind cave-adapted morphs at the end of gastrulation. Twenty percent of the transcriptome was differentially expressed. Allelic expression ratios in cave X surface hybrids showed that cis-regulatory changes are the quasi-exclusive contributors to inter-morph variations in gene expression. Among a list of 108 genes with change at the cis-regulatory level, we explored the control of expression of rx3, which is a master eye gene. We discovered that cellular rx3 levels are cis-regulated in a cell-autonomous manner, whereas rx3 domain size depends on non-autonomous Wnt and Bmp signalling. These results highlight how uncoupled mechanisms and regulatory modules control developmental gene expression and shape morphological changes. Finally, a transcriptome-wide search for fixed coding mutations and differential exon use suggested that variations in coding sequence have a minor contribution. Thus, during early embryogenesis, changes in gene expression regulation are the main drivers of cavefish developmental evolution.

A 3D molecular map of the cavefish neural plate illuminates eye-field organization and its borders in vertebrates.

The vertebrate retinas originate from a specific anlage in the anterior neural plate called the eye field. Its identity is conferred by a set of 'eye transcription factors', whose combinatorial expression has been overlooked. Here, we use the dimorphic teleost Astyanax mexicanus, which develops proper eyes in the wild type and smaller colobomatous eyes in the blind cavefish embryos, to unravel the molecular anatomy of the eye field and its variations within a species. Using a series of markers (rx3, pax6a, cxcr4b, zic1, lhx2, emx3 and nkx2.1a), we draw a comparative 3D expression map at the end of gastrulation/onset of neurulation, which highlights hyper-regionalization of the eye field into sub-territories of distinct sizes, shapes, cell identities and combinatorial gene expression levels along the three body axes. All these features show significant variations in the cavefish natural mutant. We also discover sub-domains within the prospective telencephalon and characterize cell identities at the frontiers of the eye field. We propose putative fates for some of the characterized eye-field subdivisions, and suggest the existence of a trade-off between some subdivisions in the two Astyanax morphs on a micro-evolutionary scale.

An Astyanax mexicanus mao knockout line uncovers the developmental roles of monoamine homeostasis in fish brain.

Monoaminergic systems are conserved in vertebrates, yet they present variations in neuroanatomy, genetic components and functions across species. MonoAmine Oxidase, or MAO, is the enzyme responsible for monoamine degradation. While mammals possess two genes, MAO-A and MAO-B, fish possess one single mao gene. To study the function of MAO and monoamine homeostasis on fish brain development and physiology, here we have generated a mao knockout line in Astyanax mexicanus (surface fish), by CRISPR/Cas9 technology. Homozygote mao knockout larvae died at 13 days post-fertilization. Through a time-course analysis, we report that hypothalamic serotonergic neurons undergo fine and dynamic regulation of serotonin level upon loss of mao function, in contrast to those in the raphe, which showed continuously increased serotonin levels - as expected. Dopaminergic neurons were not affected by mao loss-of-function. At behavioral level, knockout fry showed a transient decrease in locomotion that followed the variations in the hypothalamus serotonin neuronal levels. Finally, we discovered a drastic effect of mao knockout on brain progenitors proliferation in the telencephalon and hypothalamus, including a reduction in the number of proliferative cells and an increase of the cell cycle length. Altogether, our results show that MAO has multiple and varied effects on Astyanax mexicanus brain development. Mostly, they bring novel support to the idea that serotonergic neurons in the hypothalamus and raphe of the fish brain are different in nature and identity, and they unravel a link between monoaminergic homeostasis and brain growth.

Towards an integrated approach to understand Mexican cavefish evolution.

The Mexican tetra, Astyanax mexicanus, comes in two forms: a classical river-dwelling fish and a blind and depigmented cave-dwelling fish. The two morphotypes are used as models for evolutionary biology, to decipher mechanisms of morphological and behavioural evolution in response to environmental change. Over the past 40 years, insights have been obtained from genetics, developmental biology, physiology and metabolism, neuroscience, genomics, population biology and ecology. Here, we promote the idea that A. mexicanus, as a model, has reached a stage where an integrated approach or a multi-disciplinary method of analysis, whereby a phenomenon is examined from several angles, is a powerful tool that can be applied to understand general evolutionary processes. Mexican cavefish have undergone considerable selective pressure and extreme morphological evolution, an obvious advantage to contribute to our understanding of evolution through comparative analyses and to pinpoint the specific traits that may have helped their ancestors to colonize caves.

Characterisation of Pomegranate-Husk Polyphenols and Semi-Preparative Fractionation of Punicalagin.

INTRODUCTION: Pomegranate-husk is the main by-product generated from the pomegranate industry. It is a potential source of compounds highly appreciated by different costumers. Punicalagin is the main compound present in pomegranate-husk. OBJECTIVE: To characterise the pomegranate-husk total polyphenols by HPLC-ESI-MS and to establish a method for the recovery of punicalagin using a medium pressure liquid chromatography (MPLC) system. MATERIALS AND METHODS: The characterisation of total pomegranate-husk polyphenols was carried out using liquid chromatography coupled to mass spectrometry. Thus, 200 mg of pomegranate-husk polyphenols were fractionated by MPLC. The isolated punicalagin was characterised by HPLC-MS and was tested as standard reagent for the measurement of its scavenging capacity reducing DPPH and ABTS radicals. RESULTS: Twenty peaks were identified by analytical HPLC-MS analysis from the pomegranate-husk polyphenols. The main compounds were the punicalagin anomers, punicalin and ellagic acid. The MPLC method allowed three fractions to be obtained. In fraction three 39.40 ± 8.06 mg of punicalagin anomers (purity > 97.9%) were recovered. The scavenging capacity of punicalagin showed an IC50 of 109.53 and 151.50 µg/mL for DPPH and ABTS radicals, respectively. CONCLUSION: The MPLC system was an excellent tool for the separation of the main ellagitannins from pomegranate husk and for the isolation of punicalagin anomers. Fraction three was rich in high purity punicalagin anomers. The IC50 was obtained for DPPH and ABTS radicals. Copyright © 2017 John Wiley & Sons, Ltd.

Gallic acid production under anaerobic submerged fermentation by two bacilli strains.

BACKGROUND: Tannase is an enzyme that catalyses the breakdown of ester bonds in gallotannins such as tannic acid. In recent years, the interest on bacterial tannases has increased because of its wide applications. The lactic acid bacteria (LAB) plays an important role in food tannin biotransformation, it has the ability of hydrolyse tannins in ruminants intestine. The finding of tannin hydrolysis by LAB has sparked their use as tannase producer. RESULTS: The bacterial strains used in the present work were identified as Bacillus subtilis AM1 and Lactobacillus plantarum CIR1. The maximal tannase production levels were 1400 and 1239 U/L after 32 and 36 h of fermentation respectively, for B. subtilis AM1 and L. plantarum CIR1. Maximum gallic acid release was 24.16 g/L for B. subtilis AM1 and 23.73 g/L for L. plantarum CIR1. HPLC analysis showed the formation of another peaks in the retention time range of 9-14 min, which could be attributed to the formation of di or tri-galloyl glucose. CONCLUSIONS: According to database, the strains were identified as Bacillus subtilis AM1 and Lactobacillus plantarum CIR1. In conclusion, both strains had the capability to produce good titres of extracellular tannase and release gallic acid.

HIV stigma as a barrier to retention in HIV care at a general hospital in Lima, Peru: a case-control study.

HIV stigma as a barrier to retention in HIV care has not been well-studied outside the United States. We conducted a case-control study in Lima, Peru to examine this issue. Cases were out-of-care for ≥12 months (n = 66) and controls were recruited from patients in active care presenting for a clinic visit (n = 110). A previously validated HIV stigma scale with four domains was used. Associations between being out-of-care and each stigma domain were assessed using multivariable logistic regression. Stigma scores were highest for disclosure concerns. Modest associations were found for greater disclosure concerns (OR 1.16; 95 % CI 0.99, 1.36) and concerns with public attitudes (OR 1.20; 95 % CI 1.03, 1.40). Enacted stigma and negative self-image showed non-linear associations with being out-of-care that plateaued or declined, respectively, at higher levels of stigma. The threshold effect for enacted stigma warrants further exploration, while disclosure concerns may be especially amenable to intervention in this population.

Olfactory sensory system develops from coordinated movements within the neural plate.

BACKGROUND: The peripheral olfactory sensory system arises from morphologically identifiable structures called placodes. Placodes are relatively late developing structures, evident only well after the initiation of somitogenesis. Placodes are generally described as being induced from the ectoderm suggesting that their development is separate from the coordinated cell movements generating the central nervous system. RESULTS: With the advent of modern techniques it is possible to follow the development of the neurectoderm giving rise to the anterior neural tube, including the olfactory placodes. The cell movements giving rise to the optic cup are coordinated with those generating the olfactory placodes and adjacent telencephalon. The formation of the basal lamina separating the placode from the neural tube is coincident with the anterior migration of cranial neural crest. CONCLUSIONS: Olfactory placodes are transient morphological structures arising from a continuous sheet of neurectoderm that gives rise to the peripheral and central nervous system. This field of cells is specified at the end of gastrulation and not secondarily induced from ectoderm. The separation of olfactory placodes and telencephalon occurs through complex cell movements within the developing neural plate similar to that observed for the developing optic cup.

Microplate quantification of total phenolic content from plant extracts obtained by conventional and ultrasound methods.

INTRODUCTION: There is increasing interest in phenolic compounds around the world because of their potential positive impact on human health. Phenolic compounds are largely found in fruits and vegetables. Extraction of phenolic compounds is a very important step in their recovery. The newly developed technique of ultrasound-assisted extraction (UAE) appears to be an advantageous alternative compared with conventional techniques, because it is simple and environmental friendly. The potential of UAE needs to be evaluated in each plant in order to demonstrate its efficiency. OBJECTIVE: The objective of the present study was to compare a conventional method and UAE on the extraction efficiency of phenolic compounds from Jatropha dioica, Fluorensia cernua, Turnera diffusa and Eucalyptus camaldulensis plants and evaluate the in vitro anti-oxidant potential. METHODS: Validation of the new method was carried out using mixed-model methodology and regression analysis. Feasibility of this new method was shown and applied using several plants extracts obtained by different extraction methods from semi-arid Mexican plants, which were characterised by high levels of polyphenols. Additionally, the anti-oxidant potential of these extracts was determined by 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity. RESULTS: Results showed that the new microplate method can be used to determine total phenolic content in plant extracts. Additionally, an alternative extraction method by ultrasound was less efficient compared with the conventional method. CONCLUSION: The tested plants are good candidates to obtain nutraceuticals and functional food ingredients.

Advancements in Litchi chinensis Peel Processing: A Scientific Review of Drying, Extraction, and Isolation of Its Bioactive Compounds.

This article systematically reviews the advancements in processing litchi peel (Litchi chinensis), emphasizing drying, extraction, purification methods, and the potential of bioactive compounds obtained from litchi peel. This work also highlights the impact of various drying techniques on phytochemical profiles, focusing on how methods such as hot air and freeze-drying affect the preservation of bioactive compounds. The study delves into extraction methods, detailing how different solvents and techniques influence the efficiency of extracting bioactive compounds from litchi peel. Furthermore, the purification and characterization of active compounds, showcasing the role of chromatographic techniques in isolating specific bioactive molecules, is discussed. Biological properties and mechanisms of action, such as antioxidant, antihyperglycemic, cardioprotective, hepatoprotective, anti-atherosclerotic, and anticancer activities, are reviewed, providing insight into the potential health benefits of litchi peel compounds. This review highlights the importance of optimizing and selecting accurate drying and extraction methods to maximize the therapeutic effects of litchi peel and its bioactive compounds. This review also reveals the broad pharmacological potential of the isolated compounds, underscoring the need for further research to discover their specific actions and health benefits.

[Risk factors associated with virologic failure in HIV- infected patients receiving antiretroviral therapy at a public hospital in Peru]. / Factores asociados a falla virológica en pacientes infectados con VIH que reciben terapia anti-retroviral en un hospital público del Perú

OBJECTIVE: To describe clinical and biological characteristics of subjects with virologic failure who participated in the sexually transmitted diseases HIV/AIDS National Program from a Peruvian public hospital. MATERIALS AND METHODS: An exploratory descriptive study was performed with data from subjects older than 18 who started high activity antiretroviral therapy (HAART) between May 2004 and December 2009 and who had a viral load control after 24 weeks of HAART. Virologic failure was defined as a viral load value above 1000 copies/mL on follow up after 24 weeks on HAART. RESULTS: Of 1478 records of patients on HAART analyzed, the median age was 35 years [IQR, 29-41] and 69.6% were male. Also, virologic failure occurred in 24% and 3.7% died. Of subjects with virologic failure, 9.5% died. On multivariate analysis, age, history of antiretroviral use before starting HAART, change of antiretroviral therapy due to toxicity, opportunistic infections during HAART, level of CD4 + lymphocytes below 100 cells/ml at start of HAART, adherence and clinical stage were independently associated with virologic failure. In the group of patient with no history of antiretroviral use before starting HAART, age, opportunistic infections during HAART were associated with virologic failure. CONCLUSION: This study identified factors associated with virologic failure. Further studies are needed to evaluate whether the use of these factors can help to identify prospectively patients at high risk of failure, and to design interventions aimed to reduce this risk.

The Olfactory Organ Is a Unique Site for Neutrophils in the Brain.

In the vertebrate olfactory tract new neurons are continuously produced throughout life. It is widely believed that neurogenesis contributes to learning and memory and can be regulated by immune signaling molecules. Proteins originally identified in the immune system have subsequently been localized to the developing and adult nervous system. Previously, we have shown that olfactory imprinting, a specific type of long-term memory, is correlated with a transcriptional response in the olfactory organs that include up-regulation of genes associated with the immune system. To better understand the immune architecture of the olfactory organs we made use of cell-specific fluorescent reporter lines in dissected, intact adult brains of zebrafish to examine the association of the olfactory sensory neurons with neutrophils and blood-lymphatic vasculature. Surprisingly, the olfactory organs contained the only neutrophil populations observed in the brain; these neutrophils were localized in the neural epithelia and were associated with the extensive blood vasculature of the olfactory organs. Damage to the olfactory epithelia resulted in a rapid increase of neutrophils both within the olfactory organs as well as the central nervous system. Analysis of cell division during and after damage showed an increase in BrdU labeling in the neural epithelia and a subset of the neutrophils. Our results reveal a unique population of neutrophils in the olfactory organs that are associated with both the olfactory epithelia and the lymphatic vasculature suggesting a dual olfactory-immune function for this unique sensory system.

A simple quantitative method using TLC-image analysis to determine fructooligosaccharides (FOS) in food samples.

The thin-layer chromatography technique (TLC) is a simple and inexpensive analysis commonly used to identify qualitatively the presence of carbohydrates in food samples such as mono- di and oligosaccharides particularly. TLC assay could be improved using image processing software for the semiquantitative determination of this type of compound. In the present work, TLC-image analysis with Silica Gel 60 TLC plates was used for the semiquantitative determination of 6 standards of carbohydrates (glucose, fructose, sucrose, 1-kestose, nystose, and fructofuranosylnystose). Subsequently, the areas of the spots of each compound were determined by digitizing in a conventional office scanner. Then, the segmentation of the images is carried out using software for image processing. The calibration curves were plotted in the Excel software using the average of the areas of the pigmentations obtained in pixels. In this study, the technique of thin-layer chromatography was also used to quantitatively determine the presence of carbohydrates in food samples such as honey, garlic, and onion. Values of determination coefficient (R2) greater than 0.97 in all the calibration curves were obtained. This technique could be useful for detecting carbohydrates (monosaccharides, disaccharides, and oligosaccharides) in analytical assays and food samples without needing specialized analytical equipment. In this work, it was possible to determine the concentration of carbohydrates in samples of garlic and onion that showed the presence of prebiotic carbohydrates in addition to sucrose, glucose, and fructose.

The secondary metabolites from Beauveria bassiana PQ2 inhibit the growth and spore germination of Gibberella moniliformis LIA.

Fungal secondary metabolites with antimicrobial properties are used for biological pest control. Their production is influenced by several factors as environment, host, and culture conditions. In the present work, the secondary metabolites from fermented extracts of Beauveria bassiana PQ2 were tested as antifungal agents against Gibberella moniliformis LIA. The L18 (21 × 37) orthogonal array from Taguchi methodology was used to assess 8 parameters (pH, agitation, sucrose, yeast extract, KH2PO4, MgSO4, NH4NO3, and CaCl2) in B. bassiana PQ2 submerged fermentation. The ability of the fermented extracts to slow down the growth rate of G. moniliformis LIA was evaluated. The results from 18 trials were analyzed by Statistica 7 software by evaluating the signal-to-noise ratio (S/N) to find the lower-the-better condition. Optimal culture conditions were pH, 5; agitation, 250 rpm; sucrose, 37.5 g/L-1; yeast extract, 10 g/L-1; KH2PO4, 0.8 g/L-1; MgSO4, 1.2 g/L-1; NH4NO3, 0.1 g/L-1; and CaCl2, 0.4 g/L-1, being the agitation at the highest level the most significant factor. The optimal conditions were validated in a sparged bottle bioreactor resulting in a higher S/N value (12.48) compared to the estimate. The extract obtained has the capacity to inhibit the germination of G. moniliformis spores at 24 h. HPLC-ESI-MS2 allowed to identify the water-soluble red pigment as oosporein (m/z 304.9). The secondary metabolites from B. bassiana PQ2 are a suitable alternative to control the growth and sporulation of G. moniliformis.

Pescoids and Chimeras to Probe Early Evo-Devo in the Fish Astyanax mexicanus.

The fish species Astyanax mexicanus with its sighted and blind eco-morphotypes has become an original model to challenge vertebrate developmental evolution. Recently, we demonstrated that phenotypic evolution can be impacted by early developmental events starting from the production of oocytes in the fish ovaries. A. mexicanus offers an amenable model to test the influence of maternal determinants on cell fate decisions during early development, yet the mechanisms by which the information contained in the eggs is translated into specific developmental programs remain obscure due to the lack of specific tools in this emergent model. Here we describe methods for the generation of pescoids from yolkless-blastoderm explants to test the influence of embryonic and extraembryonic tissues on cell fate decisions, as well as the production of chimeric embryos obtained by intermorph cell transplantations to probe cell autonomous or non-autonomous processes. We show that Astyanax pescoids have the potential to recapitulate the main ontogenetic events observed in intact embryos, including the internalization of mesodermal progenitors and eye development, as followed with zic:GFP reporter lines. In addition, intermorph cell grafts resulted in proper integration of exogenous cells into the embryonic tissues, with lineages becoming more restricted from mid-blastula to gastrula. The implementation of these approaches in A. mexicanus will bring new light on the cascades of events, from the maternal pre-patterning of the early embryo to the evolution of brain regionalization.

Clinical diagnosis of COVID-19. A multivariate logistic regression analysis of symptoms of COVID-19 at presentation.

INTRODUCTION: The objective of this cross-sectional study was to describe the main symptoms associated with COVID-19, and their diagnostic characteristics, to aid in the clinical diagnosis. METHODS: An analysis of all patients diagnosed by RT-PCR for SARS-CoV-2 between April and May 2020 in Argentina was conducted. The data includes clinical and demographic information from all subjects at the time of presentation (n=67318, where 12% were positive for SARS-CoV-2). The study population was divided into four age groups: pediatric (0-17 years), young adults (18-44 years), adults (45-64 years), and elderly (65-103 years). Multivariate logistic regression was used to measure the association of all symptoms and to create a diagnostic model based on symptoms. RESULTS: Symptoms associated with COVID-19 were anosmia, dysgeusia, headache, low-grade fever, odynophagia, and malaise. However, the presentation of these symptoms was different between the different age groups. In turn, at the time of presentation, the symptoms associated with respiratory problems (chest pain, abdominal pain, and dyspnea) had a negative association with COVID-19 or did not present statistical relevance. On the other hand, the model based on 16 symptoms, age and sex, presented a sensitivity of 80% and a specificity of 46%. CONCLUSIONS: There were significant differences between the different age groups. Additionally, there were interactions between different symptoms that were highly associated with COVID-19. Finally, our findings showed that a regression model based on multiple factors (age, sex, interaction between symptoms) can be used as an accessory diagnostic method or a rapid screening of suspected COVID-19 cases.

Dissecting the neural divide: a continuous neurectoderm gives rise to the olfactory placode and bulb.

The olfactory epithelia arise from morphologically identifiable structures called olfactory placodes. Sensory placodes are generally described as being induced from the ectoderm suggesting that their development is separate from the coordinated cell movements generating the central nervous system. Previously, we have shown that the olfactory placodes arise from a large field of cells bordering the telencephalic precursors in the neural plate, and that cell movements, not cell division, underlie olfactory placode morphogenesis. Subsequently by image analysis, cells were tracked as they moved in the continuous sheet of neurectoderm giving rise to the peripheral (olfactory organs) and central (olfactory bulbs) nervous system (Torres-Paz and Whitlock, 2014). These studies lead to a model whereby the olfactory epithelia develop from within the border of the neural late and are a neural tube derivative, similar to the retina of the eye (Torres-Paz and Whitlock, 2014; Whitlock, 2008). Here we show that randomly generated clones of cells extend across the morphologically differentiated olfactory placodes/olfactory bulbs, and test the hypothesis that these structures are patterned by a different level of distal-less (dlx) gene expression subdividing the anterior neurectoderm into OP precursors (high Dlx expression) and OB precursors (lower Dlx expression). Manipulation of DLX protein and RNA levels resulted in morphological changes in the size of the olfactory epithelia and olfactory bulb. Thus, the olfactory epithelia and bulbs arise from a common neurectodermal region and develop in concert through coordinated morphological movements.

Structural characterization of native and oxidized procyanidins (condensed tannins) from coffee pulp (Coffea arabica) using phloroglucinolysis and thioglycolysis-HPLC-ESI-MS.

Procyanidins from coffee pulp are responsible from the limited valorization of this by-product. Information about procyanidin structure is still scarce and imprecise. The aim of this work was to study the native and oxidized procyanidins from coffee pulp with respect to composition and structure. An aqueous acetone extract from coffee pulp was purified using Sephadex LH-20. Butanolysis, phloroglucinolysis and thioglycolysis coupled to HLPC-ESI-MS were applied for the characterization of the native and oxidized procyanidins. The purification allowed to recovery three fractions (aqueous, ethanolic and acetonic) and only acetone fraction showed a high concentration of procyanidins (98%, w/w). HPLC-ESI-MS of procyanidins-rich fraction without any reaction resulted in a UV-Vis chromatogram unresolved typical of the presence of procyanidins. The extracted ion chromatogram and MS2 analysis revealed the presence from dimers to pentamers of native procyanidins. Interestingly, by first time an A-type trimeric procyanidin (m/z of 863) was observed in coffee pulp. In our study, (-)-epicatechin was the constitutive unit of procyanidins with an aDP of 6.8 (oligomeric native procyanidins) according to the phloroglucinolysis assay. Two oxidation markers useful to characterization of oxidized procyanidins were observed in the procyanidins-rich fraction after thioglycolysis, a dimer A2-ext and a molecule that corresponds to a linkage between an extension and a terminal unit. Coffee pulp procyanidins were presented with only a minor class of oxidized procyanidins. As far as we know, this is the first study about characterization of the oxidized procyanidins from coffee pulp.

Procyanidins: From Agro-Industrial Waste to Food as Bioactive Molecules.

Procyanidins are an important group of bioactive molecules known for their benefits to human health. These compounds are promising in the treatment of chronic metabolic diseases such as cancer, diabetes, and cardiovascular disease, as they prevent cell damage related to oxidative stress. It is necessary to study effective extraction methods for the recovery of these components. In this review, advances in the recovery of procyanidins from agro-industrial wastes are presented, which are obtained through ultrasound-assisted extraction, microwave-assisted extraction, supercritical fluid extraction, pressurized fluid extraction and subcritical water extraction. Current trends focus on the extraction of procyanidins from seeds, peels, pomaces, leaves and bark in agro-industrial wastes, which are extracted by ultrasound. Some techniques have been coupled with environmentally friendly techniques. There are few studies focused on the extraction and evaluation of biological activities of procyanidins. The identification and quantification of these compounds are the result of the study of the polyphenolic profile of plant sources. Antioxidant, antibiotic, and anti-inflammatory activity are presented as the biological properties of greatest interest. Agro-industrial wastes can be an economical and easily accessible source for the extraction of procyanidins.