Generation of epitope-specific hCG aptamers through a novel targeted selection approach.

Human chorionic gonadotropin (hCG) is a glycoprotein hormone used as a biomarker for several medical conditions, including pregnancy, trophoblastic and nontrophoblastic cancers. Most commercial hCG tests rely on a combination of antibodies, one of which is usually specific to the C-terminal peptide of the ß-subunit. However, cleavage of this region in many hCG degradation variants prevents rapid diagnostic tests from quantifying all hCG variants in serum and urine samples. An epitope contained within the core fragment, ß1, represents an under-researched opportunity for developing immunoassays specific to most variants of hCG. In the study described here, we report on a SELEX procedure tailored towards the identification of two pools of aptamers, one specific to the ß-subunit of hCG and another to the ß1 epitope within it. The described SELEX procedure utilized antibody-blocked targets, which is an underutilized strategy to exert negative selection pressure and in turn direct aptamer enrichment to a specific epitope. We report on the first aptamers, designated as R4_64 and R6_5, each capable of recognising two distinct sites of the hCG molecule-the ß-subunit and the (presumably) ß1-epitope, respectively. This study therefore presents a new SELEX approach and the generation of novel aptamer sequences that display potential hCG-specific biorecognition.

Natural deep eutectic solvent (NADES)-based blueberry extracts protect against ethanol-induced gastric ulcer in rats.

Blueberry is a polyphenol-rich fruit bearing great bioactive potential. Natural deep eutectic solvents (NADES) emerged as putatively biocompatible solvents that could substitute for toxic organic solvents in the extraction of fruit phenolic compounds for developing nutraceuticals or functional foods. Therefore, the aim of this study was to investigate the gastroprotective effects and the biocompatibility of a blueberry crude extract (CE) obtained using NADES and of the extract fractions (anthocyanin-rich fraction - ARF; non-anthocyanin phenolic fraction - NAPF) in a model of ethanol-induced gastric ulcer in rats. CE was the NADES-containing, ready-to-use extract that was obtained using choline chloride:glycerol:citric acid NADES (0.5:2:0.5 M ratio). ARF and NAPF were the NADES-free fractions obtained by solid phase purification of CE and were investigated to identify the bioactive fraction responsible for the effects of CE. Animals were treated for 14 days with water, NADES vehicle, CE, ARF, NAPF or lansoprazole (intragastric) and then received ethanol to induce gastric ulcer. CE decreased ulcer index and preserved the integrity of gastric mucosa. The pretreatment with CE or ARF reduced glutathione depletion and the inflammatory response. All treatments, including NADES vehicle reduced protein oxidation and nitric oxide overproduction in ethanol-treated rats. Additionally, ARF increased short-chain fatty acids in feces. These findings suggest that NADES can be used to obtain biocompatible extracts of blueberry that exhibit gastroprotective effects with no need of solvent removal. The gastroprotective effects were mainly associated to ARF but NAPF and even NADES vehicle also contributed to some protective effects.

Single-cell RNA-sequencing uncovers transcriptional states and fate decisions in haematopoiesis.

The success of marker-based approaches for dissecting haematopoiesis in mouse and human is reliant on the presence of well-defined cell surface markers specific for diverse progenitor populations. An inherent problem with this approach is that the presence of specific cell surface markers does not directly reflect the transcriptional state of a cell. Here, we used a marker-free approach to computationally reconstruct the blood lineage tree in zebrafish and order cells along their differentiation trajectory, based on their global transcriptional differences. Within the population of transcriptionally similar stem and progenitor cells, our analysis reveals considerable cell-to-cell differences in their probability to transition to another committed state. Once fate decision is executed, the suppression of transcription of ribosomal genes and upregulation of lineage-specific factors coordinately controls lineage differentiation. Evolutionary analysis further demonstrates that this haematopoietic programme is highly conserved between zebrafish and higher vertebrates.

Loss of the homologous recombination gene rad51 leads to Fanconi anemia-like symptoms in zebrafish.

RAD51 is an indispensable homologous recombination protein, necessary for strand invasion and crossing over. It has recently been designated as a Fanconi anemia (FA) gene, following the discovery of two patients carrying dominant-negative mutations. FA is a hereditary DNA-repair disorder characterized by various congenital abnormalities, progressive bone marrow failure, and cancer predisposition. In this report, we describe a viable vertebrate model of RAD51 loss. Zebrafish rad51 loss-of-function mutants developed key features of FA, including hypocellular kidney marrow, sensitivity to cross-linking agents, and decreased size. We show that some of these symptoms stem from both decreased proliferation and increased apoptosis of embryonic hematopoietic stem and progenitor cells. Comutation of p53 was able to rescue the hematopoietic defects seen in the single mutants, but led to tumor development. We further demonstrate that prolonged inflammatory stress can exacerbate the hematological impairment, leading to an additional decrease in kidney marrow cell numbers. These findings strengthen the assignment of RAD51 as a Fanconi gene and provide more evidence for the notion that aberrant p53 signaling during embryogenesis leads to the hematological defects seen later in life in FA. Further research on this zebrafish FA model will lead to a deeper understanding of the molecular basis of bone marrow failure in FA and the cellular role of RAD51.

Single-cell transcriptome analysis of fish immune cells provides insight into the evolution of vertebrate immune cell types.

The immune system of vertebrate species consists of many different cell types that have distinct functional roles and are subject to different evolutionary pressures. Here, we first analyzed conservation of genes specific for all major immune cell types in human and mouse. Our results revealed higher gene turnover and faster evolution of trans-membrane proteins in NK cells compared with other immune cell types, and especially T cells, but similar conservation of nuclear and cytoplasmic protein coding genes. To validate these findings in a distant vertebrate species, we used single-cell RNA sequencing of lck:GFP cells in zebrafish and obtained the first transcriptome of specific immune cell types in a nonmammalian species. Unsupervised clustering and single-cell TCR locus reconstruction identified three cell populations, T cells, a novel type of NK-like cells, and a smaller population of myeloid-like cells. Differential expression analysis uncovered new immune-cell-specific genes, including novel immunoglobulin-like receptors, and neofunctionalization of recently duplicated paralogs. Evolutionary analyses confirmed the higher gene turnover of trans-membrane proteins in NK cells compared with T cells in fish species, suggesting that this is a general property of immune cell types across all vertebrates.

Oxidative stability during frozen storage of fillets from silver catfish (Rhamdia quelen ) sedated with the essential oil of Aloysia triphylla during transport / Estabilidade oxidativa de filés congelados de jundiás (Rhamdia quelen) sedados com o óleo essencial de Aloysia triphylla durante o transporte

ABSTRACT: This research aimed to evaluate whether the essential oil of Aloysia triphylla (EOAT) used in vivo as a sedative in the water for transporting fish could increase the oxidative stability of silver catfish (Rhamdia quelen) fillets during frozen storage. The chemical composition of EOAT and of fillets from fish exposed to EOAT (0, 30 or 40µL L-1) were assessed. The pH and lipid oxidation parameters (conjugated dienes, CD; thiobarbituric acid-reactive-substances, TBARS) were evaluated in the fillets throughout the storage period (-18±2oC/17 months). The main compounds found in EOAT were α- and β-citral. Treatment with EOAT did not modify the proximate composition of the fillets, but 40µL L-1 EOAT reduced pH levels when compared to the control fillets (P<0.05). Compared to the control fillets, the fillets from fish treated with 30 and 40µL L-1 EOAT had higher initial CD values (P<0.05), whereas fillets from fish treated with 40µL L-1 EOAT had lower TBARS levels after 6, 9 and 17 months of storage (P<0.05). Results indicated that use of EOAT as a sedative in silver catfish transport water delays the degradation of primary oxidation products (CD) into secondary products (TBARS) in the frozen fillets. This delay in the lipid oxidation rate may increase the shelf life of frozen fillets.

RESUMO: O objetivo do trabalho foi avaliar se o uso do óleo essencial de Aloysia triphylla (OEAT) na água de transporte de peixes, in vivo como sedativo, poderia aumentar a estabilidade oxidativa de filés de jundiá (Rhamdia quelen) durante o armazenamento congelado. Avaliou-se a composição química do OEAT e dos filés dos peixes expostos ao OEAT (0, 30 ou 40µL L-1), bem como o pH e indicadores de oxidação lipídica (dienos conjugados, DC; substâncias reativas ao ácido tiobarbitúrico, TBARS) dos filés ao longo do armazenamento (-18±2oC/17 meses). O α- e o β-citral foram os compostos majoritários do OEAT. O tratamento com OEAT não modificou a composição centesimal dos filés de jundiá, mas 40µL L-1 de OEAT reduziu o pH dos filés, comparado ao controle (P<0,05). Foi observado maior teor inicial de DC nos filés dos tratamentos 30 e 40µL L-1 de OEAT e menor valor de TBARS nos filés do tratamento 40µL L-1 de OEAT após 6, 9 e 17 meses de congelamento, em comparação com os filés controle (P<0,05). Os resultados indicam que o uso do OEAT como sedativo na água de transporte de jundiás retarda a degradação de produtos primários da oxidação lipídica (DC) em produtos secundários (TBARS) nos filés congelados. Este retardo na velocidade de oxidação lipídica pode ampliar a vida útil dos filés congelados.

Single-Cell RNA-Sequencing Reveals a Continuous Spectrum of Differentiation in Hematopoietic Cells.

The transcriptional programs that govern hematopoiesis have been investigated primarily by population-level analysis of hematopoietic stem and progenitor cells, which cannot reveal the continuous nature of the differentiation process. Here we applied single-cell RNA-sequencing to a population of hematopoietic cells in zebrafish as they undergo thrombocyte lineage commitment. By reconstructing their developmental chronology computationally, we were able to place each cell along a continuum from stem cell to mature cell, refining the traditional lineage tree. The progression of cells along this continuum is characterized by a highly coordinated transcriptional program, displaying simultaneous suppression of genes involved in cell proliferation and ribosomal biogenesis as the expression of lineage specific genes increases. Within this program, there is substantial heterogeneity in the expression of the key lineage regulators. Overall, the total number of genes expressed, as well as the total mRNA content of the cell, decreases as the cells undergo lineage commitment.

The Ribosome Biogenesis Protein Nol9 Is Essential for Definitive Hematopoiesis and Pancreas Morphogenesis in Zebrafish.

Ribosome biogenesis is a ubiquitous and essential process in cells. Defects in ribosome biogenesis and function result in a group of human disorders, collectively known as ribosomopathies. In this study, we describe a zebrafish mutant with a loss-of-function mutation in nol9, a gene that encodes a non-ribosomal protein involved in rRNA processing. nol9sa1022/sa1022 mutants have a defect in 28S rRNA processing. The nol9sa1022/sa1022 larvae display hypoplastic pancreas, liver and intestine and have decreased numbers of hematopoietic stem and progenitor cells (HSPCs), as well as definitive erythrocytes and lymphocytes. In addition, ultrastructural analysis revealed signs of pathological processes occurring in endothelial cells of the caudal vein, emphasizing the complexity of the phenotype observed in nol9sa1022/sa1022 larvae. We further show that both the pancreatic and hematopoietic deficiencies in nol9sa1022/sa1022 embryos were due to impaired cell proliferation of respective progenitor cells. Interestingly, genetic loss of Tp53 rescued the HSPCs but not the pancreatic defects. In contrast, activation of mRNA translation via the mTOR pathway by L-Leucine treatment did not revert the erythroid or pancreatic defects. Together, we present the nol9sa1022/sa1022 mutant, a novel zebrafish ribosomopathy model, which recapitulates key human disease characteristics. The use of this genetically tractable model will enhance our understanding of the tissue-specific mechanisms following impaired ribosome biogenesis in the context of an intact vertebrate.

Estabilidade lipídica de filés de carpa húngara congelados tratados com extratos de Lippia Alba / Lipid stability of frozen common carp fillets treated with Lippia alba extract

O objetivo do presente estudo foi avaliar o efeito da aspersão de extratos de Lippia alba na estabilidade lipídica de filés de carpa húngara armazenados a -18±2°C. Filés não tratados (controle sem aspersão) ou aspergidos (1mL 10g-1 de filé) com água destilada (controle água destilada) ou com extratos de L. alba (0,10g mL-1) hidrometanólico ou aquoso foram analisados durante o armazenamento nos dias zero, 90 e 180. Independente do tempo de congelamento, o extrato hidrometanólico reduziu os valores de dienos conjugados (DC) dos filés em relação aos demais tratamentos, além de reduzir os valores de ácidos graxos livres aos 90 dias (P<0,05). O extrato aquoso resultou em maior teor de peróxidos após 180 dias de congelamento comparado aos demais tratamentos (P<0,05). Os extratos hidrometanólico e aquoso reduziram os valores de substâncias reativas ao ácido tiobarbitúrico (TBARS) após 180 dias, comparados aos filés tratados com água destilada e sem aspersão (P<0,05). Ambos os extratos de L. alba retardaram a oxidação lipídica, sendo que o extrato aquoso retardou a degradação de produtos primários da oxidação lipídica (peróxidos) em produtos secundários (TBARS), enquanto o extrato hidrometanólico parece ser mais eficiente, pois inibiu de forma similar tanto a formação de DC e peróxidos, quanto a sua degradação em produtos secundários (TBARS).

The effect of sprinkling with Lippia alba extracts was evaluated on the lipid stability of common carp fillets stored at -18±2°C. Fillets that received no treatment (no spray control) or that were sprayed (1mL 10g-1 fillet) with distilled water (water control) or with hydro-methanolic or aqueous extract of L. alba (0.10g mL-1) were evaluated immediately (time zero) and after 90 and 180 days. Regardless of the storage time, the hydro-methanolic extract reduced the conjugated dienes (CD) values of fillets compared to the other treatments, and reduced the free fatty acid levels at 90 days (P<0.05). The aqueous extract caused higher peroxide value after 180 days of frozen storage compared to the other treatments (P<0.05). The hydro-methanolic and aqueous extracts reduced thiobarbituric acid reactive substances (TBARS) values after 180 days compared to the non-treated fillets or to the water-sprayed fillets (P<0.05). Both extracts have delayed lipid oxidation. While the aqueous L. alba extract delayed the degradation of primary oxidation products (peroxides) into secondary products (TBARS), the hydro-methanolic extract was more efficient as it inhibited both the CD and peroxide formation and its degradation into secondary products (TBARS).

Mapping long-range promoter contacts in human cells with high-resolution capture Hi-C.

Transcriptional control in large genomes often requires looping interactions between distal DNA elements, such as enhancers and target promoters. Current chromosome conformation capture techniques do not offer sufficiently high resolution to interrogate these regulatory interactions on a genomic scale. Here we use Capture Hi-C (CHi-C), an adapted genome conformation assay, to examine the long-range interactions of almost 22,000 promoters in 2 human blood cell types. We identify over 1.6 million shared and cell type-restricted interactions spanning hundreds of kilobases between promoters and distal loci. Transcriptionally active genes contact enhancer-like elements, whereas transcriptionally inactive genes interact with previously uncharacterized elements marked by repressive features that may act as long-range silencers. Finally, we show that interacting loci are enriched for disease-associated SNPs, suggesting how distal mutations may disrupt the regulation of relevant genes. This study provides new insights and accessible tools to dissect the regulatory interactions that underlie normal and aberrant gene regulation.

Oxidative stability of refrigerated fish pates containing loquat seed extract / Estabilidade oxidativa de patês de pescado contendo extrato de semente de nêspera armazenados refrigerados

This study investigated the effects of hydroethanolic E. japonica seed extracts (EJSE) as inhibitors of lipid and protein oxidation on fish pates subjected to refrigerated storage. Five fish pate formulations were developed. These formulations included two control pates (water-control and ascorbic acid-control) and three pates with added EJSE (0.1, 0.2 and 0.4g of seed 100g-1 product, equivalent to 3.4, 6.8 or 13.6mg phenolic compounds kg-1 product), which were then stored under refrigeration for 35 days. Conjugated dienes (CD) and peroxide (PV) values increased along with the storage time; however, these values decreased and were similar among all samples at the end of 35 days of analysis (P<0.05). However, the thiobarbituric acid reactive substances levels (TBARS) did not change along the storage and were not affected by the EJSE. Additionally, there was a linear increase in the protein carbonyl content of fish pates over the storage period (P<0.05), but no effect of EJSE on protein oxidation. The results show that, at the concentrations evaluated, hydroethanolic E. japonica seed extract was unable to inhibit or reduce lipid and protein oxidation in fish pates, but the observed phenolic content emphasizes the need for further studies on the wastes of this fruit.

Este trabalho investigou os efeitos do extrato hidroetanólico de semente de E. japonica (EJSE) como inibidor da oxidação lipídica e proteica em patês a base de pescado armazenados refrigerados. Foram desenvolvidas cinco formulações de patê de pescado. Estas formulações incluíram dois patês controles (controle-água e controle-ácido ascórbico) e três adicionados de EJSE (0,1; 0,2 e 0,4g de semente 100g-1 de produto, equivalente a 3,4; 6,8 ou 13,6mg compostos fenólicos kg-1 de produto) que foram armazenados refrigerados durante 35 dias. Os valores de dienos conjugados (CD) e peróxidos (PV) aumentaram ao longo do armazenamento, contudo, CD e PV diminuíram de maneira semelhante em todas as amostras aos 35 dias de análise (P<0,05). No entanto, o conteúdo de substâncias reativas ao ácido tiobarbitúrico (TBARS) não se modificou ao longo do armazenamento e não foi afetado pelo EJSE. Também houve aumento linear no conteúdo de proteínas carboniladas dos patês de pescado ao longo do armazenamento (P<0,05), sem efeito do EJSE na oxidação proteica. Os resultados mostram que, nas concentrações avaliadas, o extrato hidroetanólico de semente de E. japonica não foi capaz de inibir ou reduzir as oxidações lipídicas e proteicas em patês de pescado, mas seu conteúdo fenólico enfatiza para a necessidade de aprofundar as pesquisas com o resíduo desta fruta.

A loss of function screen of identified genome-wide association study Loci reveals new genes controlling hematopoiesis.

The formation of mature cells by blood stem cells is very well understood at the cellular level and we know many of the key transcription factors that control fate decisions. However, many upstream signalling and downstream effector processes are only partially understood. Genome wide association studies (GWAS) have been particularly useful in providing new directions to dissect these pathways. A GWAS meta-analysis identified 68 genetic loci controlling platelet size and number. Only a quarter of those genes, however, are known regulators of hematopoiesis. To determine function of the remaining genes we performed a medium-throughput genetic screen in zebrafish using antisense morpholino oligonucleotides (MOs) to knock down protein expression, followed by histological analysis of selected genes using a wide panel of different hematopoietic markers. The information generated by the initial knockdown was used to profile phenotypes and to position candidate genes hierarchically in hematopoiesis. Further analysis of brd3a revealed its essential role in differentiation but not maintenance and survival of thrombocytes. Using the from-GWAS-to-function strategy we have not only identified a series of genes that represent novel regulators of thrombopoiesis and hematopoiesis, but this work also represents, to our knowledge, the first example of a functional genetic screening strategy that is a critical step toward obtaining biologically relevant functional data from GWA study for blood cell traits.

Using the essential oil of Aloysia triphylla (L'Her.) Britton to sedate silver catfish (Rhamdia quelen) during transport improved the chemical and sensory qualities of the fish during storage in ice.

UNLABELLED: Exposure of silver catfish to 40 µL/L of the essential oil of Aloysia triphylla (AT) during in vivo transport delayed the onset and resolution of rigor mortis as well as the degradation of IMP into HxR compared to the control. The fish that were treated with 30 or 40 µL/L of AT received lower sensory demerit scores after 10 d of storage in ice compared to the control, and the fish that were treated with 40 µL/L of AT had a longer sensory shelf life than did the control. These results indicated that using AT as a sedative in the water in which the silver catfish were transported extended their freshness and increased their shelf life during refrigerated storage. PRACTICAL APPLICATION: Interest in natural anesthetics, such as Aloysia triphylla, has increased in the field of commercial aquaculture because they reduce the number of fish lesions acquired during capture, handling, and transportation. Fish sedated with the essential oil of A. triphylla at 40 µL/L during transport before slaughter exhibited a delay in the loss of freshness and an increased shelf life in ice. In addition to improving animal welfare before slaughter, the essential oil appears to be a promising product for improving fish conservation in the food industry.

Lipid stability during the frozen storage of fillets from silver catfish exposed in vivo to the essential oil of Lippia alba (Mill.) NE Brown.

BACKGROUND: Lippia alba is effective in sedating and reducing stress to fish during transportation. Because some in vitro studies have demonstrated the antioxidant activity of L. alba, we hypothesized that its use in vivo could result in antioxidant effects post mortem. Therefore, in this study we evaluated whether the essential oil of L. alba (EO) used as sedative for fish transport would increase the lipid stability of fillets from silver catfish during frozen storage. RESULTS: The exposure to the EO in vivo did not affect conjugated diene values. However, EO (30 and 40 µL L(-1)) delayed the peak formation of peroxides (from the third to the sixth month of storage) and thiobarbituric reactive substances (from the ninth to the twelfth month of storage) when compared to control fillets. After exposure to 40 µL L(-1) EO the free fatty acid content was higher than for control at the start of fillet storage, with no differences among groups thereafter. CONCLUSION: The essential oil of L. alba used as sedative in the water to transport silver catfish can delay lipid oxidation of fillets during frozen storage. Thus L. alba may be a promising source of natural active compounds for use in aquaculture and the food industry.

Regulation of locomotion and motoneuron trajectory selection and targeting by the Drosophila homolog of Olig family transcription factors.

During the development of locomotion circuits it is essential that motoneurons with distinct subtype identities select the correct trajectories and target muscles. In vertebrates, the generation of motoneurons and myelinating glia depends on Olig2, one of the five Olig family bHLH transcription factors. We investigated the so far unknown function of the single Drosophila homolog Oli. Combining behavioral and genetic approaches, we demonstrate that oli is not required for gliogenesis, but plays pivotal roles in regulating larval and adult locomotion, and axon pathfinding and targeting of embryonic motoneurons. In the embryonic nervous system, Oli is primarily expressed in postmitotic progeny, and in particular, in distinct ventral motoneuron subtypes. oli mediates axonal trajectory selection of these motoneurons within the ventral nerve cord and targeting to specific muscles. Genetic interaction assays suggest that oli acts as part of a conserved transcription factor ensemble including Lim3, Islet and Hb9. Moreover, oli is expressed in postembryonic leg-innervating motoneuron lineages and required in glutamatergic neurons for walking. Finally, over-expression of vertebrate Olig2 partially rescues the walking defects of oli-deficient flies. Thus, our findings reveal a remarkably conserved role of Drosophila Oli and vertebrate family members in regulating motoneuron development, while the steps that require their function differ in detail.