Author Archives:

Hybrid breeding for fall armyworm resistance: Combining ability and hybrid prediction

Posted on by

Abstract

Fall armyworm (FAW, Spodoptera frugiperda) emerged as a major lepidopteran pest destroying maize in sub-Saharan Africa. A diallel mating design was used to generate 210 experimental hybrids from 21 lines. Experimental hybrids and four checks were evaluated in two locations. Commercial checks suffered higher foliar and ear damage compared to the top 15 hybrids. Mean squares associated with the genotypic variation were higher than genotype-by-environment interactions for foliar and ear damage traits. Heritabilities were moderate to high. Significant correlations were observed between grain yield (GY) with ear rot (−0.54) and ear damage (−0.45). Positive and significant GCA effects were observed for GY in seven parental lines, which were developed from multiple insect resistance breeding programmes. CKSBL10153 has the highest GCA value for GY and shows significant GCA effects for foliar and ear damage traits. These lines were identified as the ideal combiners for GY and FAW resistance and are therefore recommended for utilization as testers in the development of FAW-resistant three-way cross-hybrid maize with correlated response for increased GY. GCA and marker-based prediction correlations of GY were 0.79 and 0.96, respectively. Both GCA effects and marker-based models were effective in predicting hybrid performance for FAW resistance.

Designing optimal training sets for genomic prediction using adversarial validation with probit regression

Posted on by

Abstract

Genomic selection (GS) is a disruptive methodology that is revolutionizing animal and plant breeding. However, its practical implementation is challenging since many times there is a mismatch in the distribution of the training and testing sets. Adversarial validation is an approach popular in machine learning to detect and address the difference between the training and testing distributions. For this reason, the adversarial validation method in this research was implemented using probit regression to detect the mismatch in distributions and also to select an optimal training set. We evaluated the proposed method with 14 datasets, and the results were benchmarked regarding of using the whole reference population and simple random samples. We found that the proposed method is effective for detecting the mismatch in distributions and outperformed in prediction accuracy by 11.67% (in terms of mean square error) and by 5.35% (in terms of normalized mean square error) when the whole reference population was used as training sets. Also, in general, this outperformed some existing methods for optimal training designs in the context of GS.

Identification of potential sources of mungbean yellow mosaic India virus resistance in black gram (Vigna mungo) and expression of antioxidants and R‐genes modulating resistance response in cultivated and its two wild relatives

Posted on by

Abstract

Black gram is one of the most important short duration grain legume, which contributes significantly towards nutritional security and environmental sustainability. The virus specific primers confirms the presence of mungbean yellow mosaic India virus (MYMIV) in representative samples. A total of 27 cultivated and two wild species were found as highly resistant (HR) to MYMIV and validated through molecular markers. The start codon target (SCoT) markers analysis revealed that the SCoT loci, namely, SCoT-4 (2200 bp), SCot-9 (1150/ 1200 bp), SCoT-15 (1150/1100 bp), SCoT-16 (700 bp), SCoT-24 (2500 bp), SCoT-25 (700 bp), SCoT-33 (900/1000 bp), and SCoT-34 (600 bp), were found unique, able to distinguish HR and highly susceptible (HS) genotypes. Biochemical characterization and gene expression profiling revealed the higher expression of antioxidants and R-genes just after pathogen inoculation indicated the activation of defence mechanism in both cultivated and its wild relatives, which modulates the resistant responses in cultivated and wild accessions. These information will be really helpful in accelerating resistance breeding in black gram.

Genetic analysis and identification of SSR marker linked to Phomopsis blight resistance in eggplant (Solanum melongena L.)

Posted on by

Abstract

The present investigation was carried out to decipher inheritance of resistance and to identify linked SSR markers for Phomopsis blight resistance in eggplant. An F2 population comprising 161 plants was developed from the cross of Pusa Kranti and BR-40-7. Genetic analysis was carried out using Chi square test. Artificial inoculation of fruits was carried out using pin prick method, and scoring was done as per the standard scoring scale. The F2 plants segregated into 92 susceptible (77—highly susceptible, 15—susceptible): 69 resistant (17—highly resistant, 27—resistant, 25—moderately resistant) suggesting complimentary epistasis with ratio of 9:7. To identify the putatively linked markers to resistance gene, parental polymorphic markers were subjected to bulk segregant analysis (BSA), and two markers (emk03O04 and emf11A03) could differentiate resistant and susceptible bulk and co-segregated with resistance gene. The genetic distance between the identified markers was found to be 18.12 cM using QTL IciMapping V3.2 software depicting two new QTLs on chromosome number 6. The identified QTLs have great significant importance in marker assisted breeding programme.

In‐Cell Arrestin‐Receptor Interaction Assays

Posted on by

Abstract

G protein-coupled receptors (GPCRs) represent ∼30% of current drug targets. Ligand binding to these receptors activates G proteins and arrestins, which function in different signaling pathways. Given that functionally selective or biased ligands preferentially activate one of these two groups of pathways, they may be superior medications for certain disease states. The identification of such ligands requires robust drug screening assays for both G protein and arrestin activity. This unit describes protocols for assays that monitor reversible arrestin recruitment to GPCRs in living cells using either bioluminescence resonance energy transfer (BRET) or nanoluciferase complementation (NanoLuc). Two types of assays can be used: one configuration directly measures arrestin recruitment to a GPCR fused to a protein tag at its intracellular C-terminus, whereas the other configuration detects arrestin translocation to the plasma membrane in response to activation of an unmodified GPCR. Together, these assays are powerful tools for studying dynamic interactions between GPCRs and arrestins. © 2023 Wiley Periodicals LLC.

Basic Protocol 1: Receptor-arrestin BRET assay to measure ligand-induced recruitment of arrestin to receptors

Basic Protocol 2: Receptor-arrestin NANOBIT assay to measure ligand-induced recruitment of arrestin to receptors

Alternative Protocol 1: BRET assay to measure ligand-induced recruitment of arrestin to the plasma membrane

Alternative Protocol 2: NANOBIT assay to measure ligand-induced recruitment of arrestin to the plasma membrane

Support Protocol 1: Optimization of polyethylenimine (PEI) concentration for transfection

Current Strategies of Modeling Human Trophoblast Using Human Pluripotent Stem Cells in vitro

Posted on by

Abstract

We previously established a trophoblast differentiation protocol from primed human pluripotent stem cells (PSC). To induce this lineage, we use a combination of Bone Morphogenetic Protein-4 (BMP4) and the WNT inhibitor IWP2. This protocol has enabled us to obtain a pure population of trophectoderm (TE)-like cells that could subsequently be terminally differentiated into syncytiotrophoblasts (STB) and extravillous trophoblasts (EVT). However, the resulting TE-like cells could only be terminally differentiated to a variable mixture of STB and EVT, with a bias toward the STB lineage. Recently, methods have been developed for derivation and culture of self-renewing human trophoblast stem cells (TSC) from human embryos and early gestation placental tissues. These primary TSCs were further able to differentiate into either STB or EVT with high efficiency using the lineage specific differentiation protocols. Based partly on these protocols, we have developed methods for establishing self-renewing TSC-like cells from PSC, and for efficient lineage-specific terminal differentiation. Here, we describe in detail the protocols to derive and maintain PSC-TSC, from both embryonic stem cells (ESC) and patient-derived induced pluripotent stem cells (iPSC), and their subsequent terminal differentiation to STB and EVT. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Trophoblast Differentiation into TE-like Cells

Basic Protocol 2: Conversion of PSC-Derived TE-like Cells to TSC

Basic Protocol 3: Passaging PSC-Derived TSC in iCTB Complete Medium

Basic Protocol 4: STB Differentiation from PSC-derived TSC

Basic Protocol 5: EVT Differentiation from PSC-derived TSC

Support Protocol 1: Geltrex-coated tissue culture plate preparation

Support Protocol 2: Collagen IV-coated tissue culture plate preparation

Support Protocol 3: Fibronectin-coated tissue culture plate preparation

Stachydrine, N‐acetylornithine and trimethylamine N‐oxide levels as candidate milk biomarkers of maternal consumption of an obesogenic diet during lactation

Posted on by
Stachydrine, N-acetylornithine and trimethylamine N-oxide levels as candidate milk biomarkers of maternal consumption of an obesogenic diet during lactation

Reverting to a healthy maternal diet during lactation normalizes the altered milk metabolome found in obese rats. Stachydrine, N-acetylornithine and TMAO levels are proposed as candidate biomarkers of maternal consumption of an obesogenic diet during lactation.


Abstract

We aimed to evaluate whether improving maternal diet during lactation in diet-induced obese rats reverts the impact of western diet (WD) consumption on the metabolome of milk and offspring plasma, as well as to identify potential biomarkers of these conditions. Three groups of dams were followed: control-dams (CON-dams), fed with standard diet (SD); WD-dams, fed with WD prior and during gestation and lactation; and reversion-dams (REV-dams), fed as WD-dams but moved to SD during lactation. Metabolomic analysis was performed in milk at lactation days 5, 10, and 15, and in plasma from their male and female offspring at postnatal day 15. Milk of WD-dams presented, throughout lactation and compared to CON-dams, altered profiles of amino acids and of the carnitine pool, accompanied by changes in other polar metabolites, being stachydrine, N-acetylornithine, and trimethylamine N-oxide the most relevant and discriminatory metabolites between groups. The plasma metabolome profile was also altered in the offspring of WD-dams in a sex-dependent manner, and stachydrine, ergothioneine and the acylcarnitine C12:1 appeared as the top three most discriminating metabolites in both sexes. Metabolomic changes were largely normalized to control levels both in the milk of REV-dams and in the plasma of their offspring. We have identified a set of polar metabolites in maternal milk and in the plasma of the offspring whose alterations may indicate maternal intake of an unbalanced diet during gestation and lactation. Levels of these metabolites may also reflect the beneficial effects of implementing a healthier diet during lactation.

Skincare potential of a sustainable postbiotic extract produced through sugarcane straw fermentation by Saccharomyces cerevisiae

Posted on by
Skincare potential of a sustainable postbiotic extract produced through sugarcane straw fermentation by Saccharomyces cerevisiae

Production of a postbiotic extract using sugarcane straw as substrate of fermentation process and Saccharomyces cerevisiae as fermentative microorganism. The extract obtained showed antioxidant and anti-inflammatory activities in skin cells, as well as beneficial for skin microbiota modulation applications. This sustainable produced postbiotic extract can be used for cosmetic and skincare ingredients development


Abstract

Postbiotics are defined as a “preparation of inanimate microorganisms and/or their components that confers a health benefit on the host.” They can be produced by fermentation, using culture media with glucose (carbon source), and lactic acid bacteria of the genus Lactobacillus, and/or yeast, mainly Saccharomyces cerevisiae as fermentative microorganisms. Postbiotics comprise different metabolites, and have important biological properties (antioxidant, anti-inflammatory, etc.), thus their cosmetic application should be considered. During this work, the postbiotics production was carried out by fermentation with sugarcane straw, as a source of carbon and phenolic compounds, and as a sustainable process to obtain bioactive extracts. For the production of postbiotics, a saccharification process was carried out with cellulase at 55°C for 24 h. Fermentation was performed sequentially after saccharification at 30°C, for 72 h, using S. cerevisiae. The cells-free extract was characterized regarding its composition, antioxidant activity, and skincare potential. Its use was safe at concentrations below ~20 mg mL−1 (extract's dry weight in deionized water) for keratinocytes and ~ 7.5 mg mL−1 for fibroblasts. It showed antioxidant activity, with ABTS IC50 of 1.88 mg mL−1, and inhibited elastase and tyrosinase activities by 83.4% and 42.4%, respectively, at the maximum concentration tested (20 mg mL−1). In addition, it promoted the production of cytokeratin 14, and demonstrated anti-inflammatory activity at a concentration of 10 mg mL−1. In the skin microbiota of human volunteers, the extract inhibited Cutibacterium acnes and the Malassezia genus. Shortly, postbiotics were successfully produced using sugarcane straw, and showed bioactive properties that potentiate their use in cosmetic/skincare products.

Torpor‐responsive microRNAs in the heart of the Monito del monte, Dromiciops gliroides

Posted on by
Torpor-responsive microRNAs in the heart of the Monito del monte, Dromiciops gliroides

Monito del monte (Dromiciops gliroides) undergoes metabolic rate depression to conserve energy during torpor. Differential microRNA expression plays a key role in metabolic reorganization by regulating key signaling and metabolic pathways.


Abstract

The marsupial Monito del monte (Dromiciops gliroides) utilizes both daily and seasonal bouts of torpor to preserve energy and prolong survival during periods of cold and unpredictable food availability. Torpor involves changes in cellular metabolism, including specific changes to gene expression that is coordinated in part, by the posttranscriptional gene silencing activity of microRNAs (miRNA). Previously, differential miRNA expression has been identified in D. gliroides liver and skeletal muscle; however, miRNAs in the heart of Monito del monte remained unstudied. In this study, the expression of 82 miRNAs was assessed in the hearts of active and torpid D. gliroides, finding that 14 were significantly differentially expressed during torpor. These 14 miRNAs were then used in bioinformatic analyses to identify Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways that were predicted to be most affected by these differentially expressed miRNAs. Overexpressed miRNAs were predicted to primarily regulate glycosaminoglycan biosynthesis, along with various signaling pathways such as Phosphoinositide-3-kinase/protein kinase B and transforming growth factor-β. Similarly, signaling pathways including phosphatidylinositol and Hippo were predicted to be regulated by the underexpression of miRNAs during torpor. Together, these results suggest potential molecular adaptations that protect against irreversible tissue damage and enable continued cardiac and vascular function despite hypothermia and limited organ perfusion during torpor.

miR‐34a/SIRT1/HIF‐1α axis is involved in cardiac angiogenesis of type 2 diabetic rats: The protective effect of sodium butyrate combined with treadmill exercise

Posted on by
miR-34a/SIRT1/HIF-1α axis is involved in cardiac angiogenesis of type 2 diabetic rats: The protective effect of sodium butyrate combined with treadmill exercise

Sodium butyrate and treadmill exercise, alone or together, significantly reduced the blood glucose levels in type 2 diabetic rats. Combination of sodium butyrate and treadmill exercise, improved oral glucose tolerance test in type 2 diabetic rats. Diabetes increased miR-34a expression and FOXO1 levels, and decreased SIRT1 and HIF-1α levels that were measured by real-time PCR and ELISA method. A combination of sodium butyrate and treadmill exercise significantly decreased miR-34a expression and FOXO1 levels, and improved SIRT1 and HIF-1α levels in the heart tissue of diabetic rats. Sodium butyrate and treadmill exercises, alone or together, increased angiogenesis in the heart tissue of diabetic rats. So, those may be useful in the treatment of diabetes through the mir-34a/SIRT1/FOXO1-HIF-1α pathway.


Abstract

Type 2 diabetes mellitus (T2DM) is one of the most common metabolic disorders worldwide. Recent research has indicated that sodium butyrate (NaB) affects glucose metabolism and exercise has an anti-hyperglycemic effect in diabetes. This study aimed to evaluate the effects of NaB and treadmill exercise on heart angiogenesis through the miR-34a/SIRT1/FOXO1-HIF-1α pathway. Diabetic animals received NaB (400 mg/kg daily, orally) and treadmill exercise for 6 weeks. The effect of NaB and treadmill exercise, alone or combined, on miR-34a expression, SIRT1, FOXO1, HIF-1α levels, and angiogenesis in diabetic heart tissue was measured. Diabetes caused increased miR-34a (p < 0.01) and FOXO1 (p < 0.001) expression levels. Also, SIRT1 (p < 0.001) and HIF-1α (not significant) expression levels were reduced in diabetic rats. NaB and treadmill exercise decreased miR-34a (respectively p < 0.05 and not significant) and FOXO1 (both p < 0.001) expression levels and improved SIRT1 (both not significant) and HIF-1α (respectively p < 0.01 and p < 0.001) levels. Also, NaB combined with treadmill exercise decreased miR-34a (p < 0.001) and FOXO1 (p < 0.001) expression levels, and elevated SIRT1 (p < 0.05) and HIF-1α (p < 0.001) levels in comparison with the diabetic group. NaB and treadmill exercises modulate the expression of miR-34a and the levels of SIRT1, FOXO1, and HIF-1α proteins, thus increasing angiogenesis in the heart tissue of diabetic rats. It can be concluded that NaB and treadmill exercise, alone or combined, may be useful in the treatment of diabetes through the miR-34a/SIRT1/FOXO1-HIF-1α pathway.