A Real-Time PCR Assay to Detect and Quantify Root-Knot Nematodes from Soil Extracts.

Root-knot nematodes cause forking and stubbing of the growing carrot root tip, decreasing market value and reducing yield by up to 45%. Since crop damage by these nematodes depends on their initial population densities at planting, preplant detection of potentially low nematode numbers is critical for predicting future yield loss. The aim of this study was to overcome some of the drawbacks of the labor- and time-intensive process of root-knot nematode identification and quantification by developing and field testing a real-time PCR (qPCR) assay. Primers were designed targeting the root-knot nematode Meloidogyne incognita species complex, which includes M. incognita as well as the closely related Meloidogyne javanica and Meloidogyne arenaria. The qPCR assay successfully detected each species and showed little amplification for nontarget nematode groups except for the sister group Meloidogyne enterolobii, which is not known to occur in California. Predicted nematode densities related well with microscopic counts of nematodes from prepared solutions, as well as from solutions extracted from field soil. In a greenhouse experiment, the qPCR assay distinguished between low, medium, and high levels of M. incognita infection and qPCR predicted densities at planting were negatively related in linear models with final carrot fresh weight, length, and diameter. These results suggest that qPCR assays could be a valuable diagnostic tool to predict nematode infections and prevent crop losses.[Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Cytoglobin deficiency potentiates Crb1-mediated retinal degeneration in rd8 mice.

PURPOSE: The purpose of this study is to determine the effect of Cytoglobin (Cygb) deficiency on Crb1-related retinopathy. The Crb1 cell polarity complex is required for photoreceptor function and survival. Crb1-related retinopathies encompass a broad range of phenotypes which are not completely explained by the variability of Crb1 mutations. Genes thought to modify Crb1 function are therefore important targets of research. The biological function of Cygb involves oxygen delivery, scavenging of reactive oxygen species, and nitric oxide metabolism. However, the relationship of Cygb to diseases involving the Crb1 cell polarity complex is unknown. METHODS: Cygb knockout mice homozygous for the rd8 mutation (Cygb-/-rd8/rd8) were screened for ocular abnormalities and imaged using optical coherence tomography and fundus photography. Electroretinography was performed, as was histology and immunohistochemistry. Quantitative PCR was used to determine the effect of Cygb deficiency on transcription of Crb1 related cell polarity genes. RESULTS: Cygb-/-rd8/rd8 mice develop an abnormal retina with severe lamination abnormalities. The retina undergoes progressive degeneration with the ventral retina more severely affected than the dorsal retina. Cygb expression is in neurons of the retinal ganglion cell layer and inner nuclear layer. Immunohistochemical studies suggest that cell death predominates in the photoreceptors. Electroretinography amplitudes show reduced a- and b-waves, consistent with photoreceptor disease. Cygb deficient retinas had only modest transcriptional perturbations of Crb1-related cell polarity genes. Cygb-/- mice without the rd8 mutation did not exhibit obvious retinal abnormalities. CONCLUSIONS: Cygb is necessary for retinal lamination, maintenance of cell polarity, and photoreceptor survival in rd8 mice. These results are consistent with Cygb as a disease modifying gene in Crb1-related retinopathy. Further studies are necessary to investigate the role of Cygb in the human retina.

Ovarian cholesterol efflux: ATP-binding cassette transporters and follicular fluid HDL regulate cholesterol content in mouse oocytes†.

High density lipoproteins (HDL) take up cholesterol from peripheral tissues via ABC transporters and deliver it to the liver via scavenger receptor class B type I (SR-B1). HDL are the main lipoproteins present in follicular fluid (FF). They are thought to derive from plasma, but their origin is still controversial. SR-B1 knock-out (KO) mice have provided important evidence linking HDL metabolism and female fertility. These mice have cholesterol-rich circulating HDL and female infertility that can be restored by treating mice with the cholesterol-lowering drug probucol. Ovulated oocytes from SR-B1 KO females are dysfunctional and show excess cholesterol. The mechanisms explaining the contribution of FF HDL to oocyte cholesterol homeostasis are unknown. Here, using quantitation of filipin fluorescence we show that in SR-B1 KO ovaries, cholesterol excess is first observed in immature oocytes in antral follicles. By performing cross-transplant experiments between WT and apolipoprotein A-I deficient (ApoA-I KO) mice, which lack the main protein component of HDL, we provide evidence supporting the plasmatic origin of FF HDL. Also, we demonstrate that probucol treatment in SR-B1 KO females results in lowering of cholesterol content in their oocytes. Incubation of oocytes from SR-B1 KO mice with purified WT HDL reduces their cholesterol content, suggesting that HDL promote efflux of excess cholesterol from oocytes. In agreement with this hypothesis, we identified ABC transporters in oocytes and observed that ABCA1 KO oocytes have excess cholesterol and lower viability than WT oocytes.

Transcriptional profiling of embryos lacking the lipoprotein receptor SR-B1 reveals a regulatory circuit governing a neurodevelopmental or metabolic decision during neural tube closure.

BACKGROUND: The high-density lipoprotein receptor SR-B1 mediates cellular uptake of several lipid species, including cholesterol and vitamin E. During early mouse development, SR-B1 is located in the maternal-fetal interface, where it facilitates vitamin E transport towards the embryo. Consequently, mouse embryos lacking SR-B1 are vitamin E-deficient, and around half of them fail to close the neural tube and show cephalic neural tube defects (NTD). Here, we used transcriptomic profiling to identify the molecular determinants of this phenotypic difference between SR-B1 deficient embryos with normal morphology or with NTD. RESULTS: We used RNA-Seq to compare the transcriptomic profile of three groups of embryos retrieved from SR-B1 heterozygous intercrosses: wild-type E9.5 embryos (WT), embryos lacking SR-B1 that are morphologically normal, without NTD (KO-N) and SR-B1 deficient embryos with this defect (KO-NTD). We identified over 1000 differentially expressed genes: down-regulated genes in KO-NTD embryos were enriched for functions associated to neural development, while up-regulated genes in KO-NTD embryos were enriched for functions related to lipid metabolism. Feeding pregnant dams a vitamin E-enriched diet, which prevents NTD in SR-B1 KO embryos, resulted in mRNA levels for those differentially expressed genes that were more similar to KO-N than to KO-NTD embryos. We used gene regulatory network analysis to identify putative transcriptional regulators driving the different embryonic expression profiles, and identified a regulatory circuit controlled by the androgen receptor that may contribute to this dichotomous expression profile in SR-B1 embryos. Supporting this possibility, the expression level of the androgen receptor correlated strongly with the expression of several genes involved in neural development and lipid metabolism. CONCLUSIONS: Our analysis shows that normal and defective embryos lacking SR-B1 have divergent expression profiles, explained by a defined set of transcription factors that may explain their divergent phenotype. We propose that distinct expression profiles may be relevant during early development to support embryonic nutrition and neural tube closure.

A Quinoa Protein Hydrolysate Fractionated by Electrodialysis with Ultrafiltration Membranes Improves Maternal and Fetal Outcomes in a Mouse Model of Gestational Diabetes Mellitus.

SCOPE: Quinoa intake exerts hypoglycemic and hypolipidemic effects in animals and humans. Although peptides from quinoa inhibit key enzymes involved in glucose homeostasis in vitro, their in vivo antidiabetic properties have not been investigated. METHODS AND RESULTS: This study evaluated the effect of oral administration of a quinoa protein hydrolysate (QH) produced through enzymatic hydrolysis and fractionation by electrodialysis with ultrafiltration membrane (EDUF) (FQH) on the metabolic and pregnancy outcomes of Lepdb/+ pregnant mice, a preclinical model of gestational diabetes mellitus. The 4-week pregestational consumption of 2.5 mg mL-1 of QH in water prevented glucose intolerance and improves hepatic insulin signaling in dams, also reducing fetal weights. Sequencing and bioinformatic analyses of the defatted FQH (FQHD) identified 11 peptides 6-10 amino acids long that aligned with the quinoa proteome and exhibited putative anti-dipeptidyl peptidase-4 (DPP-IV) activity, confirmed in vitro in QH, FQH, and FDQH fractions. Peptides homologous to mouse and human proteins enriched for biological processes related to glucose metabolism are also identified. CONCLUSION: Processing of quinoa protein may be used to develop a safe and effective nutritional intervention to control glucose intolerance during pregnancy. Further studies are required to confirm if this nutritional intervention is applicable to pregnant women.

Implications of High-Density Cholesterol Metabolism for Oocyte Biology and Female Fertility.

Cholesterol is an essential component of animal cells. Different regulatory mechanisms converge to maintain adequate levels of this lipid because both its deficiency and excess are unfavorable. Low cell cholesterol content promotes its synthesis and uptake from circulating lipoproteins. In contrast, its excess induces the efflux to high-density lipoproteins (HDL) and their transport to the liver for excretion, a process known as reverse cholesterol transport. Different studies suggest that an abnormal HDL metabolism hinders female fertility. HDL are the only lipoproteins detected in substantial amounts in follicular fluid (FF), and their size and composition correlate with embryo quality. Oocytes obtain cholesterol from cumulus cells via gap junctions because they cannot synthesize cholesterol de novo and lack HDL receptors. Recent evidence has supported the possibility that FF HDL play a major role in taking up excess unesterified cholesterol (UC) from the oocyte. Indeed, genetically modified mouse models with disruptions in reverse cholesterol transport, some of which show excessive circulating UC levels, exhibit female infertility. Cholesterol accumulation can affect the egg´s viability, as reported in other cell types, and activate the plasma membrane structure and activity of membrane proteins. Indeed, in mice deficient for the HDL receptor Scavenger Class B Type I (SR-B1), excess circulating HDL cholesterol and UC accumulation in oocytes impairs meiosis arrest and hinders the developmental capacity of the egg. In other cells, the addition of cholesterol activates calcium channels and dysregulates cell death/survival signaling pathways, suggesting that these mechanisms may link altered HDL cholesterol metabolism and infertility. Although cholesterol, and lipids in general, are usually not evaluated in infertile patients, one study reported high circulating UC levels in women showing longer time to pregnancy as an outcome of fertility. Based on the evidence described above, we propose the existence of a well-regulated and largely unexplored system of cholesterol homeostasis controlling traffic between FF HDL and oocytes, with significant implications for female fertility.

Expression of RPRM/rprm in the Olfactory System of Embryonic Zebrafish (Danio rerio).

The Reprimo (RPRM) family is composed of highly conserved single-exon genes. The expression pattern of this gene family has been recently described during zebrafish (Danio rerio) embryogenesis, and primarily locates in the nervous system. Its most characterized member, RPRM, which duplicated to give rise rprma and rprmb in the fish lineage, is known to act as a tumor-suppressor gene in mammalian models. Here, we describe in detail the spatiotemporal expression of three rprm genes (rprma, rprmb, and rprml) within distinct anatomical structures in the developing peripheral and central nervous system. In the zebrafish, rprma mRNA is expressed in the olfactory placodes (OP) and olfactory epithelium (OE), rprmb is observed in the tectum opticum (TeO) and trigeminal ganglion (Tg), whereas rprml is found primarily in the telencephalon (Tel). At protein level, RPRM is present in a subset of cells in the OP, and neurons in the OE, TeO, hindbrain and sensory peripheral structures. Most importantly, the expression of RPRM has been conserved between teleosts and mammals. Thus, we provide a reference dataset describing the expression patterns of RPRM gene products during zebrafish and mouse development as a first step to approach the physiological role of the RPRM gene family.

Deficient Vitamin E Uptake During Development Impairs Neural Tube Closure in Mice Lacking Lipoprotein Receptor SR-BI.

SR-BI is the main receptor for high density lipoproteins (HDL) and mediates the bidirectional transport of lipids, such as cholesterol and vitamin E, between these particles and cells. During early development, SR-BI is expressed in extraembryonic tissue, specifically in trophoblast giant cells in the parietal yolk sac. We previously showed that approximately 50% of SR-BI-/- embryos fail to close the anterior neural tube and develop exencephaly, a perinatal lethal condition. Here, we evaluated the role of SR-BI in embryonic vitamin E uptake during murine neural tube closure. Our results showed that SR-BI-/- embryos had a very low vitamin E content in comparison to SR-BI+/+ embryos. Whereas SR-BI-/- embryos with closed neural tubes (nSR-BI-/-) had high levels of reactive oxygen species (ROS), intermediate ROS levels between SR-BI+/+ and nSR-BI-/- embryos were detected in SR-BI-/- with NTD (NTD SR-BI-/-). Reduced expression of Pax3, Alx1 and Alx3 genes was found in NTD SR-BI-/- embryos. Maternal α-tocopherol dietary supplementation prevented NTD almost completely (from 54% to 2%, p < 0.001) in SR-BI-/- embryos and normalized ROS and gene expression levels. In sum, our results suggest the involvement of SR-BI in the maternal provision of embryonic vitamin E to the mouse embryo during neural tube closure.

Early onset intrauterine growth restriction in a mouse model of gestational hypercholesterolemia and atherosclerosis.

The susceptibility to develop atherosclerosis is increased by intrauterine growth restriction and prenatal exposure to maternal hypercholesterolemia. Here, we studied whether mouse gestational hypercholesterolemia and atherosclerosis affected fetal development and growth at different stages of gestation. Female LDLR KO mice fed a proatherogenic, high cholesterol (HC) diet for 3 weeks before conception and during pregnancy exhibited a significant increase in non-HDL cholesterol and developed atherosclerosis. At embryonic days 12.5 (E12.5), E15.5, and E18.5, maternal gestational hypercholesterolemia and atherosclerosis were associated to a 22-24% reduction in male and female fetal weight without alterations in fetal number/litter or morphology nor placental weight or structure. Feeding the HC diet exclusively at the periconceptional period did not alter fetal growth, suggesting that maternal hypercholesterolemia affected fetal weight only after implantation. Vitamin E supplementation (1,000 UI of α-tocopherol/kg) of HC-fed females did not change the mean weight of E18.5 fetuses but reduced the percentage of fetuses exhibiting body weights below the 10th percentile of weight (HC: 90% vs. HC/VitE: 68%). In conclusion, our results showed that maternal gestational hypercholesterolemia and atherosclerosis in mice were associated to early onset fetal growth restriction and that dietary vitamin E supplementation had a beneficial impact on this condition.