Search Results (25)

ASYMMETRIC LEAVES1 (AS1) and AS2 play essential roles in regulating leaf development in plants. However, their functional roles in soybean remain poorly understood. Here, we identified two members of the soybean AS1 gene family, GmAS1a and GmAS1c, which exhibit high expression levels in stem and leaf tissues. Using the CRISPR/Cas9 system, we targeted four GmAS1 and three GmAS2 genes, generating mutant lines with distinct leaf development phenotypes, including wrinkling (refers to fine lines and creases on the leaf surface, like aged skin texture), curling (describes the inward or outward rolling of leaf edges, deviating from the typical flat shape), and narrow. We found that functional redundancy exists among the four GmAS1 genes in soybean. GmAS1 and GmAS2 cooperatively regulate leaf curling, leaf crinkling phenotypes, and leaf width in soybean, with functional redundancy also observed between these two genes. Transcriptome sequencing analysis of w3 mutant (as1b as1c as1d as2a as2b as2c) identified 1801 differentially expressed genes (DEGs), including 192 transcription factors (TFs). Gene ontology enrichment analysis revealed significant enrichment of DEGs in pathways associated with plant hormone biosynthesis and signal transduction. A detailed examination of the DEGs showed several genes involved in the development of leaf lateral organs, such as KNOX (SHOOT MERISTEMLESS (STM), KNAT1, KNAT2, and KNAT6), LOB (LBD25, LBD30), and ARP5, were down-regulated in w3/WT (wild-type) comparison. CRISPR/Cas9-mediated targeted mutagenesis of the GmAS1/2 genes significantly impairs leaf development and polarity establishment in soybean, providing valuable germplasm resources and a theoretical framework for future studies on leaf morphogenesis. Full article

Galectin-3 is a multifunctional protein that is associated with diseases of the chorioretinal interface, in which the retinal pigment epithelium (RPE) plays a central role in disease development and progression. Since galectin-3 can function extracellularly as well as intracellularly via different mechanisms, we developed an immortalized human RPE cell line (ARPE-19) with a knockdown for galectin-3 expression (ARPE-19/LGALS3^+/−) using a sgRNA/Cas9 all-in-one expression vector. By Western blot analysis, a reduced galectin-3 expression of approximately 48 to 60% in heterozygous ARPE-19/LGALS3^+/− cells was observed when compared to native controls. Furthermore, ARPE-19/LGALS3^+/− cells displayed a flattened, elongated phenotype with decreased E-cadherin as well as enhanced N-cadherin and α-smooth muscle actin mRNA expression, indicating an epithelial–mesenchymal transition of the cells. Compared to wildtype controls, ARPE-19/LGALS3^+/− cells had significantly reduced metabolic activity to 86% and a substantially decreased proliferation to 73%. Furthermore, an enhanced cell adhesion and a diminished migration of immortalized galectin-3 knockdown RPE cells was observed compared to native ARPE-19 cells. Finally, by Western blot analysis, reduced pAKT, pERK1/2, and β-catenin signaling were detected in ARPE-19/LGALS3^+/− cells when compared to wildtype controls. In summary, in RPE cells, endogenous galectin-3 appears to be essential for maintaining the epithelial phenotype as well as cell biological functions such as metabolism, proliferation, or migration, effects that might be mediated via a decreased activity of the AKT, ERK1/2, and β-catenin signaling pathways. Full article

Lipid metabolism and oxidative stress are major contributors to ocular diseases, including drusen formation and photoreceptor damage. Aster-B, encoded by GRAMD1B, mediates the non-vesicular transport of cholesterol and carotenoids and is highly expressed in the human eye, though its specific ocular functions remain unknown. We investigated Aster-B’s role in ARPE-19 cells, a model of the retinal pigment epithelium (RPE), using CRISPR/dCas9 to generate an Aster-B-expressing cell line. Aster-B expression significantly improved cell survival under oxidative stress induced by hydrogen peroxide (H₂O₂) and was associated with the activation of the p53 and TGFβ signaling pathways, indicating a role in modulating stress responses. To confirm its lipid transport activity, we treated cholesterol-depleted cells with carotenoids and tracked their localization. In Aster-B-expressing cells, carotenoids accumulated in mitochondria, while in control cells, they remained in other cellular compartments. Under oxidative stress, mitochondrial carotenoid levels declined in Aster-B-expressing cells but not in control cells. Interestingly, carotenoids enhanced survival in control cells exposed to H₂O₂ but had a detrimental effect in Aster-B-expressing cells, suggesting that carotenoid function is context and location dependent. These findings highlight Aster-B’s role in coordinating lipid transport and stress responses in the RPE, with implications for oxidative stress-related eye diseases. Full article

Glucose-sensing ChREBP and MondoA are transcriptional factors involved in the lipogenic, inflammatory, and insulin signaling pathways implicated in metabolic disorders; however, limited ocular studies have been conducted on these proteins. We aimed to investigate the potential role of ChREBP in the pathogenesis of diabetic retinopathy (DR). We used diabetic human and mouse retinal cryosections analyzed by immunohistochemistry. qRT-PCR was performed to quantify gene expression. To explore the role of ChREBP in rods, we generated caChREBP^RP mice with constitutively active (ca) ChREBP. These mice underwent retinal functional testing, which was followed by proteomic analysis using LC-MS. Furthermore, ARPE-19 cells were infected with lentiviral particles expressing human ChREBP (ARPE-19^ChREBP) and subjected to global proteomics. Our results demonstrate that both proteins were expressed across the retina, although with distinct distribution patterns: MondoA was more prominently expressed in cones, while ChREBP was broadly expressed throughout the retina. Elevated expression of both proteins was observed in DR. This may have contributed to rod photoreceptor degeneration, as we observed diminished scotopic ERG amplitudes in caChREBP^RP mice at P35. The retinal proteomic landscape revealed a decline in the KEGG pathways associated with phototransduction, amino acid metabolism, and cell adhesion. Furthermore, rod-specific caChREBP induced TXNIP expression. Consistent with altered retinal proteomics, ARPE-19^ChREBP cells exhibit a metabolic shift toward increased glyoxylate signaling, sugar metabolism, and lysosomal activation. Our study demonstrates that ChREBP overexpression causes significant metabolic reprogramming triggering retinal functional loss in mice. Full article

Pepper (Capsicum annuum L.) suffers severe quality and yield loss from oomycete diseases caused by Phytophthora capsici. CaSGT1 was previously determined to positively regulate the immune response of pepper plants against P. capsici, but by which mechanism remains elusive. In the present study, the potential interacting proteins of CaSGT1 were isolated from pepper using a yeast two-hybrid system, among which CaARP1 was determined to interact with CaSGT1 via bimolecular fluorescence complementation (BiFC) and microscale thermophoresis (MST) assays. CaARP1 belongs to the auxin-repressed protein family, which is well-known to function in modulating plant growth. The transcriptional and protein levels of CaARP1 were both significantly induced by infection with P. capsici. Silencing of CaARP1 promotes the vegetative growth of pepper plants and attenuates its disease resistance to P. capsici, as well as compromising the hypersensitive response-like cell death in pepper leaves induced by PcINF1, a well-characterized typical PAMP from P. capsici. Chitin-induced transient expression of CaARP1 in pepper leaves enhanced its disease resistance to P. capsici, which is amplified by CaSGT1 co-expression as a positive regulator. Taken together, our result revealed that CaARP1 plays a dual role in the pepper, negatively regulating the vegetative growth and positively regulating plant immunity against P. capsici in a manner associated with CaSGT1. Full article

The superconductivity of CaC₆ as a function of pressure and Ca isotopic composition was revisited using DFT calculations on a 2c–double hexagonal superlattice. The introduction of superlattices was motivated by previous synchrotron absorption and Raman spectroscopy results on other superconductors that showed evidence of superlattice vibrations at low (THz) frequencies. For CaC₆, superlattices have previously been invoked to explain the ARPES data. A superlattice along the hexagonal c-axis direction is also illustrative of atomic orbital symmetry and periodicity, including bonding and antibonding s-orbital character implied by cosine-modulated electronic bands. Inspection of the cosine band revealed that the cosine function has a small (meV) energy difference between the bonding and antibonding regions, relative to a midpoint non-bonding energy. Fermi surface nesting was apparent in an appropriately folded Fermi surface using a superlattice construct. Nesting relationships identified phonon vectors for the conservation of energy and for phase coherency between coupled electrons at opposite sides of the Fermi surface. A detailed analysis of this Fermi surface nesting provided accurate estimates of the superconducting gaps for CaC₆ with the change in applied pressure. The recognition of superlattices within a rhombohedral or hexagonal representation provides consistent mechanistic insight on superconductivity and electron−phonon coupling in CaC₆. Full article

Breast cancer develops upon sequential acquisition of driver mutations in mammary epithelial cells; however, how these mutations collaborate to transform normal cells remains unclear in most cases. We aimed to reconstitute this process in a particular case. To this end, we combined the activated form of the PI 3-kinase harboring the H1047R mutation with the inactivation of the histone lysine methyl-transferase KMT2D in the non-tumorigenic human mammary epithelial cell line MCF10A. We found that PI 3-kinase activation promoted cell-cycle progression, especially when growth signals were limiting, as well as cell migration, both in a collective monolayer and as single cells. Furthermore, we showed that KMT2D inactivation had relatively little influence on these processes, except for single-cell migration, which KMT2D inactivation promoted in synergy with PI 3-kinase activation. The combination of these two genetic alterations induced expression of the ARPC5L gene that encodes a subunit of the Arp2/3 complex. ARPC5L depletion fully abolished the enhanced migration persistence exhibited by double-mutant cells. Our reconstitution approach in MCF10A has thus revealed both the cell function and the single-cell migration, and the underlying Arp2/3-dependent mechanism, which are synergistically regulated when KMT2D inactivation is combined with the activation of the PI 3-kinase. Full article

In the eye, an increase in galectin-1 is associated with various chorioretinal diseases, in which retinal pigment epithelium (RPE) cells play a crucial role in disease development and progression. Since little is known about the function of endogenous galectin-1 in these cells, we developed a galectin-1-deficient immortalized RPE cell line (ARPE-19-LGALS1^−/−) using a sgRNA/Cas9 all-in-one expression vector and investigated its cell biological properties. Galectin-1 deficiency was confirmed by Western blot analysis and immunocytochemistry. Cell viability and proliferation were significantly decreased in ARPE-19-LGALS1^−/− cells when compared to wild-type controls. Further on, an increased attachment of galectin-1-deficient RPE cells was observed by cell adhesion assay when compared to control cells. The diminished viability and proliferation, as well as the enhanced adhesion of galectin-1-deficient ARPE-19 cells, could be blocked, at least in part, by the additional treatment with human recombinant galectin-1. In addition, a significantly reduced migration was detected in ARPE-19-LGALS1^−/− cells. In comparison to control cells, galectin-1-deficient RPE cells had enhanced expression of sm-α-actin and N-cadherin, whereas expression of E-cadherin showed no significant alteration. Finally, a compensatory expression of galectin-8 mRNA was observed in ARPE-19-LGALS1^−/− cells. In conclusion, in RPE cells, endogenous galectin-1 has crucial functions for various cell biological processes, including viability, proliferation, migration, adherence, and retaining the epithelial phenotype. Full article

Diabetic retinopathy (DR), a complication of diabetes mellitus (DM), can cause severe visual loss. The retinal pigment epithelium (RPE) plays a crucial role in retinal physiology but is vulnerable to oxidative damage. We investigated the protective effects of selenium (Se) on retinal pigment epithelium (ARPE-19) and primary human retinal microvascular endothelial (ACBRI 181) cells against high glucose (HG)-induced oxidative stress and apoptotic cascade. To achieve this objective, we utilized varying concentrations of D-glucose (ranging from 5 to 80 mM) to induce the HG model. HG-induced oxidative stress in ARPE-19 and ACBRI 181 cells and the apoptotic cascade were evaluated by determining Ca²⁺ overload, mitochondrial membrane depolarization, caspase-3/-9 activation, intracellular reactive oxygen species (ROS), lipid peroxidation (LP), glutathione (GSH), glutathione peroxidase (GSH-Px), vascular endothelial growth factor (VEGF) and apoptosis levels. A cell viability assay utilizing MTT was conducted to ascertain the optimal concentration of Se to be employed. The quantification of MTT, ROS, VEGF levels, and caspase-3 and -9 activation was accomplished using a plate reader. To quantitatively assess LP and GSH levels, GSH-Px activities were utilized by spectrophotometer and apoptosis, mitochondrial membrane depolarization, and the release of Ca²⁺ from intracellular stores were evaluated by spectrofluorometer. Our investigation revealed a significant augmentation in oxidative stress induced by HG, leading to cellular damage through modulation of mitochondrial membrane potential, ROS levels, and intracellular Ca²⁺ release. Incubation with Se resulted in a notable reduction in ROS production induced by HG, as well as a reduction in apoptosis and the activation of caspase-3 and -9. Additionally, Se incubation led to decreased levels of VEGF and LP while concurrently increasing levels of GSH and GSH-Px. The findings from this study strongly suggest that Se exerts a protective effect on ARPE-19 and ACBRI 181 cells against HG-induced oxidative stress and apoptosis. This protective mechanism is partially mediated through the intracellular Ca²⁺ signaling pathway. Full article

Because of their exposure to air, eyes can come into contact with air pollutants such as particulate matter (PM), which may cause severe ocular pathologies. Prolonged ocular PM exposure may increase inflammation and endoplasmic reticulum stress in the retina. Herein, we investigated whether PM exposure induces ocular inflammation and endoplasmic reticulum (ER) stress-related cellular responses in human retinal epithelium-19 (ARPE-19) cells. To understand how PM promotes ocular inflammation, we monitored the activation of the mitogen-activated protein kinase (MAPK)/nuclear factor kappa beta (NFκB) axis and the expression of key inflammatory mRNAs. We also measured the upregulation of signature components for the ER-related unfolded protein response (UPR) pathways, as well as intracellular calcium ([Ca²⁺]_i) levels, as readouts for ER stress induction following PM exposure. Ocular PM exposure significantly elevated the expression of multiple cytokine mRNAs and increased phosphorylation levels of NFκB-MAPK axis in a PM dose-dependent manner. Moreover, incubation with PM significantly increased [Ca²⁺]_i levels and the expression of UPR-related proteins, which indicated ER stress resulting from cell hypoxia, and upregulation of hypoxic adaptation mechanisms such as the ER-associated UPR pathways. Our study demonstrated that ocular PM exposure increased inflammation in ARPE-19 cells, by activating the MAPK/NFκB axis and cytokine mRNA expression, while also inducing ER stress and stress adaptation responses. These findings may provide helpful insight into clinical and non-clinical research examining the role of PM exposure in ocular pathophysiology and delineating its underlying molecular mechanisms. Full article

Age-related macular degeneration (AMD) is a blinding disease characterised by dysfunction of the retinal pigmented epithelium (RPE) which culminates in disruption or loss of the neurosensory retina. Genome-wide association studies have identified >60 genetic risk factors for AMD; however, the expression profile and functional role of many of these genes remain elusive in human RPE. To facilitate functional studies of AMD-associated genes, we developed a human RPE model with integrated CRISPR interference (CRISPRi) for gene repression by generating a stable ARPE19 cell line expressing dCas9-KRAB. We performed transcriptomic analysis of the human retina to prioritise AMD-associated genes and selected TMEM97 as a candidate gene for knockdown study. Using specific sgRNAs, we showed that knockdown of TMEM97 in ARPE19 reduced reactive oxygen species (ROS) levels and exerted a protective effect against oxidative stress-induced cell death. This work provides the first functional study of TMEM97 in RPE and supports a potential role of TMEM97 in AMD pathobiology. Our study highlights the potential for using CRISPRi to study AMD genetics, and the CRISPRi RPE platform generated here provided a useful in vitro tool for functional studies of AMD-associated genes. Full article

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system still lacking a cure. Treatment typically focuses on slowing the progression and managing MS symptoms. Single-cell transcriptomics allows the investigation of the immune system—the key player in MS onset and development—in great detail increasing our understanding of MS mechanisms and stimulating the discovery of the targets for potential therapies. Still, de novo drug development takes decades; however, this can be reduced by drug repositioning. A promising approach is to select potential drugs based on activated or inhibited genes and pathways. In this study, we explored the public single-cell RNA data from an experiment with six patients on single-cell RNA peripheral blood mononuclear cells (PBMC) and cerebrospinal fluid cells (CSF) of patients with MS and idiopathic intracranial hypertension. We demonstrate that AIM2 inflammasome, SMAD2/3 signaling, and complement activation pathways are activated in MS in different CSF and PBMC immune cells. Using genes from top-activated pathways, we detected several promising small molecules to reverse MS immune cells’ transcriptomic signatures, including AG14361, FGIN-1-27, CA-074, ARP 101, Flunisolide, and JAK3 Inhibitor VI. Among these molecules, we also detected an FDA-approved MS drug Mitoxantrone, supporting the reliability of our approach. Full article

Earlier researchers have highlighted the utilization of salt eustress for boosting the nutritional and phenolic acid (PA) profiles and antiradical potential (ARP) of vegetables, which eventually boost food values for nourishing human diets. Amaranth is a rapidly grown, diversely acclimated C₄ leafy vegetable with climate resilience and salinity resistance. The application of salinity eustress in amaranth has a great scope to augment the nutritional and PA profiles and ARP. Therefore, the A. gangeticus genotype was evaluated in response to salt eustress for nutrients, PA profile, and ARP. Antioxidant potential and high-yielding genotype (LS1) were grown under four salt eustresses (control, 25 mM, 50 mM, 100 mM NaCl) in a randomized completely block design (RCBD) in four replicates. Salt stress remarkably augmented microelements, proximate, macro-elements, phytochemicals, PA profiles, and ARP of A. gangeticus leaves in this order: control < low sodium chloride stress (LSCS) < moderate sodium chloride stress (MSCS) < severe sodium chloride stress (SSCS). A large quantity of 16 PAs, including seven cinnamic acids (CAs) and nine benzoic acids (BAs) were detected in A. gangeticus genotypes. All the microelements, proximate, macro-elements, phytochemicals, PA profiles, and ARP of A. gangeticus under MSCS, and SSCS levels were much higher in comparison with the control. It can be utilized as preferential food for our daily diets as these antiradical compounds have strong antioxidants. Salt-treated A. gangeticus contributed to excellent quality in the end product in terms of microelements, proximate, macro-elements, phytochemicals, PA profiles, and ARP. A. gangeticus can be cultivated as an encouraging substitute crop in salt-affected areas of the world. Full article

Microglia are resident immune cells in the central nervous system (CNS). Microglial activation plays a prominent role in neuroinflammation and CNS diseases. However, the underlying mechanisms of microglial activation are not well understood. Here, we report that the transcription factor interferon regulatory factor 1 (IRF1) plays critical roles in microglial activation and retinal inflammation by regulating pro- and anti-inflammatory gene expression. IRF1 expression was upregulated in activated retinal microglia compared to those at the steady state. IRF1 knockout (KO) in BV2 microglia cells (BV2^ΔIRF1) created by CRISPR/Cas9 genome-editing technique causes decreased microglia proliferation, migration, and phagocytosis. IRF1-KO decreased pro-inflammatory M1 marker gene expression induced by lipopolysaccharides (LPS), such as IL-6, COX-2, and CCL5, but increased anti-inflammatory M2 marker gene expression by IL-4/13, such as Arg-1, CD206, and TGF-β. Compared to the wild-type cells, microglial-conditioned media (MCM) of activated BV2^ΔIRF1 cell cultures reduced toxicity or death to several retinal cells, including mouse cone photoreceptor-like 661 W cells, rat retinal neuron precursor R28 cells, and human ARPE-19 cells. IRF1 knockdown by siRNA alleviated microglial activation and retinal inflammation induced by LPS in mice. Together, the findings suggest that IRF1 plays a vital role in regulating microglial activation and retinal inflammation and, therefore, may be targeted for treating inflammatory and degenerative retinal diseases. Full article

The application of PGPB is considered a surrogate approach to reducing the amounts of phosphorus fertilizers applied in addition to its role in improving nutrient availability under stress conditions. The objective of this study was to evaluate five levels of calcium superphosphate (CSP); ultimately, CSP was applied in five levels: CSP₂₀, CSP₄₀, CSP₆₀, CSP₈₀, and CSP₁₀₀ were applied at 69, 138, 207, 276, and 345 kg ha⁻¹, respectively, and two treatments of Bacillus megaterium DSM 2894 (with and without) were applied on sweet potato (Beauregard cv.) plants grown in calcareous soils in the 2019 and 2020 seasons in Egypt. Some macro- and micronutrient (i.e., nitrogen (N), phosphorus (P), calcium (Ca), iron (Fe), manganese (Mn), zinc (Zn), and copper (Cu)) uptake, antiradical power (ARP), and protein and total root yields (TRYs) were determined. The plants inoculated with B. megaterium DSM 2894 had increased leaf N, P, and Mn contents in both seasons; in addition, Ca was increased in the second season. Furthermore, all of the root nutrient contents (except N) as well as the ARP and TRY were increased in both seasons as compared with those of the noninoculated plants. On the other hand, the maximum values of the leaf Ca, Fe, and Cu contents and the root Ca, Fe, and Zn contents were recorded with the CSP₂₀ treatment in both seasons. CSP₆₀ was the superior treatment for N (in the leaves), Mn (in the roots), ARP, protein contents, and TRY in both seasons and for the leaf Zn content in the 2019 season. The application of the CSP₁₀₀ treatment gave the highest values for the leaf and root P contents and the root Cu contents in both seasons as well as for the leaf Mn content in the first season and the root N content in the 2020 growth season. Thus, it was concluded that the application of CSP₂₀, CSP₆₀, and CSP₁₀₀ treatments with the B. megaterium DSM2894 mixture gave the best values compared to the use of CSP or DSM2894 individually to attenuate CaCO₃-induced damage. Full article