CRISPR/Cas9-Based Knock-Out of the PMR4 Gene Reduces Susceptibility to Late Blight in Two Tomato Cultivars

Phytophthora infestans, the causal agent of late blight (LB) in tomato (Solanum lycopersicum L.), is a devastating disease and a serious concern for plant productivity. The presence of susceptibility (S) genes in plants facilitates pathogen proliferation; thus, disabling these genes may help provide a broad-spectrum and durable type of tolerance/resistance. Previous studies on Arabidopsis and tomato have highlighted that knock-out mutants of the PMR4 susceptibility gene are tolerant to powdery mildew. Moreover, PMR4 knock-down in potato has been shown to confer tolerance to LB. To verify the same effect in tomato in the present study, a CRISPR–Cas9 vector containing four single guide RNAs (sgRNAs: sgRNA1, sgRNA6, sgRNA7, and sgRNA8), targeting as many SlPMR4 regions, was introduced via Agrobacterium-tumefaciens-mediated transformation into two widely grown Italian tomato cultivars: ‘San Marzano’ (SM) and ‘Oxheart’ (OX). Thirty-five plants (twenty-six SM and nine OX) were selected and screened to identify the CRISPR/Cas9-induced mutations. The different sgRNAs caused mutation frequencies ranging from 22.1 to 100% and alternatively precise insertions (sgRNA6) or deletions (sgRNA7, sgRNA1, and sgRNA8). Notably, sgRNA7 induced in seven SM genotypes a −7 bp deletion in the homozygous status, whereas sgRNA8 led to the production of fifteen SM genotypes with a biallelic mutation (−7 bp and −2 bp). Selected edited lines were inoculated with P. infestans, and four of them, fully knocked out at the PMR4 locus, showed reduced disease symptoms (reduction in susceptibility from 55 to 80%) compared to control plants. The four SM lines were sequenced using Illumina whole-genome sequencing for deeper characterization without exhibiting any evidence of mutations in the candidate off-target regions. Our results showed, for the first time, a reduced susceptibility to Phytophtora infestans in pmr4 tomato mutants confirming the role of KO PMR4 in providing broad-spectrum protection against pathogens.     

A Novel Model Using AAV9-Cre to Knockout Adult Leydig Cell Gene Expression Reveals a Physiological Role of Glucocorticoid Receptor Signalling in Leydig Cell Function

Glucocorticoids are steroids involved in key physiological processes such as development, metabolism, inflammatory and stress responses and are mostly used exogenously as medications to treat various inflammation-based conditions. They act via the glucocorticoid receptor (GR) expressed in most cells. Exogenous glucocorticoids can negatively impact the function of the Leydig cells in the testis, leading to decreased androgen production. However, endogenous glucocorticoids are produced by the adrenal and within the testis, but whether their action on GR in Leydig cells regulates steroidogenesis is unknown. This study aimed to define the role of endogenous GR signalling in adult Leydig cells. We developed and compared two models; an inducible Cre transgene driven by expression of the Cyp17a1 steroidogenic gene (Cyp17-iCre) that depletes GR during development and a viral vector-driven Cre (AAV9-Cre) to deplete GR in adulthood. The delivery of AAV9-Cre ablated GR in adult mouse Leydig cells depleted Leydig cell GR more efficiently than the Cyp17-iCre model. Importantly, adult depletion of GR in Leydig cells caused reduced expression of luteinising hormone receptor (Lhcgr) and of steroidogenic enzymes required for normal androgen production. These findings reveal that Leydig cell GR signalling plays a physiological role in the testis and highlight that a normal balance of glucocorticoid activity in the testis is important for steroidogenesis.     

Dual Mode of Mitochondrial ROS Action during Reprogramming to Pluripotency

Essential changes in cell metabolism and redox signaling occur during the reprogramming of somatic cells into induced pluripotent stem cells (iPSCs). In this paper, using genetic and pharmacological approaches, we have investigated the role of electron transport chain (ETC) complex-I (CI) of mitochondria in the process of cell reprogramming to pluripotency. Knockdown of NADH-ubiquinone oxidoreductase core subunits S1 (Ndufs1) or subunit B10 (Ndufb10) of the CI or inhibition of this complex with rotenone during mouse embryonic fibroblast (MEF) reprogramming resulted in a significantly decreased number of induced pluripotent stem cells (iPSCs). We have found that mitochondria and ROS levels due course of the reprogramming tightly correlate with each other, both reaching peak by day 3 and significantly declining by day 10 of the process. The transient augmentation of mitochondrial reactive oxygen species (ROS) could be attenuated by antioxidant treatment, which ameliorated overall reprogramming. However, ROS scavenging after day 3 or during the entire course of reprogramming was suppressive for iPSC formation. The ROS scavenging within the CI-deficient iPSC-precursors did not improve, but further suppressed the reprogramming. Our data therefore point to distinct modes of mitochondrial ROS action during the early versus mid and late stages of reprogramming. The data further substantiate the paradigm that balanced levels of oxidative phosphorylation have to be maintained on the route to pluripotency.     

The Flavonol Quercitrin Hinders GSK3 Activity and Potentiates the Wnt/β-Catenin Signaling Pathway

The Wnt/β-catenin signaling pathway dictates cell proliferation and differentiation during embryonic development and tissue homeostasis. Its deregulation is associated with many pathological conditions, including neurodegenerative disease, frequently downregulated. The lack of efficient treatment for these diseases, including Alzheimer’s disease (AD), makes Wnt signaling an attractive target for therapies. Interestingly, novel Wnt signaling activating compounds are less frequently described than inhibitors, turning the quest for novel positive modulators even more appealing. In that sense, natural compounds are an outstanding source of potential drug leads. Here, we combine different experimental models, cell-based approaches, neuronal culture assays, and rodent behavior tests with Xenopus laevis phenotypic analysis to characterize quercitrin, a natural compound, as a novel Wnt signaling potentiator. We find that quercitrin potentiates the signaling in a concentration-dependent manner and increases the occurrence of the Xenopus secondary axis phenotype mediated by Xwnt8 injection. Using a GSK3 biosensor, we describe that quercitrin impairs GSK3 activity and increases phosphorylated GSK3β S9 levels. Treatment with XAV939, an inhibitor downstream of GSK3, impairs the quercitrin-mediated effect. Next, we show that quercitrin potentiates the Wnt3a-synaptogenic effect in hippocampal neurons in culture, which is blocked by XAV939. Quercitrin treatment also rescues the hippocampal synapse loss induced by intracerebroventricular injection of amyloid-β oligomers (AβO) in mice. Finally, quercitrin rescues AβO-mediated memory impairment, which is prevented by XAV939. Thus, our study uncovers a novel function for quercitrin as a Wnt/β-catenin signaling potentiator, describes its mechanism of action, and opens new avenues for AD treatments.     

Physiological and Pathological Remodeling of Cerebral Microvessels

There is growing evidence that the remodeling of cerebral microvessels plays an important role in plastic changes in the brain associated with development, experience, learning, and memory consolidation. At the same time, abnormal neoangiogenesis, and deregulated regulation of microvascular regression, or pruning, could contribute to the pathogenesis of neurodevelopmental diseases, stroke, and neurodegeneration. Aberrant remodeling of microvesselsis associated with blood–brain barrier breakdown, development of neuroinflammation, inadequate microcirculation in active brain regions, and leads to the dysfunction of the neurovascular unit and progressive neurological deficits. In this review, we summarize current data on the mechanisms of blood vessel regression and pruning in brain plasticity and in Alzheimer’s-type neurodegeneration. We discuss some novel approaches to modulating cerebral remodeling and preventing degeneration-coupled aberrant microvascular activity in chronic neurodegeneration.     

Reaction of Corroles with Sarcosine and Paraformaldehyde: A New Facet of Corrole Chemistry

Details on the unexpected formation of two new (dimethylamino)methyl corrole isomers from the reaction of 5,10,15-tris(pentafluorophenyl)corrolatogallium(III) with sarcosine and paraformaldehyde are presented. Semi-empirical calculations on possible mechanism pathways seem to indicate that the new compounds are probably formed through a Mannich-type reaction. The extension of the protocol to the free-base 5,10,15-tris(pentafluorophenyl)corrole afforded an unexpected new seven-membered ring corrole derivative, confirming the peculiar behavior of corroles towards known reactions when compared to the well-behaved porphyrin counterparts.     

Two Subgroups within the GH43_36 α-l-Arabinofuranosidase Subfamily Hydrolyze Arabinosyl from Either Mono-or Disubstituted Xylosyl Units in Wheat Arabinoxylan

Fungal arabinofuranosidases (ABFs) catalyze the hydrolysis of arabinosyl substituents (Ara) and are key in the interplay with other glycosyl hydrolases to saccharify arabinoxylans (AXs). Most characterized ABFs belong to GH51 and GH62 and are known to hydrolyze the linkage of α-(1→2)-Ara and α-(1→3)-Ara in monosubstituted xylosyl residues (Xyl) (ABF-m2,3). Nevertheless, in AX a substantial number of Xyls have two Aras (i.e., disubstituted), which are unaffected by ABFs from GH51 and GH62. To date, only two fungal enzymes have been identified (in GH43_36) that specifically release the α-(1→3)-Ara from disubstituted Xyls (ABF-d3). In our research, phylogenetic analysis of available GH43_36 sequences revealed two major clades (GH43_36a and GH43_36b) with an expected substrate specificity difference. The characterized fungal ABF-d3 enzymes aligned with GH43_36a, including the GH43_36 from Humicola insolens (HiABF43_36a). Hereto, the first fungal GH43_36b (from Talaromyces pinophilus) was cloned, purified, and characterized (TpABF43_36b). Surprisingly, TpABF43_36b was found to be active as ABF-m2,3, albeit with a relatively low rate compared to other ABFs tested, and showed minor xylanase activity. Novel specificities were also discovered for the HiABF43_36a, as it also released α-(1→2)-Ara from a disubstitution on the non-reducing end of an arabinoxylooligosaccharide (AXOS), and it was active to a lesser extent as an ABF-m2,3 towards AXOS when the Ara was on the second xylosyl from the non-reducing end. In essence, this work adds new insights into the biorefinery of agricultural residues.     

Retinal Cyclic Nucleotide-Gated Channel Regulation by Calmodulin

Retinal cyclic nucleotide-gated (CNG) ion channels bind to intracellular cGMP and mediate visual phototransduction in photoreceptor rod and cone cells. Retinal rod CNG channels form hetero-tetramers comprised of three CNGA1 and one CNGB1 protein subunits. Cone CNG channels are similar tetramers consisting of three CNGA3 and one CNGB3 subunits. Calmodulin (CaM) binds to two distinct sites (CaM1: residues 565–587 and CaM2: residues 1120–1147) within the cytosolic domains of rod CNGB1. The binding of Ca²⁺-bound CaM to CNGB1 promotes the Ca²⁺-induced desensitization of CNG channels in retinal rods that may be important for photoreceptor light adaptation. Mutations that affect Ca²⁺-dependent CNG channel function are responsible for inherited forms of blindness. In this review, we propose structural models of the rod CNG channel bound to CaM that suggest how CaM might cause channel desensitization and how dysregulation of the channel may lead to retinal disease.     

Somatostatin,Cortistatin and Their Receptors Exert Antitumor Actions in Androgen-Independent Prostate Cancer Cells: Critical Role of Endogenous Cortistatin

Somatostatin (SST), cortistatin (CORT), and their receptors (SSTR1-5/sst5TMD4-TMD5) comprise a multifactorial hormonal system involved in the regulation of numerous pathophysiological processes. Certain components of this system are dysregulated and play critical roles in the development/progression of different endocrine-related cancers. However, the presence and therapeutic role of this regulatory system in prostate cancer (PCa) remain poorly explored. Accordingly, we performed functional (proliferation/migration/colonies-formation) and mechanistic (Western-blot/qPCR/microfluidic-based qPCR-array) assays in response to SST and CORT treatments and CORT-silencing (using specific siRNA) in different PCa cell models [androgen-dependent (AD): LNCaP; androgen-independent (AI)/castration-resistant PCa (CRPC): 22Rv1 and PC-3], and/or in the normal-like prostate cell-line RWPE-1. Moreover, the expression of SST/CORT system components was analyzed in PCa samples from two different patient cohorts [internal (n = 69); external (Grasso, n = 88)]. SST and CORT treatment inhibited key functional/aggressiveness parameters only in AI-PCa cells. Mechanistically, antitumor capacity of SST/CORT was associated with the modulation of oncogenic signaling pathways (AKT/JNK), and with the significant down-regulation of critical genes involved in proliferation/migration and PCa-aggressiveness (e.g., MKI67/MMP9/EGF). Interestingly, CORT was highly expressed, while SST was not detected, in all prostate cell-lines analyzed. Consistently, endogenous CORT was overexpressed in PCa samples (compared with benign-prostatic-hyperplasia) and correlated with key clinical (i.e., metastasis) and molecular (i.e., SSTR2/SSTR5 expression) parameters. Remarkably, CORT-silencing drastically enhanced proliferation rate and blunted the antitumor activity of SST-analogues (octreotide/pasireotide) in AI-PCa cells. Altogether, we provide evidence that SST/CORT system and SST-analogues could represent a potential therapeutic option for PCa, especially for CRPC, and that endogenous CORT could act as an autocrine/paracrine regulator of PCa progression.