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.     

Decay-Accelerating Factor Creates an Organ-Protective Phenotype after Hemorrhage in Conscious Rats

Preclinical and clinical studies have shown that traumatic hemorrhage (TH) induces early complement cascade activation, leading to inflammation-associated multiple-organ dysfunction syndrome (MODS). Several previous studies have demonstrated the beneficial effects of complement inhibition in anesthetized (unconscious) animal models of hemorrhage. Anesthetic agents profoundly affect the immune response, microcirculation response, and coagulation patterns and thereby may confound the TH research data acquired. However, no studies have addressed the effect of complement inhibition on inflammation-driven MODS in a conscious model of hemorrhage. This study investigated whether early administration of decay-accelerating factor (CD55/DAF, a complement C3/C5 inhibitor) alleviates hemorrhage-induced organ damage and how DAF modulates hemorrhage-induced organ damage. DAF was administered to unanesthetized male Sprague Dawley rats subjected to pressure-controlled hemorrhage followed by a prolonged (4 h) hypotensive resuscitation with or without lactated Ringer’s (LR). We assessed DAF effects on organ protection, tissue levels of complement synthesis and activation, T lymphocyte infiltration, fluid resuscitation requirements, and metabolic acidosis. Hemorrhage with (HR) or without (H) LR resuscitation resulted in significantly increased C3, C5a, and C5b-9 deposition in the lung and intestinal tissues. HR rats had significantly higher tissue levels of complement activation/deposition (particularly C5a and C5b-9 in the lung tissues), a higher but not significant amount of C3 and C5b-9 pulmonary microvascular deposition, and relatively severe injury in the lung and intestinal tissues compared to H rats. DAF treatment significantly reduced tissue C5b-9 formation and C3 deposition in the H or HR rats and decreased tissue levels of C5a and C3 mRNA in the HR rats. This treatment prevented the injury of these organs, improved metabolic acidosis, reduced fluid resuscitation requirements, and decreased T-cell infiltration in lung tissues. These findings suggest that DAF has the potential as an organ-protective adjuvant treatment for TH during prolonged damage control resuscitation.     

Thyroxine Regulates the Opening of the Organ of Corti through Affecting P-Cadherin and Acetylated Microtubule

Different serum thyroxine levels may influence the morphology of the inner ear during development. A well-developed organ of Corti (OC) is considered to be critical to the function of hearing. In our study, we treated mice with triiodothyronine (T3) and found that the opening of the OC occurred sooner than in control mice. We also observed an increased formation of acetylated microtubules and a decrease in the adhesion junction molecule P-cadherin the during opening of the OC. Our investigation indicates that thyroxin affects P-cadherin expression and microtubule acetylation to influence the opening of the OC.     

Factors and Pathways Modulating Endothelial Cell Senescence in Vascular Aging

Aging causes a progressive decline in the structure and function of organs. With advancing age, an accumulation of senescent endothelial cells (ECs) contributes to the risk of developing vascular dysfunction and cardiovascular diseases, including hypertension, diabetes, atherosclerosis, and neurodegeneration. Senescent ECs undergo phenotypic changes that alter the pattern of expressed proteins, as well as their morphologies and functions, and have been linked to vascular impairments, such as aortic stiffness, enhanced inflammation, and dysregulated vascular tone. Numerous molecules and pathways, including sirtuins, Klotho, RAAS, IGFBP, NRF2, and mTOR, have been implicated in promoting EC senescence. This review summarizes the molecular players and signaling pathways driving EC senescence and identifies targets with possible therapeutic value in age-related vascular diseases.     

Identification of Potential New Aedes aegypti Juvenile Hormone Inhibitors from N-Acyl Piperidine Derivatives: A Bioinformatics Approach

Aedes aegypti mosquitoes transmit several human pathogens that cause millions of deaths worldwide, mainly in Latin America. The indiscriminate use of insecticides has resulted in the development of species resistance to some such compounds. Piperidine, a natural alkaloid isolated from Piper nigrum, has been used as a hit compound due to its larvicidal activity against Aedes aegypti. In the present study, piperidine derivatives were studied through in silico methods: pharmacophoric evaluation (PharmaGist), pharmacophoric virtual screening (Pharmit), ADME/Tox prediction (Preadmet/Derek 10.0^®), docking calculations (AutoDock 4.2) and molecular dynamics (MD) simulation on GROMACS-5.1.4. MP-416 and MP-073 molecules exhibiting ΔG binding (MMPBSA −265.95 ± 1.32 kJ/mol and −124.412 ± 1.08 kJ/mol, respectively) and comparable to holo (ΔG binding = −216.21 ± 0.97) and pyriproxyfen (a well-known larvicidal, ΔG binding= −435.95 ± 2.06 kJ/mol). Considering future in vivo assays, we elaborated the theoretical synthetic route and made predictions of the synthetic accessibility (SA) (SwissADME), lipophilicity and water solubility (SwissADME) of the promising compounds identified in the present study. Our in silico results show that MP-416 and MP-073 molecules could be potent insecticides against the Aedes aegypti mosquitoes.     

Variable Expression of GABAA Receptor Subunit Gamma 2 Mutation in a Nuclear Family Displaying Developmental and Encephalopathic Phenotype

Mutations in GABA_A receptor subunit genes (GABRs) are a major etiology for developmental and epileptic encephalopathies (DEEs). This article reports a case of a genetic abnormality in GABRG2 and updates the pathophysiology and treatment development for mutations in DEEs based on recent advances. Mutations in GABRs, especially in GABRA1, GABRB2, GABRB3, and GABRG2, impair GABAergic signaling and are frequently associated with DEEs such as Dravet syndrome and Lennox–Gastaut syndrome, as GABAergic signaling is critical for early brain development. We here present a novel association of a microdeletion of GABRG2 with a diagnosed DEE phenotype. We characterized the clinical phenotype and underlying mechanisms, including molecular genetics, EEGs, and MRI. We then compiled an update of molecular mechanisms of GABR mutations, especially the mutations in GABRB3 and GABRG2 attributed to DEEs. Genetic therapy is also discussed as a new avenue for treatment of DEEs through employing antisense oligonucleotide techniques. There is an urgent need to define treatment targets and explore new treatment paradigms for the DEEs, as early deployment could alleviate long-term disabilities and improve quality of life for patients. This study highlights biomolecular targets for future therapeutic interventions, including via both pharmacological and genetic approaches.     

Improved Bladder Tumor RNA Isolation from Archived Tissues Using Methylene Blue for Normalization,Multiplex RNA Hybridization,Sequencing and Subtyping

Methylene blue (MB) is a dye used for histology with clinical importance and intercalates into nucleic acids. After MB staining of formalin fixed paraffin embedded (FFPE) muscle invasive bladder cancer (MIBC) and normal urothelium, specific regions could be microdissected. It is not known if MB influences RNA used for gene expression studies. Therefore, we analyzed MIBC using five different RNA isolation methods comparing patient matched FFPE and fresh frozen (FF) tissues pre-stained with or without MB. We demonstrate a positive impact of MB on RNA integrity with FF tissues using real time PCR with no interference of its chemical properties. FFPE tissues showed no improvement of RNA integrity, which we propose is due to formalin induced nucleotide crosslinks. Using direct multiplex RNA hybridization the best genes for normalization of MIBC and control tissues were identified from 34 reference genes. In addition, 5SrRNA and 5.8SrRNA were distinctive reference genes detecting <200 bp fragments important for mRNA analyses. Using these normalized RNAs from MB stained MIBC and applying multiplex RNA hybridization and mRNA sequencing, a minimal gene expression panel precisely identified luminal and basal MIBC tumor subtypes, important for diagnosis, prognosis and chemotherapy response.