Relations of Social Maturity, Executive Function, and Self-Efficacy Among Deaf University Students

This study explored possible associations of social maturity, executive function (EF), self-efficacy, and communication variables among deaf university students, both cochlear implant (CI) users and nonusers. Previous studies have demonstrated differences between deaf and hearing children and young adults in EF and EF-related social and cognitive functioning. EF differences also have been demonstrated between hearing children and deaf children who use CIs. Long-term influences of cochlear implantation in the social domain largely have not been explored, but were examined in the present study in terms of social maturity, as it might be related to EF and communication variables. Replicating and extending recent findings, social maturity was found to be related to somewhat different aspects of EF in CI users, deaf nonusers, and hearing students, but unrelated to hearing status, CI use, or deaf students' use of sign language versus spoken language. Self-efficacy proved a predictor of self-reported socially mature and immature behaviours for all groups. Individuals' beliefs about their parents' views of such behaviours was a potent predictor of behaviours for deaf CI users and those deaf students who reported sign language as their best form of communication

Short Palate, Lung, and Nasal Epithelial Clone 1 Has Antimicrobial and Antibiofilm Activities against the Burkholderia cepacia Complex

ABSTRACT The opportunistic bacteria of the Burkholderia cepacia complex (Bcc) are extremely pathogenic to cystic fibrosis (CF) patients, and acquisition of Bcc bacteria is associated with a significant increase in mortality. Treatment of Bcc infections is difficult because the bacteria are multidrug resistant and able to survive in biofilms. Short palate, lung, and nasal epithelial clone 1 (SPLUNC1) is an innate defense protein that is secreted by the upper airways and pharynx. While SPLUNC1 is known to have antimicrobial functions, its effects on Bcc strains are unclear. We therefore tested the hypothesis that SPLUNC1 is able to impair Bcc growth and biofilm formation. We found that SPLUNC1 exerted bacteriostatic effects against several Bcc clinical isolates, including B. cenocepacia strain J2315 (50% inhibitory concentration [IC 50 ] = 0.28 μM), and reduced biofilm formation and attachment (IC 50 = 0.11 μM). We then determined which domains of SPLUNC1 are responsible for its antimicrobial activity. Deletions of SPLUNC1's N terminus and α6 helix did not affect its function. However, deletion of the α4 helix attenuated antimicrobial activity, while the corresponding α4 peptide displayed antimicrobial activity. Chronic neutrophilia is a hallmark of CF lung disease, and neutrophil elastase (NE) cleaves SPLUNC1. However, we found that the ability of SPLUNC1 to disrupt biofilm formation was significantly potentiated by NE pretreatment. While the impact of CF on SPLUNC1-Bcc interactions is not currently known, our data suggest that understanding this interaction may have important implications for CF lung disease

Active site flexibility revealed in crystal structures of Parabacteroides merdae β-glucuronidase from the human gut microbiome

β-Glucuronidase (GUS) enzymes in the gastrointestinal tract are involved in maintaining mammalian-microbial symbiosis and can play key roles in drug efficacy and toxicity. Parabacteroides merdae GUS was identified as an abundant mini-Loop 2 (mL2) type GUS enzyme in the Human Microbiome Project gut metagenomic database. Here, we report the crystal structure of P. merdae GUS and highlight the differences between this enzyme and extant structures of gut microbial GUS proteins. We find that P. merdae GUS exhibits a distinct tetrameric quaternary structure and that the mL2 motif traces a unique path within the active site, which also includes two arginines distinctive to this GUS. We observe two states of the P. merdae GUS active site; a loop repositions itself by more than 50 Å to place a functionally-relevant residue into the enzyme's catalytic site. Finally, we find that P. merdae GUS is able to bind to homo and heteropolymers of the polysaccharide alginic acid. Together, these data broaden our understanding of the structural and functional diversity in the GUS family of enzymes present in the human gut microbiome and point to specialization as an important feature of microbial GUS orthologs

Acetylation of lysine 109 modulates pregnane X receptor DNA binding and transcriptional activity

Pregnane X receptor (PXR) is a major transcriptional regulator of xenobiotic metabolism and transport pathways in the liver and intestines, which are critical for protecting organisms against potentially harmful xenobiotic and endobiotic compounds. Inadvertent activation of drug metabolism pathways through PXR is known to contribute to drug resistance, adverse drug–drug interactions, and drug toxicity in humans. In both humans and rodents, PXR has been implicated in non-alcoholic fatty liver disease, diabetes, obesity, inflammatory bowel disease, and cancer. Because of PXR's important functions, it has been a therapeutic target of interest for a long time. More recent mechanistic studies have shown that PXR is modulated by multiple PTMs. Herein we provide the first investigation of the role of acetylation in modulating PXR activity. Through LC–MS/MS analysis, we identified lysine 109 (K109) in the hinge as PXR's major acetylation site. Using various biochemical and cell-based assays, we show that PXR's acetylation status and transcriptional activity are modulated by E1A binding protein (p300) and sirtuin 1 (SIRT1). Based on analysis of acetylation site mutants, we found that acetylation at K109 represses PXR transcriptional activity. The mechanism involves loss of RXRα dimerization and reduced binding to cognate DNA response elements. This mechanism may represent a promising therapeutic target using modulators of PXR acetylation levels

Structural Insights into Endobiotic Reactivation by Human Gut Microbiome-Encoded Sulfatases

Phase II drug metabolism inactivates xenobiotics and endobiotics through the addition of either a glucuronic acid or sulfate moiety prior to excretion, often via the gastrointestinal tract. While the human gut microbial β-glucuronidase enzymes that reactivate glucuronide conjugates in the intestines are becoming well characterized and even controlled by targeted inhibitors, the sulfatases encoded by the human gut microbiome have not been comprehensively examined. Gut microbial sulfatases are poised to reactivate xenobiotics and endobiotics, which are then capable of undergoing enterohepatic recirculation or exerting local effects on the gut epithelium. Here, using protein structure-guided methods, we identify 728 distinct microbiome-encoded sulfatase proteins from the 4.8 million unique proteins present in the Human Microbiome Project Stool Sample database and 1766 gut microbial sulfatases from the 9.9 million sequences in the Integrated Gene Catalogue. We purify a representative set of these sulfatases, elucidate crystal structures, and pinpoint unique structural motifs essential to endobiotic sulfate processing. Gut microbial sulfatases differentially process sulfated forms of the neurotransmitters serotonin and dopamine, and the hormones melatonin, estrone, dehydroepiandrosterone, and thyroxine in a manner dependent both on variabilities in active site architecture and on markedly distinct oligomeric states. Taken together, these data provide initial insights into the structural and functional diversity of gut microbial sulfatases, providing a path toward defining the roles these enzymes play in health and disease

Targeting Regorafenib-Induced Toxicity through Inhibition of Gut Microbial β-Glucuronidases

Regorafenib (Stivarga) is an oral small molecule kinase inhibitor used to treat metastatic colorectal cancer, hepatocellular carcinomas, and gastrointestinal stromal tumors. Diarrhea is one of the most frequently observed adverse reactions associated with regorafenib. This toxicity may arise from the reactivation of the inactive regorafenib-glucuronide to regorafenib by gut microbial β-glucuronidase (GUS) enzymes in the gastrointestinal tract. We sought to unravel the molecular basis of regorafenib-glucuronide processing by human intestinal GUS enzymes and to examine the potential inhibition of these enzymes. Using a panel of 31 unique gut microbial GUS enzymes derived from the 279 mapped from the human gut microbiome, we found that only four were capable of regorafenib-glucuronide processing. Using crystal structures as a guide, we pinpointed the molecular features unique to these enzymes that confer regorafenib-glucuronide processing activity. Furthermore, a pilot screen identified the FDA-approved drug raloxifene as an inhibitor of regorafenib reactivation by the GUS proteins discovered. Novel synthetic raloxifene analogs exhibited improved potency in both in vitro and ex vivo studies. Taken together, these data establish that regorafenib reactivation is exclusively catalyzed by gut microbial enzymes and that these enzymes are amenable to targeted inhibition. Our results unravel key molecular details of regorafenib reactivation in the GI tract and provide a potential pathway to improve clinical outcomes with regorafenib

Structural Features Essential to the Antimicrobial Functions of Human SPLUNC1

SPLUNC1 is an abundantly secreted innate immune protein in the mammalian respiratory tract that exerts bacteriostatic and antibiofilm effects, binds to lipopolysaccharide (LPS), and acts as a fluid-spreading surfactant. Here, we unravel the structural elements essential for the surfactant and antimicrobial functions of human SPLUNC1 (short palate lung nasal epithelial clone 1). A unique α-helix (α4) that extends from the body of SPLUNC1 is required for the bacteriostatic, surfactant, and LPS binding activities of this protein. Indeed, we find that mutation of just four leucine residues within this helical motif to alanine is sufficient to significantly inhibit the fluid spreading abilities of SPLUNC1, as well as its bacteriostatic actions against Gram-negative pathogens Burkholderia cenocepacia and Pseudomonas aeruginosa. Conformational flexibility in the body of SPLUNC1 is also involved in the bacteriostatic, surfactant, and LPS binding functions of the protein as revealed by disulfide mutants introduced into SPLUNC1. In addition, SPLUNC1 exerts antibiofilm effects against Gram-negative bacteria, although α4 is not involved in this activity. Interestingly, though, the introduction of surface electrostatic mutations away from α4 based on the unique dolphin SPLUNC1 sequence, and confirmed by crystal structure, is shown to impart antibiofilm activity against Staphylococcus aureus, the first SPLUNC1-dependent effect against a Gram-positive bacterium reported to date. Together, these data pinpoint SPLUNC1 structural motifs required for the antimicrobial and surfactant actions of this protective human protein

Substrate preference of Gen endonucleases highlights the importance of branched structures as DNA damage repair intermediates

Human GEN1 and yeast Yen1 are endonucleases with the ability to cleave Holliday junctions (HJs), which are proposed intermediates in recombination. In vivo, GEN1 and Yen1 function secondarily to Mus81, which has weak activity on intact HJs. We show that the genetic relationship is reversed in Drosophila, with Gen mutants having more severe defects than mus81 mutants. In vitro, DmGen, like HsGEN1, efficiently cleaves HJs, 5΄ flaps, splayed arms, and replication fork structures. We find that the cleavage rates for 5΄ flaps are significantly higher than those for HJs for both DmGen and HsGEN1, even in vast excess of enzyme over substrate. Kinetic studies suggest that the difference in cleavage rates results from a slow, rate-limiting conformational change prior to HJ cleavage: formation of a productive dimer on the HJ. Despite the stark difference in vivo that Drosophila uses Gen over Mus81 and humans use MUS81 over GEN1, we find the in vitro activities of DmGen and HsGEN1 to be strikingly similar. These findings suggest that simpler branched structures may be more important substrates for Gen orthologs in vivo, and highlight the utility of using the Drosophila model system to further understand these enzymes