N6-Methyladenosine Reader YTHDF1 Promotes ARHGEF2 Translation and RhoA Signaling in Colorectal Cancer.

BACKGROUND & AIMS: N6-methyladenosine (m6A) governs the fate of RNAs through m6A readers. Colorectal cancer (CRC) exhibits aberrant m6A modifications and expression of m6A regulators. However, how m6A readers interpret oncogenic m6A methylome to promote malignant transformation remains to be illustrated. METHODS: YTH N6-methyladenosine RNA binding protein 1 (Ythdf1) knockout mouse was generated to determine the effect of Ythdf1 in CRC tumorigenesis in vivo. Multiomic analysis of RNA-sequencing, m6A methylated RNA immunoprecipitation sequencing, YTHDF1 RNA immunoprecipitation sequencing, and proteomics were performed to unravel targets of YTHDF1 in CRC. The therapeutic potential of targeting YTHDF1-m6A-Rho/Rac guanine nucleotide exchange factor 2 (ARHGEF2) was evaluated using small interfering RNA (siRNA) encapsulated by lipid nanoparticles (LNP). RESULTS: DNA copy number gain of YTHDF1 is a frequent event in CRC and contributes to its overexpression. High expression of YTHDF1 is significantly associated with metastatic gene signature in patient tumors. Ythdf1 knockout in mice dampened tumor growth in an inflammatory CRC model. YTHDF1 promotes cell growth in CRC cell lines and primary organoids and lung and liver metastasis in vivo. Integrative multiomics analysis identified RhoA activator ARHGEF2 as a key downstream target of YTHDF1. YTHDF1 binds to m6A sites of ARHGEF2 messenger RNA, resulting in enhanced translation of ARHGEF2. Ectopic expression of ARHGEF2 restored impaired RhoA signaling, cell growth, and metastatic ability both in vitro and in vivo caused by YTHDF1 loss, verifying that ARHGEF2 is a key target of YTHDF1. Finally, ARHGEF2 siRNA delivered by LNP significantly suppressed tumor growth and metastasis in vivo. CONCLUSIONS: We identify a novel oncogenic epitranscriptome axis of YTHDF1-m6A-ARHGEF2, which regulates CRC tumorigenesis and metastasis. siRNA-delivering LNP drug validated the therapeutic potential of targeting this axis in CRC.

A detergent-free strategy for the reconstitution of active enzyme complexes from native biological membranes into nanoscale discs.

BACKGROUND: The reconstitution of membrane proteins and complexes into nanoscale lipid bilayer structures has contributed significantly to biochemical and biophysical analyses. Current methods for performing such reconstitutions entail an initial detergent-mediated step to solubilize and isolate membrane proteins. Exposure to detergents, however, can destabilize many membrane proteins and result in a loss of function. Amphipathic copolymers have recently been used to stabilize membrane proteins and complexes following suitable detergent extraction. However, the ability of these copolymers to extract proteins directly from native lipid bilayers for subsequent reconstitution and characterization has not been explored. RESULTS: The styrene-maleic acid (SMA) copolymer effectively solubilized membranes of isolated mitochondria and extracted protein complexes. Membrane complexes were reconstituted into polymer-bound nanoscale discs along with endogenous lipids. Using respiratory Complex IV as a model, these particles were shown to maintain the enzymatic activity of multicomponent electron transporting complexes. CONCLUSIONS: We report a novel process for reconstituting fully operational protein complexes directly from cellular membranes into nanoscale lipid bilayers using the SMA copolymer. This facile, single-step strategy obviates the requirement for detergents and yields membrane complexes suitable for structural and functional studies.

Agreement between mothers and children with malocclusion in rating children's oral health-related quality of life.

INTRODUCTION: The aim of this study was to compare the assessment of oral health-related quality of life (OH-QoL) between children with malocclusion and their mothers, by using responses to the child perceptions questionnaire and the parental-caregivers perceptions questionnaire. METHODS: The study was conducted in 90 children, aged 11 to 14 years, with a malocclusion grade of 4 or 5 according to the index of orthodontic treatement need dental health component. The children and their mothers completed the questionnaires independently. RESULTS: The mean ratings were similar for total scores (children, 20.4; mothers, 20.1), oral symptoms (children, 5.2; mothers, 4.7), and social well-being (children, 4.3; mothers, 4.8). However, the mothers group had a lower mean score for functional limitations (children, 5.3; mothers, 3.6) and a higher mean score for emotional well-being (children, 5.6; mothers, 7.1). The correlations between children's and mothers' responses ranged from rs = 0.545 for total score and emotional well-being to rs = 0.357 for functional limitations. There were good correlations between their responses to global (rs = 0.466) and life overall (rs = 0.427) questions, but poor correlations between the 2 questions, suggesting that these concepts were considered differently. CONCLUSIONS: Maternal opinions were similar to those of their children for the overall impact on OH-QoL of malocclusion, but mothers were more dissatisfied with the appearance of their children's teeth and overestimated the emotional impact of malocclusion. It would be useful to develop a specific measure to assess OH-QoL in children with malocclusion.

A comparison of hydrophobic polyurethane and polyurethane peripherally inserted central catheter: results from a feasibility randomized controlled trial.

BACKGROUND: To evaluate the feasibility of an efficacy trial comparing a hydrophobic polyurethane peripherally inserted central catheter (PICC) with a standard polyurethane PICC. METHODS: This pilot randomised controlled trial (RCT) was conducted between May 2017 and February 2018. Adult participants (n = 111) were assigned to hydrophobic polyurethane PICC with proximal valve (intervention) or a polyurethane PICC with external clamp (standard care). Primary outcome was trial feasibility including PICC failure. Secondary outcomes were central line-associated bloodstream infection, local infection, occlusion, thrombosis, fracture and dislodgement, phlebitis, local or systemic allergic reaction, and PICC dwell time. RESULTS: All feasibility outcomes were achieved, apart from eligibility criteria. In total, 338 patients were screened, 138 were eligible (41%), and of these 111 were randomised (80%). Patients received the allocated PICC in 106 (95%) insertions. No patients withdrew from the study and there was no missing data. PICC failure was 24% (13/55) in the intervention group and 22% (12/55) in the standard care group (p = 0.820). PICC failure per 1000 PICC days was 16.3 in the intervention group and 18.4 in the control group (p = 0.755). The average dwell time was 12 days in the intervention and 8 days in the control group. CONCLUSIONS: This study demonstrates the feasibility of an efficacy trial of PICC materials in an adult population, once adjustments were made to include not only in-patients, but also patients being discharged to the Hospital in the Home service. TRIAL REGISTRATION: Australia and New Zealand Clinical Trials Registry ACTRN12616001578493 . Prospectively registered on 16 November 2016. The trial protocol was published a priori (Kleidon et al., Vasc Access 3:15-21, 2017).

Quality of clinical photographs taken by orthodontists, professional photographers, and orthodontic auxiliaries.

INTRODUCTION: A survey of the members of the Angle Society of Europe showed that 60% of orthodontists took their own clinical photographs, 35% assigned the task to an auxiliary, and 5% hired professional clinical photographers. It is always useful to ensure that orthodontists' time is used to maximum effect. Clinical photography could be delegated to auxiliary staff. In this study, we assessed the quality of photographs taken by orthodontists to see whether those taken by orthodontic auxiliaries and clinical photographers are of comparable quality. METHODS: Fifty sets of orthodontic photographs were collected from each of 3 types of photographers: orthodontists, orthodontic auxiliaries, and professional clinical photographers. Four assessors scored each set for quality and detailed errors. The results were compared to determine whether there were differences between the quality of the photographs taken by the different groups. RESULTS AND CONCLUSIONS: Most of the photos taken by the 3 groups of photographers were judged to be good or acceptable. The results for extraoral photographs showed no statistically significant differences between the 3 groups for good (P = 0.398) and acceptable (P = 0.398) images. The results for intraoral photographs did not differ significantly for acceptable and unacceptable photographs, but orthodontists produced significantly more good-quality intraoral photographs (P = 0.046).

Disinfection of transboundary animal disease viruses on surfaces used in pork packing plants.

In the event of an intentional or accidental incursion of a transboundary animal disease (TAD) virus into the US, a major concern to the meat industry would be the potential contamination of packing plants by processing infected animals. TAD agents such as foot and mouth disease virus (FMDV), African swine fever virus (ASFV) and classical swine fever virus (CSFV) are found in swine products such as blood and feces and are present in the tissues of infected animals. To test the disinfection of TAD viruses in a pork-packing environment, a previously developed disinfection assay was used to test two biocides currently used by industry sanitarians, against TAD viruses dried on industry relevant surfaces in saline or swine products. With the exception of one virus, both commercial disinfectants tested were effective against the TAD viruses dried on steel, plastic, and sealed concrete surfaces in the absence of the swine products. Disinfectant activity was greatly inhibited in the presence of dried blood and meat juices. The acidic disinfectants were able to inactivate the viruses in swine feces whereas fecal material generally inhibited sodium hypochlorite-based disinfectants. These results highlight the importance of manufacturer-recommended pre-cleaning steps to remove gross soil before surface disinfection. Taken together, these data support the use of acid- and surfactant-containing commercial products for packing plant disinfection during a TAD virus outbreak event.

The cell-free integration of a polytopic mitochondrial membrane protein into liposomes occurs cotranslationally and in a lipid-dependent manner.

The ADP/ATP Carrier (AAC) is the most abundant transporter of the mitochondrial inner membrane. The central role that this transporter plays in cellular energy production highlights the importance of understanding its structure, function, and the basis of its pathologies. As a means of preparing proteoliposomes for the study of membrane proteins, several groups have explored the use of cell-free translation systems to facilitate membrane protein integration directly into preformed unilamellar vesicles without the use of surfactants. Using AAC as a model, we report for the first time the detergent-free reconstitution of a mitochondrial inner membrane protein into liposomes using a wheat germ-based in vitro translation system. Using a host of independent approaches, we demonstrate the efficient integration of AAC into vesicles with an inner membrane-mimetic lipid composition and, more importantly, that the integrated AAC is functionally active in transport. By adding liposomes at different stages of the translation reaction, we show that this direct integration is obligatorily cotranslational, and by synthesizing stable ribosome-bound nascent chain intermediates, we show that the nascent AAC polypeptide interacts with lipid vesicles while ribosome-bound. Finally, we show that the presence of the phospholipid cardiolipin in the liposomes specifically enhances AAC translation rate as well as the efficiency of vesicle association and integration. In light of these results, the possible mechanisms of liposome-assisted membrane protein integration during cell-free translation are discussed with respect to the mode of integration and the role of specific lipids.