Small Open Reading Frames, How to Find Them and Determine Their Function

Advances in genomics and molecular biology have revealed an abundance of small open reading frames (sORFs) across all types of transcripts. While these sORFs are often assumed to be non-functional, many have been implicated in physiological functions and a significant number of sORFs have been described in human diseases. Thus, sORFs may represent a hidden repository of functional elements that could serve as therapeutic targets. Unlike protein-coding genes, it is not necessarily the encoded peptide of an sORF that enacts its function, sometimes simply the act of translating an sORF might have a regulatory role. Indeed, the most studied sORFs are located in the 5′UTRs of coding transcripts and can have a regulatory impact on the translation of the downstream protein-coding sequence. However, sORFs have also been abundantly identified in non-coding RNAs including lncRNAs, circular RNAs and ribosomal RNAs suggesting that sORFs may be diverse in function. Of the many different experimental methods used to discover sORFs, the most commonly used are ribosome profiling and mass spectrometry. These can confirm interactions between transcripts and ribosomes and the production of a peptide, respectively. Extensions to ribosome profiling, which also capture scanning ribosomes, have further made it possible to see how sORFs impact the translation initiation of mRNAs. While high-throughput techniques have made the identification of sORFs less difficult, defining their function, if any, is typically more challenging. Together, the abundance and potential function of many of these sORFs argues for the necessity of including sORFs in gene annotations and systematically characterizing these to understand their potential functional roles. In this review, we will focus on the high-throughput methods used in the detection and characterization of sORFs and discuss techniques for validation and functional characterization.publishedVersio

Endodontic sealers after exposure to chlorhexidine digluconate:An assessment of physicochemical properties

Objectives Final root canal irrigation should ideally maintain the physicochemical stability of root canal sealers. We seek to assess the effect of contact with 2% chlorhexidine digluconate (CHX) on the physicochemical properties of AH Plus, BioRoot™ RCS, and Pulp Canal Sealer (PCS). Methods Mixed sealers were placed in cylindrical teflon molds and allowed to set for 1.5x the manufacturers’ setting time. Half of the specimens had their free surface in contact with CHX for the first minute of their setting period. Solubility, radiopacity, surface roughness, microhardness and wettability of the sealers were assessed up to 28 days after setting. Elemental analysis of sealer surfaces and their leachates together with pH measurements were also performed. Appropriate parametric and non-parametric analysis with post hoc tests were performed (p < 0.05). Results Exposure to CHX had no effect on solubility and radiopacity of all sealers. CHX altered the surface roughness of PCS and BioRoot RCS (p < 0.05). Contact with CHX reduced the microhardness of AH Plus and PCS (p < 0.05). AH Plus was more hydrophilic after CHX contact, whereas PCS became more hydrophobic (p < 0.05). AH Plus and PCS surfaces appeared to adsorb CHX as exhibited by chlorine peaks after contact with CHX. Sealer leachates’ alkalinity was not affected. CHX increased elution of silicon and zirconium for BioRoot and zinc for PCS leachates. Significance In our study, CHX affected sealers’ physicochemical properties to various extents. Further studies are needed to confirm the obtained results by investigating various final irrigation strategies and correlating to biological properties

CHOPCHOP v3: expanding the CRISPR web toolbox beyond genome editing

The CRISPR–Cas system is a powerful genome editing tool that functions in a diverse array of organisms and cell types. The technology was initially developed to induce targeted mutations in DNA, but CRISPR–Cas has now been adapted to target nucleic acids for a range of purposes. CHOPCHOP is a web tool for identifying CRISPR–Cas single guide RNA (sgRNA) targets. In this major update of CHOPCHOP, we expand our toolbox beyond knockouts. We introduce functionality for targeting RNA with Cas13, which includes support for alternative transcript isoforms and RNA accessibility predictions. We incorporate new DNA targeting modes, including CRISPR activation/repression, targeted enrichment of loci for long-read sequencing, and prediction of Cas9 repair outcomes. Finally, we expand our results page visualization to reveal alternative isoforms and downstream ATG sites, which will aid users in avoiding the expression of truncated proteins. The CHOPCHOP web tool now supports over 200 genomes and we have released a command-line script for running larger jobs and handling unsupported genomes. CHOPCHOP v3 can be found at https://chopchop.cbu.uib.nopublishedVersio

Effect of chlorhexidine digluconate on antimicrobial activity, cell viability and physicochemical properties of three endodontic sealers

ObjectiveAssess the biological and physicochemical properties of AH Plus, BioRoot RCS and Pulp Canal Sealer (PCS) leachates with and without chlorhexidine (CHX).MethodsThe sealers were studied in no contact and 1-minute contact with CHX. For biological properties (antibacterial activity and cytotoxicity), leachates were formed in saline of freshly mixed, 1-, 7- and 28 days set sealers. The antibacterial properties of sealer leachates were investigated for planktonic and biofilm growth of E. faecalis, S. mutans, S.epidermidis and S.aureus. The 3-(4,5 dimethylthiazolyl-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to evaluate murine fibroblast cell viability after exposure to the leachates. The physical properties (water uptake, sorption, solubility, porosity, surface characteristics) of sealers and the pH of the immersion liquid (saline or distilled water) were also assessed over a 28-days period.ResultsCHX improved the antibacterial properties of the sealer leachates and reduced cell viability for all sealer leachates, except for freshly mixed PCS. BioRoot RCS leachates presented the highest antibacterial properties and cell viability with and without CHX contact. PCS was the material most affected by CHX in terms of physical properties, whereas for AH Plus, solubility was increased. CHX did not affect the physical properties of BioRoot RCS, except for solubility that was decreased. CHX contact did not change sealers’ alkalinity in distilled water whereas it increased it for AH Plus and BioRoot RCS in saline.SignificanceCHX improved the antibacterial efficacy of sealer leachates and either compromised or did not affect cell viability. CHX affected to various extent sealers’ physicochemical properties

Estimating molar-incisor-hypomineralization among 8-year-olds based on 15-year public oral health practice-based data

Background:A wide range in the prevalence of molar-incisor-hypomineralization (MIH) has been reported. Population-based studies are recommended. However, such studies are expensive and time-consuming. Objectives:To estimate the magnitude of MIH condition among 8-year-olds based on routine oral health examinations and to associate first permanent molar (FPM) affection with that of other permanent teeth over time. Materials and methods:This retrospective study, with cross-sectional and longitudinal components, was based on electronic oral health records; all 8-year-olds examined between 2002 and 2016 were included. Results:The average estimated prevalence of MIH was 8.3%; yearly range was 4.8-15.9%. The mean number of affected teeth was 1.4; 62% had one affected tooth. One-surface defects were the most frequent (66%). Asymmetric distribution of affected teeth was observed. In follow-up, 10.7%, 8.4%, and 11.2% had at least one affected permanent canine, premolar, or second permanent molar, respectively. The proportion of children with other MIH-affected permanent teeth was higher in the group with >= 2 MIH-affected teeth than in group with one affected FPM at the age of 8. Conclusions:The average prevalence of MIH was comparable to that reported elsewhere. The number of MIH-affected teeth at early mixed dentition predicts the affection of other permanent teeth over time, mainly that of permanent canines. Further screening of children with MIH is recommended to improve individually tailored early preventive and restorative dental care.Peer reviewe

Survival of primary molars with pulpotomy interventions : public oral health practice-based study in Helsinki

Objectives To assess long-term survival of pulpotomized primary molars and factors related to the survival of the teeth. Materials and methods This retrospective longitudinal study was based on electronic dental health records of children who attended public health service. Eligible children werePeer reviewe

Estimating molar-incisor-hypomineralization among 8-year-olds based on 15-year public oral health practice-based data

Background:A wide range in the prevalence of molar-incisor-hypomineralization (MIH) has been reported. Population-based studies are recommended. However, such studies are expensive and time-consuming. Objectives:To estimate the magnitude of MIH condition among 8-year-olds based on routine oral health examinations and to associate first permanent molar (FPM) affection with that of other permanent teeth over time. Materials and methods:This retrospective study, with cross-sectional and longitudinal components, was based on electronic oral health records; all 8-year-olds examined between 2002 and 2016 were included. Results:The average estimated prevalence of MIH was 8.3%; yearly range was 4.8-15.9%. The mean number of affected teeth was 1.4; 62% had one affected tooth. One-surface defects were the most frequent (66%). Asymmetric distribution of affected teeth was observed. In follow-up, 10.7%, 8.4%, and 11.2% had at least one affected permanent canine, premolar, or second permanent molar, respectively. The proportion of children with other MIH-affected permanent teeth was higher in the group with >= 2 MIH-affected teeth than in group with one affected FPM at the age of 8. Conclusions:The average prevalence of MIH was comparable to that reported elsewhere. The number of MIH-affected teeth at early mixed dentition predicts the affection of other permanent teeth over time, mainly that of permanent canines. Further screening of children with MIH is recommended to improve individually tailored early preventive and restorative dental care.Peer reviewe

Profiling of Small Ribosomal Subunits Reveals Modes and Regulation of Translation Initiation

Translation initiation is often attributed as the rate-determining step of eukaryotic protein synthesis and key to gene expression control. Despite this centrality, the series of steps involved in this process is poorly understood. Here, we capture the transcriptome-wide occupancy of ribosomes across all stages of translation initiation, enabling us to characterize the transcriptome-wide dynamics of ribosome recruitment to mRNAs, scanning across 5′ UTRs and stop codon recognition, in a higher eukaryote. We provide mechanistic evidence for ribosomes attaching to the mRNA by threading the mRNA through the small subunit. Moreover, we identify features that regulate the recruitment and processivity of scanning ribosomes and redefine optimal initiation contexts. Our approach enables deconvoluting translation initiation into separate stages and identifying regulators at each step.publishedVersio