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The Fanconi anemia group C protein (FANCC) is one of the several proteins that comprise the Fanconi anemia (FA) network involved in genomic surveillance. FANCC is mainly cytoplasmic and has many functions, including apoptosis suppression through caspase-mediated proteolytic processing. Here, we examined the role of FANCC proteolytic fragments by identifying their binding partners. We performed a yeast two-hybrid screen with caspase-mediated FANCC cleavage products and identified the dependence receptor uncoordinated-5A (UNC5A) protein. Here, we show that FANCC physically interacts with UNC5A, a pro-apoptotic dependence receptor. FANCC interaction occurs through the UNC5A intracellular domain, specifically via its death domain. FANCC modulates cell sensitivity to UNC5A-mediated apoptosis; we observed reduced UNC5A-mediated apoptosis in the presence of FANCC and increased apoptosis in FANCC-depleted cells. Our results show that FANCC interferes with UNC5A's functions in apoptosis and suggest that FANCC may participate in developmental processes through association with the dependence receptor UNC5A. [ABSTRACT FROM AUTHOR]

To gain insight into presenilin-1 (PS1) structural aspects, we explored the structure–function relationship of its N- and C-terminal (NTF and CTF, respectively) complexes. We demonstrated that both NTF and CTF act as independent but inter-changing binding units capable of binding each other (NTF/CTF) or their homologues (NTF/NTF; CTF/CTF). The Alzheimer’s disease-associated PS1 mutations Y115H and M146L do not affect their ability to hetero- and/or homodimerize, thus conserving their basic integrity and function(s). These results suggest that PS1 associates intra-molecularly to form higher order complexes, which may be needed for endoproteolytic cleavage and/or γ-secretase-associated activity. [Copyright &y& Elsevier]

A growing body of evidence indicates that presenilins could exist and be active as oligomeric complexes. Using yeast two-hybrid and cell culture analysis, we provide evidence that presenilin-1 (PS1) may self-oligomerize giving rise to specific full-length/full-length homodimers. When expressed in N2A and HEK239T cultured cells, full-length PS1-wt and 5′myc-PS1-wt form specific homodimers corresponding to twice their molecular weight. The Alzheimer’s disease-associated PS1 mutations Y115H, M146L, L392V, ΔE10(PS11–289/320–467), the γ-secretase dominant negative mutant D257A, and the PS1 polymorphism mutant E318G do not affect their ability to self-oligomerize. Under non-denaturing conditions, endogenous PS1 forms specific homo-oligomers in human cultured cells. The results obtained herein suggest that PS1 associates intramolecularly to form higher order complexes, which may be needed for endoproteolytic cleavage and/or γ-secretase-associated activity. [Copyright &y& Elsevier]

The function of the Fanconi anemia group C protein (FANCC) is still unknown, though many studies point to a role in damage response signaling. Unlike other known FA proteins, FANCC is mainly localized to the cytoplasm and is thought to act as a messenger of cellular damage rather than an effector of repair. FANCC has been shown to interact with several cytoplasmic and nuclear proteins and to delay the onset of apoptosis through redox regulation of GSTP1. We investigated the fate and function of FANCC during apoptosis. Here we show that FANCC undergoes proteolytic modification by a caspase into a predominant 47-kDa ubiquitinated protein fragment. Lack of proteolytic modification at the putative cleavage site delays apoptosis but does not affect MMC complementation. These results suggest that FANCC function is regulated through proteolytic processing. [ABSTRACT FROM AUTHOR]

Antimicrobial resistance (AMR) is a public health threat worldwide. Next-generation sequencing (NGS) has opened unprecedented opportunities to accelerate AMR mechanism discovery and diagnostics. Here, we present an integrative approach to investigate trimethoprim (TMP) resistance in the key pathogen Streptococcus pneumoniae . We explored a collection of 662 S . pneumoniae genomes by conducting a genome-wide association study (GWAS), followed by functional validation using resistance reconstruction experiments, combined with machine learning (ML) approaches to predict TMP minimum inhibitory concentration (MIC). Our study showed that multiple additive mutations in the folA and sulA loci are responsible for TMP non-susceptibility in S. pneumoniae and can be used as key features to build ML models for digital MIC prediction, reaching an average accuracy within ±1 twofold dilution factor of 86.3%. Our roadmap of in silico analysis-wet-lab validation-diagnostic tool building could be adapted to explore AMR in other combinations of bacteria-antibiotic., Importance: In the age of next-generation sequencing (NGS), while data-driven methods such as genome-wide association study (GWAS) and machine learning (ML) excel at finding patterns, functional validation can be challenging due to the high numbers of candidate variants. We designed an integrative approach combining a GWAS on S. pneumoniae clinical isolates, followed by whole-genome transformation coupled with NGS to functionally characterize a large set of GWAS candidates. Our study validated several phenotypic folA mutations beyond the standard Ile100Leu mutation, and showed that the overexpression of the sulA locus produces trimethoprim (TMP) resistance in Streptococcus pneumoniae . These validated loci, when used to build ML models, were found to be the best inputs for predicting TMP minimal inhibitory concentrations. Integrative approaches can bridge the genotype-phenotype gap by biological insights that can be incorporated in ML models for accurate prediction of drug susceptibility., Competing Interests: The authors declare no conflict of interest.

Two strains of Streptococcus pneumoniae, one expressing the methyltransferase Erm(B) and the other negative for erm (B), were selected for solithromycin resistance in vitro either with direct drug selection or with chemical mutagenesis followed by drug selection. We obtained a series of mutants that we characterized by next-generation sequencing. We found mutations in various ribosomal proteins (L3, L4, L22, L32, and S4) and in the 23S rRNA. We also found mutations in subunits of the phosphate transporter, in the DEAD box helicase CshB, and in the erm (B)L leader peptide. All mutations were shown to decrease solithromycin susceptibility when transformed into sensitive isolates. Some of the genes derived from our in vitro screens were found to be mutated also in clinical isolates with decreased susceptibility to solithromycin. While many mutations were in coding sequences, some were found in regulatory regions. These included novel phenotypic mutations in the intergenic regions of the macrolide resistance locus mef (E)/ mel and in the vicinity of the ribosome binding site of erm (B). Our screens highlighted that macrolide-resistant S. pneumoniae can easily acquire resistance to solithromycin, and they revealed many new phenotypic mutations., Competing Interests: The authors declare no conflict of interest.

Summary: It has been previously demonstrated that the Notch1 signalling pathway is impaired in presenilin-1 null cells. This observation suggests a role for presenilin-1 in the Notch1 developmental pathway, possibly through physical interaction. Here, we show that presenilin-1 and Notch1 do not interact directly with each other but are associated in the cell. These findings raise the possibility that the gamma-secretase cleavage occurs via a presenilin complex in association with a putative co-factor specific for the molecule that is being cleaved (e.g. Notch1, (beta-amyloid precursor protein, E-cadherin and ErbB-4, all of which are gamma-secretase substrates).

Light-emitting sources are currently used to activate the setting reaction of restorative composite resins. It is well known that the polymerization extent of the resins will highly influence their clinical behavior. The monomer-polymer transformation will mostly depend on the chemical nature of the photo-initiators used and on the luminous energy given by the curing device. The aims of this study were to characterize three light curing devices and to compare their efficiency to polymerize a composite resin (SureFil). For that purpose, the emission spectra and the irradiance of a plasma arc (Flipo), a QTH visible light (Elipar Trilight), and a LEDs curing device (Elipar Freelight) were measured. Then, the depth of cure and the Vickers hardness of composite samples were evaluated, for each curing device, as a function of the exposure time. The emission spectra obtained showed the irradiance of the visible light emitted as a function of the wavelength, which was different for each light-curing device. The maximum cured depth (approximately 4 mm) give only a qualitative indication of the extent of the polymerization, as the hardness of the composite samples diminished as a function of the depth. Moreover, it strongly depended on the exposure time. Hence, to obtain a hardness of approximately 100 HV 0.5 at 2 mm depth, the illumination time must be at least of 3 s with the plasma arc Flipo, 10 s with the LEDs Elipar Freelight and 20 s with the QTH Elipar Trilight. In order to efficiently polymerize a composite resin, the emission spectra of the luminous source shall correspond with the absorption spectra of the photo-initiators. The present study showed that the LEDs Elipar Freelight is the most efficient curing device when the camphorquinone is used, as 92% of the emitted energy will be absorbed by the initiator. However, the power of this source is relatively low, hence higher exposure time shall be used.

The objectives of this study were (1) to investigate the effect of operator variability on the shear bond strength of adhesives to dentin and (2) to determine the effectiveness of education on bonding performance for different types of adhesives. Thirty general practitioners were recruited for a CE course by a regional mailing. They used bovine dentin as a substrate for bonding the adhesive system they routinely used in practice and two other materials (no previous experience). Each adhesive OptiBond FL, ScotchBond Multipurpose Plus, ScotchBond 1 and Clearfil SE was applied according to manufacturer's instructions and immediately tested using a shear bond strength test. Shear bond strengths between adhesives and dentin were compared before and after a 90 min lecture on bonding principles and materials. For dentists with and without previous experience with a material, there were no statistically significant differences seen before and after the lecture (paired t-tests, p < or = 0.05). However, in every case, the bond strengths after the lecture were higher than those before (range of improvement from 15 to 150%). For dentists with routine experience with a particular material, all materials were statistically equivalent after the lecture, although the OptiBond FL was the highest. For dentists who had no previous experience with a material, the ScotchBond 1 had lower bond strengths than the other materials after the lecture. There was a large range in the ability of dentists to manipulate adhesive systems correctly. However, if a dentist has sufficient experience and receives sufficient education, any of these materials can give reasonable results.