Acetobacter pomorum in the Drosophila gut microbiota buffers against host metabolic impacts of dietary preservative formula and batch variation in dietary yeast.

Gut microbiota are fundamentally important for healthy function in animal hosts. Drosophila melanogaster is a powerful system for understanding host-microbiota interactions, with modulation of the microbiota inducing phenotypic changes that are conserved across animal taxa. Qualitative differences in diet, such as preservatives and dietary yeast batch variation, may affect fly health indirectly via microbiota, and may potentially have hitherto uncharacterized effects directly on the fly. These factors are rarely considered, controlled, and are not standardized among laboratories. Here, we show that the microbiota's impact on fly triacylglyceride (TAG) levels-a commonly-measured metabolic index-depends on both preservatives and yeast, and combinatorial interactions among the three variables. In studies of conventional, axenic, and gnotobiotic flies, we found that microbial impacts were apparent only on specific yeast-by-preservative conditions, with TAG levels determined by a tripartite interaction of the three experimental factors. When comparing axenic and conventional flies, we found that preservatives caused more variance in host TAG than microbiota status, and certain yeast-preservative combinations even reversed effects of microbiota on TAG. Preservatives had major effects in axenic flies, suggesting either direct effects on the fly or indirect effects via media. However, Acetobacter pomorum buffers the fly against this effect, despite the preservatives inhibiting growth, indicating that this bacterium benefits the host in the face of mutual environmental toxicity. Our results suggest that antimicrobial preservatives have major impacts on host TAG, and that microbiota modulates host TAG dependent on the combination of the dietary factors of preservative formula and yeast batch. IMPORTANCE Drosophila melanogaster is a premier model for microbiome science, which has greatly enhanced our understanding of the basic biology of host-microbe interactions. However, often overlooked factors such as dietary composition, including yeast batch variability and preservative formula, may confound data interpretation of experiments within the same lab and lead to different findings when comparing between labs. Our study supports this notion; we find that the microbiota does not alter host TAG levels independently. Rather, TAG is modulated by combinatorial effects of microbiota, yeast batch, and preservative formula. Specific preservatives increase TAG even in germ-free flies, showing that a commonplace procedure in fly husbandry alters metabolic physiology. This work serves as a cautionary tale that fly rearing methodology can mask or drive microbiota-dependent metabolic changes and also cause microbiota-independent changes.

Mitonuclear interactions shape both direct and parental effects of diet on fitness and involve a SNP in mitoribosomal 16s rRNA.

Nutrition is a primary determinant of health, but responses to nutrition vary with genotype. Epistasis between mitochondrial and nuclear genomes may cause some of this variation, but which mitochondrial loci and nutrients participate in complex gene-by-gene-by-diet interactions? Furthermore, it remains unknown whether mitonuclear epistasis is involved only in the immediate responses to changes in diet, or whether mitonuclear genotype might modulate sensitivity to variation in parental nutrition, to shape intergenerational fitness responses. Here, in Drosophila melanogaster, we show that mitonuclear epistasis shapes fitness responses to variation in dietary lipids and amino acids. We also show that mitonuclear genotype modulates the parental effect of dietary lipid and amino acid variation on offspring fitness. Effect sizes for the interactions between diet, mitogenotype, and nucleogenotype were equal to or greater than the main effect of diet for some traits, suggesting that dietary impacts cannot be understood without first accounting for these interactions. Associating phenotype to mtDNA variation in a subset of populations implicated a C/T polymorphism in mt:lrRNA, which encodes the 16S rRNA of the mitochondrial ribosome. This association suggests that directionally different responses to dietary changes can result from variants on mtDNA that do not change protein coding sequence, dependent on epistatic interactions with variation in the nuclear genome.

The importance of reaction norms in dietary restriction and ageing research.

Ageing research has progressed rapidly through our ability to modulate the ageing process. Pharmacological and dietary treatments can increase lifespan and have been instrumental in our understanding of the mechanisms of ageing. Recently, several studies have reported genetic variance in response to these anti-ageing interventions, questioning their universal application and making a case for personalised medicine in our field. As an extension of these findings the response to dietary restriction was found to not be repeatable when the same genetic mouse lines were retested. We show here that this effect is more widespread with the response to dietary restriction also showing low repeatability across genetic lines in the fly (Drosophila melanogaster). We further argue that variation in reaction norms, the relationship between dose and response, can explain such conflicting findings in our field. We simulate genetic variance in reaction norms and show that such variation can: 1) lead to over- or under-estimation of treatment responses, 2) dampen the response measured if a genetically heterogeneous population is studied, and 3) illustrate that genotype-by-dose-by-environment interactions can lead to low repeatability of DR and potentially other anti-ageing interventions. We suggest that putting experimental biology and personalised geroscience in a reaction norm framework will aid progress in ageing research.

Transcriptional memory of dFOXO activation in youth curtails later-life mortality through chromatin remodeling and Xbp1.

A transient, homeostatic transcriptional response can result in transcriptional memory, programming subsequent transcriptional outputs. Transcriptional memory has great but unappreciated potential to alter animal aging as animals encounter a multitude of diverse stimuli throughout their lifespan. Here we show that activating an evolutionarily conserved, longevity-promoting transcription factor, dFOXO, solely in early adulthood of female fruit flies is sufficient to improve their subsequent health and survival in midlife and late life. This youth-restricted dFOXO activation causes persistent changes to chromatin landscape in the fat body and requires chromatin remodelers such as the SWI/SNF and ISWI complexes to program health and longevity. Chromatin remodeling is accompanied by a long-lasting transcriptional program that is distinct from that observed during acute dFOXO activation and includes induction of Xbp1. We show that this later-life induction of Xbp1 is sufficient to curtail later-life mortality. Our study demonstrates that transcriptional memory can profoundly alter how animals age.

Multiscale analysis reveals that diet-dependent midgut plasticity emerges from alterations in both stem cell niche coupling and enterocyte size.

The gut is the primary interface between an animal and food, but how it adapts to qualitative dietary variation is poorly defined. We find that the Drosophila midgut plastically resizes following changes in dietary composition. A panel of nutrients collectively promote gut growth, which sugar opposes. Diet influences absolute and relative levels of enterocyte loss and stem cell proliferation, which together determine cell numbers. Diet also influences enterocyte size. A high sugar diet inhibits translation and uncouples intestinal stem cell proliferation from expression of niche-derived signals, but, surprisingly, rescuing these effects genetically was not sufficient to modify diet's impact on midgut size. However, when stem cell proliferation was deficient, diet's impact on enterocyte size was enhanced, and reducing enterocyte-autonomous TOR signaling was sufficient to attenuate diet-dependent midgut resizing. These data clarify the complex relationships between nutrition, epithelial dynamics, and cell size, and reveal a new mode of plastic, diet-dependent organ resizing.

Alternative monomer for BisGMA-free resin composites formulations.

OBJECTIVE: Water sorption, high volumetric shrinkage, polymerization stress, and potential estrogenic effects triggered by leached compounds are some of the major concerns related to BisGMA-TEGDMA co-monomer systems used in dental composites. These deficiencies call for the development of alternative organic matrices in order to maximize the clinical lifespan of resin composite dental restorations. This study proposes BisGMA-free systems based on the combination of UDMA and a newly synthesized diurethane dimethacrylate, and evaluates key mechanical and physical properties of the resulting materials. METHODS: 2EMATE-BDI (2-hydroxy-1-ethyl methacrylate) was synthesized by the reaction between 2-hydroxy-1-ethyl methacrylate with a difunctional isocyanate (1.3-bis (1- isocyanato-1-methylethylbenzene) - BDI). The compound was copolymerized with UDMA (urethane dimethacrylate) at 40 and 60wt%. UDMA copolymerizations with 40 and 60wt% TEGDMA (triethylene glycol dimethacrylate) were tested as controls, as well as a formulation based in BisGMA (bisphenol A-glycidyl methacrylate)-TEGDMA 60:40% (BT). The organic matrices were made polymerizable by the addition of DMPA (2.2-dimethoxyphenoxy acetophenone) and DPI-PF6 (diphenyliodonium hexafluorophosphate) at 0.2 and 0.4wt%, respectively. Formulations were tested as composite with the addition of 70wt% inorganic content consisting of barium borosilicate glass (0.7µm) and fumed silica mixed in 95 and 5wt%, respectively. All photocuring procedures were carried out by a mercury arc lamp filtered to 320-500nm at 800mW/cm2. The experimental resin composites were tested for kinetics of polymerization and polymerization stress in real time. Flexural strength, elastic modulus, water sorption, and solubility were assessed according to ISO 4049. Biofilm formation was analyzed after 24h by luciferase assay. Data were statistically analyzed by one-way ANOVA and Tukey's test (α≤0.05). RESULTS: In general, the addition of 2EMATE-BDI into the formulations decreased the maximum rate of polymerization (RPMAX), the degree of conversion at RPMAX (DC at RPMAX), and the final degree of conversion (final DC). However, these reductions did not compromise mechanical properties, which were comparable to the BT controls, especially after 7-day water incubation. The incorporation of 60wt% 2EMATE-BDI reduced water sorption of the composite. 2EMATE-BDI containing formulations showed reduction in polymerization stress of 30% and 50% in comparison to BT control and TEGDMA copolymerizations, respectively. Biofilm formation was similar among the tested groups. SIGNIFICANCE: The use of the newly synthesized diurethane dimethacrylate as co-monomer in dental resin composite formulations seems to be a promising option to develop polymers with low-shrinkage and potentially decreased water degradation.

The neuronal receptor tyrosine kinase Alk is a target for longevity.

Inhibition of signalling through several receptor tyrosine kinases (RTKs), including the insulin-like growth factor receptor and its orthologues, extends healthy lifespan in organisms from diverse evolutionary taxa. This raises the possibility that other RTKs, including those already well studied for their roles in cancer and developmental biology, could be promising targets for extending healthy lifespan. Here, we focus on anaplastic lymphoma kinase (Alk), an RTK with established roles in nervous system development and in multiple cancers, but whose effects on aging remain unclear. We find that several means of reducing Alk signalling, including mutation of its ligand jelly belly (jeb), RNAi knock-down of Alk, or expression of dominant-negative Alk in adult neurons, can extend healthy lifespan in female, but not male, Drosophila. Moreover, reduced Alk signalling preserves neuromuscular function with age, promotes resistance to starvation and xenobiotic stress, and improves night sleep consolidation. We find further that inhibition of Alk signalling in adult neurons modulates the expression of several insulin-like peptides, providing a potential mechanistic link between neuronal Alk signalling and organism-wide insulin-like signalling. Finally, we show that TAE-684, a small molecule inhibitor of Alk, can extend healthy lifespan in Drosophila, suggesting that the repurposing of Alk inhibitors may be a promising direction for strategies to promote healthy aging.

Toward Stimuli-Responsive Dynamic Thermosets through Continuous Development and Improvements in Covalent Adaptable Networks (CANs).

Covalent adaptable networks (CANs), unlike typical thermosets or other covalently crosslinked networks, possess a unique, often dormant ability to activate one or more forms of stimuli-responsive, dynamic covalent chemistries as a means to transition their behavior from that of a viscoelastic solid to a material with fluid-like plastic flow. Upon application of a stimulus, such as light or other irradiation, temperature, or even a distinct chemical signal, the CAN responds by transforming to a state of temporal plasticity through activation of either reversible addition or reversible bond exchange, either of which allows the material to essentially re-equilibrate to an altered set of conditions that are distinct from those in which the original covalently crosslinked network is formed, often simultaneously enabling a new and distinct shape, function, and characteristics. As such, CANs span the divide between thermosets and thermoplastics, thus offering unprecedented possibilities for innovation in polymer and materials science. Without attempting to comprehensively review the literature, recent developments in CANs are discussed here with an emphasis on the most effective dynamic chemistries that render these materials to be stimuli responsive, enabling features that make CANs more broadly applicable.

A randomised controlled trial of bag-valve-mask teaching techniques.

BACKGROUND: Bag-valve-mask (BVM) ventilation is a vital skill in the management of the collapsed patient; however, the quality of BVM ventilation is a cause for concern. Modified techniques, designed to be easier for the novice practitioner, offer an opportunity to improve quality. One such modification is the 'LASOO' (Lift, Apply, Slide, Oppose, Observe) approach, which offers theoretical benefits over the traditionally taught 'CE' (finger shapes) technique. We conducted a randomised controlled trial (RCT) to determine whether LASOO was superior to CE in terms of tidal volume, when taught to novices in the skills-lab setting. We conducted a randomised controlled trial (RCT) to determine whether LASOO was superior to CE in terms of tidal volume, when taught to novices in the skills-lab setting METHODS: A total of 76 undergraduate health care students received a manikin-based teaching session on LASOO or CE. They then delivered 20 breaths (10 with each hand) to a modified airway manikin. The primary outcome was mean tidal volume; secondary outcomes were the proportion of breaths that achieved 150-mL and 400-mL threshold volumes. Subgroup analyses and statistical modelling were conducted for time-point, hand dominance and hand size. RESULTS: The mean tidal volume was 320 mL for CE and 304 mL for LASOO. The median percentage of attempts that exceeded 150 mL was 85 for CE and 82.5 for LASOO. The median percentage of attempts that exceeded 400 mL was 20 for CE and 20 for LASOO. The differences recorded between the techniques were not statistically significant. There was a small, statistically significant increase in tidal volume across both techniques with time-point and holding the mask with the non-dominant hand. DISCUSSION: LASOO is a viable alternative to CE. Educators may opt to teach either or both techniques, allowing students to choose the technique that they prefer.

Combined Dynamic Network and Filler Interface Approach for Improved Adhesion and Toughness in Pressure-Sensitive Adhesives.

Of importance for adhesive materials, particularly pressure-sensitive adhesive (PSA) systems, is the ability to increase bulk toughness without reduction of adhesion. Previous approaches for increasing PSA durability sacrifice permanent cross-linking or adhesive potential, limiting performance. In this work, covalent adaptable networks (CANs) derived from thiol-thioester exchange (TTE) are utilized as a basis for adhesive films. Tensile and single-lap shear tests were conducted for adhesive materials containing no filler, 15 wt % nanoparticles functionalized with thioester-containing acrylate, or 15 wt % nanoparticles functionalized with nonthioester-containing acrylate. Additionally, fatigue experiments were conducted on unfilled adhesives. Results indicate that TTE improves toughness, adhesion, and fatigue in unfilled materials. Filled adhesives with activated TTE showed a nearly fourfold increase in adhesion with slightly reduced toughness compared to uncatalyzed filled specimens. This work has implications in many industries, from biomedical to automotive, as toughness and fatigue resistance are important considerations for adhesive applications.

Vinyl sulfonamide based thermosetting composites via thiol-Michael polymerization.

OBJECTIVE: To assess the performance of thiol Michael photocurable composites based on ester-free thiols and vinyl sulfonamides of varying monomer structures and varied filler loadings and to contrast the properties of the prototype composites with conventional BisGMA-TEGDMA methacrylate composite. METHODS: Synthetic divinyl sulfonamides and ester-free tetrafunctional thiol monomers were utilized for thiol-Michael composite development with the incorporation of thiolated microfiller. Polymerization kinetics was investigated using FTIR spectroscopy. Resin viscosities were assessed with rheometry. Water uptake properties were assessed according to standardized methods. Thermomechanical properties were analyzed by dynamic mechanical analysis. Flexural modulus/strength and flexural toughness were measured on a universal testing machine in three-point bending testing mode. RESULTS: The vinyl sulfonamide-based thiol-Michael resin formulation demonstrated a wide range of viscosities with a significant increase in the functional group conversion when compared to the BisGMA-TEGDMA system. The two different types of vinyl sulfonamide under investigation demonstrated significant differences towards the water sorption. Tertiary vinyl sulfonamide did not undergo visible swelling whereas the secondary vinyl sulfonamide composite swelled extensively in water. With the introduction of rigid monomer into the polymer matrix the glass transition temperature increased and so increased the toughness. Glassy thiol-Michael composites were obtained by ambient curing. SIGNIFICANCE: Employing the newly developed step-growth thiol-Michael resins in dental composites will provide structural uniformity, improved stability and lower water sorption.

Dynamic covalent chemistry (DCC) in dental restorative materials: Implementation of a DCC-based adaptive interface (AI) at the resin-filler interface for improved performance.

OBJECTIVE: Dental restorative composites have been extensively studied with a goal to improve material performance. However, stress induced microcracks from polymerization shrinkage, thermal and other stresses along with the low fracture toughness of methacrylate-based composites remain significant problems. Herein, the study focuses on applying a dynamic covalent chemistry (DCC)-based adaptive interface to conventional BisGMA/TEGDMA (70:30) dental resins by coupling moieties capable of thiol-thioester (TTE) DCC to the resin-filler interface as a means to induce interfacial stress relaxation and promote interfacial healing. METHODS: Silica nanoparticles (SNP) are functionalized with TTE-functionalized silanes to covalently bond the interface to the network while simultaneously facilitating relaxation of the filler-matrix interface via DCC. The functionalized particles were incorporated into the otherwise static conventional BisGMA/TEGDMA (70:30) dental resins. The role of interfacial bond exchange to enhance dental composite performance in response to shrinkage and other stresses, flexural modulus and toughness was investigated. Shrinkage stress was monitored with a tensometer coupled with FTIR spectroscopy. Flexural modulus/strength and flexural toughness were characterized in three-point bending on a universal testing machine. RESULTS: A reduction of 30% in shrinkage stress was achieved when interfacial TTE bond exchange was activated while not only maintaining but also enhancing mechanical properties of the composite. These enhancements include a 60% increase in Young's modulus, 33% increase in flexural strength and 35% increase in the toughness, relative to composites unable to undergo DCC but otherwise identical in composition. Furthermore, by combining interfacial DCC with resin-based DCC, an 80% reduction of shrinkage-induced stress is observed in a thiol-ene system "equipped" with both types of DCC mechanisms relative to the composite without DCC in either the resin or at the resin-filler interface. SIGNIFICANCE: This behavior highlights the advantages of utilizing the DCC at the resin-filler interface as a stress-relieving mechanism that is compatible with current and future developments in the field of dental restorative materials, nearly independent of the type of resin improvements and types that will be used, as it can dramatically enhance their mechanical performance by reducing both polymerization and mechanically applied stresses throughout the composite lifetime.

Antibacterial, ester-free monomers: Polymerization kinetics, mechanical properties, biocompatibility and anti-biofilm activity.

OBJECTIVES: Quaternary ammonium (QA) methacrylate monomers have been extensively investigated and demonstrate excellent antibacterial properties. However, the presence of ester bonds makes them prone to degradation in the oral cavity. In this study, ester-free QA monomers based on meth-acrylamides were synthesized and screened for polymerization kinetics, mechanical properties and antibacterial effects. MATERIALS AND METHODS: Tertiary quaternary ammonium acrylamides (AM) and methacrylamides (MAM) with alkyl side chain lengths of 9 and 14 carbons (C9 and C14) were synthesized and incorporated at 10 wt% into experimental composites based on BisGMA:TEGDMA (1:1), camphorquinone/ethyl-4-dimethylaminobenzoate (0.2/0.8 wt%) and 70 wt% barium glass fillers. Analogous methacrylate versions (MA) were used as controls. Degree of conversion (DC) and rate of polymerization (RP) during photoactivation (800 mW/cm2) were followed in real-time with near-IR. Flexural Strength (FS) and Modulus (E) were measured on 2 × 2 × 25 mm bars in 3-point bending after 24 h dry storage and 7-day storage in water at 37 °C. Antimicrobial properties and biofilm adhesion (fouling) were evaluated by bioluminescence (Luciferase Assay) and biofilm removal by water spray microjet impingement test, respectively. Cytotoxicity was assessed by MTT assay on dental pulp stem cells (DPSC). Data were analyzed with one-way ANOVA/Tukey's test (α = 0.05). RESULTS: DC was similar for all groups tested (∼70%). Both MAMs and C14-AM presented significantly lower RP. Under dry conditions, FS (110-120 MPa) and E (8-9 GPa) were similar for all groups. After water storage, all materials presented FS/E similar to the control, except for C14-AM (for FS) and C14-MAM (for E), which were lower. All C14 versions were strongly antibacterial, decreasing the titer counts of biofilm by more than two orders of magnitude in comparison to the control. C9 monomers did not present significant antibacterial nor antifouling properties. And biofilms had approximately equivalent adhesion on the C9 composites as on the control. Cytotoxicity did not show significant differences between the MA and AM versions and the control group. CONCLUSIONS: C14-QA monomers based on methacrylates and meth-acrylamides present strong antibacterial properties, and in general, similar conversion/mechanical properties compared to the methacrylate control. STATEMENT OF SIGNIFICANCE: This work demonstrates the viability of methacrylamides and acrylamides as potential components in dental restorative materials with antimicrobial properties. The use of ester-free polymerizable functionalities has the potential of improving the degradation resistance of these materials long-term. The use of (meth)acrylamides did not interfere with the antimicrobial potential of quaternary ammonium-based materials.

Effect of residual solvent on performance of acrylamide-containing dental materials.

OBJECTIVE: Methacrylamide-based monomers are being pursued as novel, hydrolytically stable materials for use in dental adhesives. The impact of residual solvents, due to the chemical synthesis procedures or the need for solvated adhesives systems, on the kinetics of polymerization and mechanical properties was the aim of the present investigation. METHODS: Two base monomers (70wt% BisGMA or HEMAM-BDI - newly synthesized secondary methacrylamide) were combined with 30wt% N,N-dimethylacrylamide. Eethyl acetate (EtOAc), or 75vol% ethanol/25vol% water (EtOH/H2O) were added as solvents in concentrations of 2, 5, 15 and 20wt%. The resins were made polymerizable by the addition of 0.2wt% 2,2-dimethoxy-2-phenyl acetophenone (DMPA) and 0.4wt% diphenyliodonium hexafluorophosphate (DPI-PF6). Specimens (n=3) were photoactivated with a mercury arc lamp (Acticure 4000, 320-500nm, 250mW/cm2) for 5min. Degree of conversion (DC, %) was tracked in near-IR spectroscopy in real time and yield strength and modulus of elasticity were measured in three-point bending after dry and wet storage (n=6). The data was subject to one-way ANOVA/Tukey's Test (p≤0.05), or Student's t-test (p≤0.001). RESULTS: In all groups for both BisGMA and HEMAM-BDI-based materials, DC and DC at Rpmax increased and maximum rate of polymerization decreased as solvent concentration increased. Despite the increased DC, BisGMA mixtures showed a decrease in FS starting at 5wt% EtOAc or 15wt% EtOH/H2O. Yield strength for the HEMAM-BDI groups was overall lower than that of the BisGMA groups, but the modulus of elasticity was significantly higher. SIGNIFICANCE: The presence of residual solvent, from manufacturing or from practitioner's handling, affects polymerization kinetics and mechanical properties of resins. Methacrylates appear to be more strongly influenced than methacrylamides.

Longevity is determined by ETS transcription factors in multiple tissues and diverse species.

Ageing populations pose one of the main public health crises of our time. Reprogramming gene expression by altering the activities of sequence-specific transcription factors (TFs) can ameliorate deleterious effects of age. Here we explore how a circuit of TFs coordinates pro-longevity transcriptional outcomes, which reveals a multi-tissue and multi-species role for an entire protein family: the E-twenty-six (ETS) TFs. In Drosophila, reduced insulin/IGF signalling (IIS) extends lifespan by coordinating activation of Aop, an ETS transcriptional repressor, and Foxo, a Forkhead transcriptional activator. Aop and Foxo bind the same genomic loci, and we show that, individually, they effect similar transcriptional programmes in vivo. In combination, Aop can both moderate or synergise with Foxo, dependent on promoter context. Moreover, Foxo and Aop oppose the gene-regulatory activity of Pnt, an ETS transcriptional activator. Directly knocking down Pnt recapitulates aspects of the Aop/Foxo transcriptional programme and is sufficient to extend lifespan. The lifespan-limiting role of Pnt appears to be balanced by a requirement for metabolic regulation in young flies, in which the Aop-Pnt-Foxo circuit determines expression of metabolic genes, and Pnt regulates lipolysis and responses to nutrient stress. Molecular functions are often conserved amongst ETS TFs, prompting us to examine whether other Drosophila ETS-coding genes may also affect ageing. We show that five out of eight Drosophila ETS TFs play a role in fly ageing, acting from a range of organs and cells including the intestine, adipose and neurons. We expand the repertoire of lifespan-limiting ETS TFs in C. elegans, confirming their conserved function in ageing and revealing that the roles of ETS TFs in physiology and lifespan are conserved throughout the family, both within and between species.

Use of (meth)acrylamides as alternative monomers in dental adhesive systems.

OBJECTIVES: Methacrylamides are proposed as components for dental adhesive systems with enhanced resistance to hydrolytic and enzymatic degradation. The specific objective of this study was to evaluate the polymerization kinetics, water sorption and solubility, pH-derived degradation and microtensile bond strength of various monofunctional acrylamides and meth(acrylamides) when copolymerized with dimethacrylates. METHODS: Base monomers were added at 60 wt%, and included either BisGMA or UDMA. Monofunctional monomers were added at 40 wt%, including one (meth)acrylate as the control, two secondary methacrylamides and two tertiary acrylamides. DMPA (0.2 wt%) and DPI-PF6 (0.4 wt%)/BHT (0.1 wt%) were added as initiators/inhibitor. Polymerization kinetics wwere followed with near-IR spectroscopy in real time. Water sorption (WS) and solubility (SL) were measured following ISO 4049. Monomer degradation at different pH levels was assessed with 1H NMR. Microtensile bond strength (MTBS) was assessed in caries-free human third molars 48 h and 3 weeks after restorations were placed using solvated BisGMA-based adhesives (40 vol% ethanol). Data were analyzed with one-way ANOVA/Tukey's test (α = 0.05). RESULTS: As expected, rate of polymerization and final degree of conversion (DC) were higher for the acryl versions of each monomer, and decreased with increasing steric hindrance around the vinyl group for each molecule. In general, UDMA copolymerizations were more rapid and extensive than for BisGMA, but this was dependent upon the specific monofunctional monomer added. WS/SL were in general higher for the (meth)acrylamides compared to the (meth)acrylates, except for the tertiary acrylamide, which showed the lowest values. One of the secondary methacrylamides was significantly more stable than the methacrylate control, but the alpha substitutions decreased stability to degradation in acid pH. MTBS in general was higher for the (meth)acrylates. While for all materials the MTBS values at 3 weeks decreased in relation to the 24 h results, the tertiary acrylamide showed no reduction in bond strength. SIGNIFICANCE: This study highlights the importance of considering steric and electronic factors when designing monomers for applications where rapid polymerizations are needed, especially when co-polymerizations with other base monomers are required to balance mechanical properties, as is the case with dental adhesives. The results of this investigation will be used to design fully formulated adhesives to be tested in clinically-relevant conditions.

Tissue-specific transcriptome profiling of Drosophila reveals roles for GATA transcription factors in longevity by dietary restriction.

Dietary restriction (DR) extends animal lifespan, but imposes fitness costs. This phenomenon depends on dietary essential amino acids (EAAs) and TOR signalling, which exert systemic effects. However, the roles of specific tissues and cell-autonomous transcriptional regulators in diverse aspects of the DR phenotype are unknown. Manipulating relevant transcription factors (TFs) specifically in lifespan-limiting tissues may separate the lifespan benefits of DR from the early-life fitness costs. Here, we systematically analyse transcription across organs of Drosophila subjected to DR or low TOR and predict regulatory TFs. We predict and validate roles for the evolutionarily conserved GATA family of TFs, and identify conservation of this signal in mice. Importantly, restricting knockdown of the GATA TF srp to specific fly tissues recapitulated the benefits but not the costs of DR. Together, our data indicate that the GATA TFs mediate effects of dietary amino acids on lifespan, and that by manipulating them in specific tissues it is possible to reap the fitness benefits of EAAs, decoupled from a cost to longevity.

The Drosophila melanogaster Gut Microbiota Provisions Thiamine to Its Host.

The microbiota of Drosophila melanogaster has a substantial impact on host physiology and nutrition. Some effects may involve vitamin provisioning, but the relationships between microbe-derived vitamins, diet, and host health remain to be established systematically. We explored the contribution of microbiota in supplying sufficient dietary thiamine (vitamin B1) to support D. melanogaster at different stages of its life cycle. Using chemically defined diets with different levels of available thiamine, we found that the interaction of thiamine concentration and microbiota did not affect the longevity of adult D. melanogaster Likewise, this interplay did not have an impact on egg production. However, we determined that thiamine availability has a large impact on offspring development, as axenic offspring were unable to develop on a thiamine-free diet. Offspring survived on the diet only when the microbiota was present or added back, demonstrating that the microbiota was able to provide enough thiamine to support host development. Through gnotobiotic studies, we determined that Acetobacter pomorum, a common member of the microbiota, was able to rescue development of larvae raised on the no-thiamine diet. Further, it was the only microbiota member that produced measurable amounts of thiamine when grown on the thiamine-free fly medium. Its close relative Acetobacter pasteurianus also rescued larvae; however, a thiamine auxotrophic mutant strain was unable to support larval growth and development. The results demonstrate that the D. melanogaster microbiota functions to provision thiamine to its host in a low-thiamine environment.IMPORTANCE There has been a long-standing assumption that the microbiota of animals provides their hosts with essential B vitamins; however, there is not a wealth of empirical evidence supporting this idea, especially for vitamin B1 (thiamine). To determine whether this assumption is true, we used Drosophila melanogaster and chemically defined diets with different thiamine concentrations as a model. We found that the microbiota does provide thiamine to its host, enough to allow the development of flies on a thiamine-free diet. The power of the Drosophila-microbiota system allowed us to determine that one microbiota member in particular, Acetobacter pomorum, is responsible for the thiamine provisioning. Thereby, our study verifies this long-standing hypothesis. Finally, the methods used in this work are applicable for interrogating the underpinnings of other aspects of the tripartite interaction between diet, host, and microbiota.

RNA polymerase III limits longevity downstream of TORC1.

Three distinct RNA polymerases transcribe different classes of genes in the eukaryotic nucleus. RNA polymerase (Pol) III is the essential, evolutionarily conserved enzyme that generates short, non-coding RNAs, including tRNAs and 5S rRNA. The historical focus on transcription of protein-coding genes has left the roles of Pol III in organismal physiology relatively unexplored. Target of rapamycin kinase complex 1 (TORC1) regulates Pol III activity, and is also an important determinant of longevity. This raises the possibility that Pol III is involved in ageing. Here we show that Pol III limits lifespan downstream of TORC1. We find that a reduction in Pol III extends chronological lifespan in yeast and organismal lifespan in worms and flies. Inhibiting the activity of Pol III in the gut of adult worms or flies is sufficient to extend lifespan; in flies, longevity can be achieved by Pol III inhibition specifically in intestinal stem cells. The longevity phenotype is associated with amelioration of age-related gut pathology and functional decline, dampened protein synthesis and increased tolerance of proteostatic stress. Pol III acts on lifespan downstream of TORC1, and limiting Pol III activity in the adult gut achieves the full longevity benefit of systemic TORC1 inhibition. Hence, Pol III is a pivotal mediator of this key nutrient-signalling network for longevity; the growth-promoting anabolic activity of Pol III mediates the acceleration of ageing by TORC1. The evolutionary conservation of Pol III affirms its potential as a therapeutic target.