Antibody agonists trigger immune receptor signaling through local exclusion of receptor-type protein tyrosine phosphatases.

Antibodies can block immune receptor engagement or trigger the receptor machinery to initiate signaling. We hypothesized that antibody agonists trigger signaling by sterically excluding large receptor-type protein tyrosine phosphatases (RPTPs) such as CD45 from sites of receptor engagement. An agonist targeting the costimulatory receptor CD28 produced signals that depended on antibody immobilization and were sensitive to the sizes of the receptor, the RPTPs, and the antibody itself. Although both the agonist and a non-agonistic anti-CD28 antibody locally excluded CD45, the agonistic antibody was more effective. An anti-PD-1 antibody that bound membrane proximally excluded CD45, triggered Src homology 2 domain-containing phosphatase 2 recruitment, and suppressed systemic lupus erythematosus and delayed-type hypersensitivity in experimental models. Paradoxically, nivolumab and pembrolizumab, anti-PD-1-blocking antibodies used clinically, also excluded CD45 and were agonistic in certain settings. Reducing these agonistic effects using antibody engineering improved PD-1 blockade. These findings establish a framework for developing new and improved therapies for autoimmunity and cancer.

Modulating Phase Behavior in Fatty Acid-Modified Elastin-like Polypeptides (FAMEs): Insights into the Impact of Lipid Length on Thermodynamics and Kinetics of Phase Separation.

Although post-translational lipidation is prevalent in eukaryotes, its impact on the liquid-liquid phase separation of disordered proteins is still poorly understood. Here, we examined the thermodynamic phase boundaries and kinetics of aqueous two-phase system (ATPS) formation for a library of elastin-like polypeptides modified with saturated fatty acids of different chain lengths. By systematically altering the physicochemical properties of the attached lipids, we were able to correlate the molecular properties of lipids to changes in the thermodynamic phase boundaries and the kinetic stability of droplets formed by these proteins. We discovered that increasing the chain length lowers the phase separation temperature in a sigmoidal manner due to alterations in the unfavorable interactions between protein and water and changes in the entropy of phase separation. Our kinetic studies unveiled remarkable sensitivity to lipid length, which we propose is due to the temperature-dependent interactions between lipids and the protein. Strikingly, we found that the addition of just a single methylene group is sufficient to allow tuning of these interactions as a function of temperature, with proteins modified with C7-C9 lipids exhibiting non-Arrhenius dependence in their phase separation, a behavior that is absent for both shorter and longer fatty acids. This work advances our theoretical understanding of protein-lipid interactions and opens avenues for the rational design of lipidated proteins in biomedical paradigms, where precise control over the phase separation is pivotal.

Mechanisms of adaptive hypertrophic cardiac remodeling in a large animal model of premature ventricular contraction-induced cardiomyopathy.

Frequent premature ventricular contractions (PVCs) promoted eccentric cardiac hypertrophy and reduced ejection fraction (EF) in a large animal model of PVC-induced cardiomyopathy (PVC-CM), but the molecular mechanisms and markers of this hypertrophic remodeling remain unexplored. Healthy mongrel canines were implanted with pacemakers to deliver bigeminal PVCs (50% burden with 200-220 ms coupling interval). After 12 weeks, left ventricular (LV) free wall samples were studied from PVC-CM and Sham groups. In addition to reduced LV ejection fraction (LVEF), the PVC-CM group showed larger cardiac myocytes without evident ultrastructural alterations compared to the Sham group. Biochemical markers of pathological hypertrophy, such as store-operated Ca2+ entry, calcineurin/NFAT pathway, ß-myosin heavy chain, and skeletal type α-actin were unaltered in the PVC-CM group. In contrast, pro-hypertrophic and antiapoptotic pathways including ERK1/2 and AKT/mTOR were activated and/or overexpressed in the PVC-CM group, which appeared counterbalanced by an overexpression of protein phosphatase 1 and a borderline elevation of the anti-hypertrophic factor atrial natriuretic peptide. Moreover, the potent angiogenic and pro-hypertrophic factor VEGF-A and its receptor VEGFR2 were significantly elevated in the PVC-CM group. In conclusion, a molecular program is in place to keep this structural remodeling associated with frequent PVCs as an adaptive pathological hypertrophy.

Alterations of sarcoplasmic reticulum-mediated Ca2+ uptake in a model of premature ventricular contraction (PVC)-induced cardiomyopathy.

Premature ventricular contractions (PVCs) are the most frequent ventricular arrhythmias in the overall population. PVCs are known to acutely enhance contractility by the post-extrasystolic potentiation phenomenon, but over time persistent PVCs promote PVC-induced cardiomyopathy (PVC-CM), characterized by a reduction of the left ventricular (LV) ejection fraction. Ca2+ cycling in myocytes commands muscle contraction and in this process, SERCA2 leads the Ca2+ reuptake into the sarcoplasmic reticulum (SR) shaping cytosolic Ca2+ signal decay and muscle relaxation. Altered Ca2+ reuptake can contribute to the contractile dysfunction observed in PVC-CM. To better understand Ca2+ handling using our PVC-CM model (canines with 50% PVC burden for 12 weeks), SR-Ca2+ reuptake was investigated by measuring Ca2+ dynamics and analyzing protein expression. Kinetic analysis of Ca2+ reuptake in electrically paced myocytes showed a ~ 21 ms delay in PVC-CM compared to Sham in intact isolated myocytes, along with a ~ 13% reduction in SERCA2 activity assessed in permeabilized myocytes. Although these trends were not statistically significant between groups using hierarchical statistics, relaxation of myocytes following contraction was significantly slower in PVC-CM vs Sham myocytes. Western blot analyses indicate a 22% reduction in SERCA2 expression, a 23% increase in phospholamban (PLN) expression, and a 50% reduction in PLN phosphorylation in PVC-CM samples vs Sham. Computational analysis simulating a 20% decrease in SR-Ca2+ reuptake resulted in a ~ 22 ms delay in Ca2+ signal decay, consistent with the experimental result described above. In conclusion, SERCA2 and PLB alterations described above have a modest contribution to functional adaptations observed in PVC-CM.

Adaptive Recombinant Nanoworms from Genetically Encodable Star Amphiphiles.

Recombinant nanoworms are promising candidates for materials and biomedical applications ranging from the templated synthesis of nanomaterials to multivalent display of bioactive peptides and targeted delivery of theranostic agents. However, molecular design principles to synthesize these assemblies (which are thermodynamically favorable only in a narrow region of the phase diagram) remain unclear. To advance the identification of design principles for the programmable assembly of proteins into well-defined nanoworms and to broaden their stability regimes, we were inspired by the ability of topologically engineered synthetic macromolecules to acess rare mesophases. To test this design principle in biomacromolecular assemblies, we used post-translational modifications (PTMs) to generate lipidated proteins with precise topological and compositional asymmetry. Using an integrated experimental and computational approach, we show that the material properties (thermoresponse and nanoscale assembly) of these hybrid amphiphiles are modulated by their amphiphilic architecture. Importantly, we demonstrate that the judicious choice of amphiphilic architecture can be used to program the assembly of proteins into adaptive nanoworms, which undergo a morphological transition (sphere-to-nanoworms) in response to temperature stimuli.

Long-term outcome of 200 patients referred to a first seizure clinic.

OBJECTIVES: To determine what proportion of our First Seizure referrals reflected true unprovoked first seizures or epilepsy, and to assess the long-term diagnostic accuracy of our First Seizure Clinic (FSC) by quantifying the risk of subsequent seizures in our FSC cohort. METHODS: We prospectively collected data of 200 adult patients referred to the FSC between May 2014 and December 2015. We reviewed clinical notes, electroencephalography (EEG) data and performed telephone follow-up at 28-month post-diagnosis. RESULTS: Of the 200 patients referred to the FSC, 181 attended. At the initial assessment, 39 of these patients were diagnosed with epilepsy, with most of these patients (59%) found to have a history of previous seizures. Fifty patients were diagnosed with a first seizure, of which 28% were labelled as provoked seizures. Sixty nine of the patients received another diagnosis and 23 were labelled as indeterminable. At 28 months follow-up, 11 (22%) of patients who received a diagnosis of first seizure subsequently received a diagnosis of epilepsy. In the remaining groups, only 5 (5%) patients were diagnosed with epilepsy (of these three were in the indeterminable group). CONCLUSIONS: Our study shows that 50% of the patients referred to a FSC had not experienced a seizure but were given an alternative diagnosis. Secondly, our study indicates that the risk of seizure recurrence following a first seizure is quite low (22%). This is because a substantial proportion of the patients were diagnosed with epilepsy already at the first assessment. The high proportion of patients being diagnosed with epilepsy was mainly due to a history of previous seizures. Thirdly, patients who were given an alternative diagnosis at the first assessment had a low probability (5%) for seizure recurrence.

Short-Range Forecasting of COVID-19 During Early Onset at County, Health District, and State Geographic Levels Using Seven Methods: Comparative Forecasting Study.

BACKGROUND: Forecasting methods rely on trends and averages of prior observations to forecast COVID-19 case counts. COVID-19 forecasts have received much media attention, and numerous platforms have been created to inform the public. However, forecasting effectiveness varies by geographic scope and is affected by changing assumptions in behaviors and preventative measures in response to the pandemic. Due to time requirements for developing a COVID-19 vaccine, evidence is needed to inform short-term forecasting method selection at county, health district, and state levels. OBJECTIVE: COVID-19 forecasts keep the public informed and contribute to public policy. As such, proper understanding of forecasting purposes and outcomes is needed to advance knowledge of health statistics for policy makers and the public. Using publicly available real-time data provided online, we aimed to evaluate the performance of seven forecasting methods utilized to forecast cumulative COVID-19 case counts. Forecasts were evaluated based on how well they forecast 1, 3, and 7 days forward when utilizing 1-, 3-, 7-, or all prior-day cumulative case counts during early virus onset. This study provides an objective evaluation of the forecasting methods to identify forecasting model assumptions that contribute to lower error in forecasting COVID-19 cumulative case growth. This information benefits professionals, decision makers, and the public relying on the data provided by short-term case count estimates at varied geographic levels. METHODS: We created 1-, 3-, and 7-day forecasts at the county, health district, and state levels using (1) a naïve approach, (2) Holt-Winters (HW) exponential smoothing, (3) a growth rate approach, (4) a moving average (MA) approach, (5) an autoregressive (AR) approach, (6) an autoregressive moving average (ARMA) approach, and (7) an autoregressive integrated moving average (ARIMA) approach. Forecasts relied on Virginia's 3464 historical county-level cumulative case counts from March 7 to April 22, 2020, as reported by The New York Times. Statistically significant results were identified using 95% CIs of median absolute error (MdAE) and median absolute percentage error (MdAPE) metrics of the resulting 216,698 forecasts. RESULTS: The next-day MA forecast with 3-day look-back length obtained the lowest MdAE (median 0.67, 95% CI 0.49-0.84, P<.001) and statistically significantly differed from 39 out of 59 alternatives (66%) to 53 out of 59 alternatives (90%) at each geographic level at a significance level of .01. For short-range forecasting, methods assuming stationary means of prior days' counts outperformed methods with assumptions of weak stationarity or nonstationarity means. MdAPE results revealed statistically significant differences across geographic levels. CONCLUSIONS: For short-range COVID-19 cumulative case count forecasting at the county, health district, and state levels during early onset, the following were found: (1) the MA method was effective for forecasting 1-, 3-, and 7-day cumulative case counts; (2) exponential growth was not the best representation of case growth during early virus onset when the public was aware of the virus; and (3) geographic resolution was a factor in the selection of forecasting methods.

A knottin scaffold directs the CXC-chemokine-binding specificity of tick evasins.

Tick evasins (EVAs) bind either CC- or CXC-chemokines by a poorly understood promiscuous or "one-to-many" mechanism to neutralize inflammation. Because EVAs potently inhibit inflammation in many preclinical models, highlighting their potential as biological therapeutics for inflammatory diseases, we sought to further unravel the CXC-chemokine-EVA interactions. Using yeast surface display, we identified and characterized 27 novel CXC-chemokine-binding evasins homologous to EVA3 and defined two functional classes. The first, which included EVA3, exclusively bound ELR+ CXC-chemokines, whereas the second class bound both ELR+ and ELR- CXC-chemokines, in several cases including CXC-motif chemokine ligand 10 (CXCL10) but, surprisingly, not CXCL8. The X-ray crystal structure of EVA3 at a resolution of 1.79 Å revealed a single antiparallel ß-sheet with six conserved cysteine residues forming a disulfide-bonded knottin scaffold that creates a contiguous solvent-accessible surface. Swapping analyses identified distinct knottin scaffold segments necessary for different CXC-chemokine-binding activities, implying that differential ligand positioning, at least in part, plays a role in promiscuous binding. Swapping segments also transferred chemokine-binding activity, resulting in a hybrid EVA with dual CXCL10- and CXCL8-binding activities. The solvent-accessible surfaces of the knottin scaffold segments have distinctive shape and charge, which we suggest drives chemokine-binding specificity. These studies provide structural and mechanistic insight into how CXC-chemokine-binding tick EVAs achieve class specificity but also engage in promiscuous binding.

What I wish I'd learned as an orthodontic trainee: an online survey of British Orthodontic Society members concerning postgraduate training experiences.

OBJECTIVE: To survey the opinion of recently qualified and established orthodontists on the perceived value of their training and to identify specific areas which which were considered to be deficient, adequately covered or focussed on excessively. DESIGN: Descriptive cross-sectional survey. SETTING: Online electronic questionnaire. PARTICIPANTS: Members of the British Orthodontic Society (BOS). METHODS: An electronic questionnaire was circulated to members of the BOS focusing on dental education history, and opinions concerning orthodontic teaching generally and specific clinical and non-clinical subjects. Information was also obtained in terms of possible need for improvement, modification or removal of teaching on focused academic and clinical aspects. RESULTS: A total of 217 responses were received from 1080 emailed invitations resulting in a response rate of 20.1%. Respondents were generally satisfied with their training both in relation to theoretical, academic and practical aspects. However, training was regarded as deficient by some respondents in respect of temporary anchorage devices (38%), bonded retainers (6%), experience with lingual appliances (47%), removable aligners (44%), inter-proximal reduction (24%) and adult orthodontics (16%), working with therapists (32%), and NHS contracts (47%) and commissioning (47%). CONCLUSION: The overall satisfaction of BOS members with postgraduate orthodontic training is generally high, although both recently qualified and established practitioners emphasised the need for better exposure to training in specific practical aspects and practice management within the NHS.

Enterococcus faecalis Demonstrates Pathogenicity through Increased Attachment in an Ex Vivo Polymicrobial Pulpal Infection.

This study investigated the host response to a polymicrobial pulpal infection consisting of Streptococcus anginosus and Enterococcus faecalis, bacteria commonly implicated in dental abscesses and endodontic failure, using a validated ex vivo rat tooth model. Tooth slices were inoculated with planktonic cultures of S. anginosus or E. faecalis alone or in coculture at S. anginosus/E. faecalis ratios of 50:50 and 90:10. Attachment was semiquantified by measuring the area covered by fluorescently labeled bacteria. Host response was established by viable histological cell counts, and inflammatory response was measured using reverse transcription-quantitative PCR (RT-qPCR) and immunohistochemistry. A significant reduction in cell viability was observed for single and polymicrobial infections, with no significant differences between infection types (∼2,000 cells/mm2 for infected pulps compared to ∼4,000 cells/mm2 for uninfected pulps). E. faecalis demonstrated significantly higher levels of attachment (6.5%) than S. anginosus alone (2.3%) and mixed-species infections (3.4% for 50:50 and 2.3% for 90:10), with a remarkable affinity for the pulpal vasculature. Infections with E. faecalis demonstrated the greatest increase in tumor necrosis factor alpha (TNF-α) (47.1-fold for E. faecalis, 14.6-fold for S. anginosus, 60.1-fold for 50:50, and 25.0-fold for 90:10) and interleukin 1ß (IL-1ß) expression (54.8-fold for E. faecalis, 8.8-fold for S. anginosus, 54.5-fold for 50:50, and 39.9-fold for 90:10) compared to uninfected samples. Immunohistochemistry confirmed this, with the majority of inflammation localized to the pulpal vasculature and odontoblast regions. Interestingly, E. faecalis supernatant and heat-killed E. faecalis treatments were unable to induce the same inflammatory response, suggesting E. faecalis pathogenicity in pulpitis is linked to its greater ability to attach to the pulpal vasculature.

Considerations for best practices in studies of fiber or other dietary components and the intestinal microbiome.

A 2-day workshop organized by the National Institutes of Health and U.S. Department of Agriculture included 16 presentations focused on the role of diet in alterations of the gastrointestinal microbiome, primarily that of the colon. Although thousands of research projects have been funded by U.S. federal agencies to study the intestinal microbiome of humans and a variety of animal models, only a minority addresses dietary effects, and a small subset is described in sufficient detail to allow reproduction of a study. Whereas there are standards being developed for many aspects of microbiome studies, such as sample collection, nucleic acid extraction, data handling, etc., none has been proposed for the dietary component; thus this workshop focused on the latter specific point. It is important to foster rigor in design and reproducibility of published studies to maintain high quality and enable designs that can be compared in systematic reviews. Speakers addressed the influence of the structure of the fermentable carbohydrate on the microbiota and the variables to consider in design of studies using animals, in vitro models, and human subjects. For all types of studies, strengths and weaknesses of various designs were highlighted, and for human studies, comparisons between controlled feeding and observational designs were discussed. Because of the lack of published, best-diet formulations for specific research questions, the main recommendation is to describe dietary ingredients and treatments in as much detail as possible to allow reproduction by other scientists.

N-linked glycan stabilization of the VWF A2 domain.

Shear forces in the blood trigger a conformational transition in the von Willebrand factor (VWF) A2 domain, from its native folded to an unfolded state, in which the cryptic scissile bond (Y1605-M1606) is exposed and can then be proteolysed by ADAMTS13. The conformational transition depends upon a Ca(2+)binding site and a vicinal cysteine disulfide bond. Glycosylation at N1574 has previously been suggested to modulate VWF A2 domain interaction with ADAMTS13 through steric hindrance by the bulky carbohydrate structure. We investigated how the N-linked glycans of the VWF A2 domain affect thermostability and regulate both the exposure of the ADAMTS13 binding sites and the scissile bond. We show by differential scanning fluorimetry that the N-linked glycans thermodynamically stabilize the VWF A2 domain. The essential component of the glycan structure is the first sugar residue (GlcNAc) at the N1574 attachment site. From its crystal structures, N1574-GlcNAc is predicted to form stabilizing intradomain interactions with Y1544 and nearby residues. Substitution of the surface-exposed Y1544 to aspartic acid is able to stabilize the domain in the absence of glycosylation and protect against ADAMTS13 proteolysis in both the VWF A2 domain and FLVWF. Glycan stabilization of the VWF A2 domain acts together with the Ca(2+)binding site and vicinal cysteine disulfide bond to control unfolding and ADAMTS13 proteolysis.

Low-fat versus ketogenic diet in Parkinson's disease: A pilot randomized controlled trial.

BACKGROUND: Preliminary evidence suggests that diet manipulation may influence motor and nonmotor symptoms in PD, but conflict exists regarding the ideal fat to carbohydrate ratio. OBJECTIVES: We designed a pilot randomized, controlled trial to compare the plausibility, safety, and efficacy of a low-fat, high-carbohydrate diet versus a ketogenic diet in a hospital clinic of PD patients. METHODS: We developed a protocol to support PD patients in a diet study and randomly assigned patients to a low-fat or ketogenic diet. Primary outcomes were within- and between-group changes in MDS-UPDRS Parts 1 to 4 over 8 weeks. RESULTS: We randomized 47 patients, of which 44 commenced the diets and 38 completed the study (86% completion rate for patients commencing the diets). The ketogenic diet group maintained physiological ketosis. Both groups significantly decreased their MDS-UPDRS scores, but the ketogenic group decreased more in Part 1 (-4.58 ± 2.17 points, representing a 41% improvement in baseline Part 1 scores) compared to the low-fat group (-0.99 ± 3.63 points, representing an 11% improvement) (P < 0.001), with the largest between-group decreases observed for urinary problems, pain and other sensations, fatigue, daytime sleepiness, and cognitive impairment. There were no between-group differences in the magnitude of decrease for Parts 2 to 4. The most common adverse effects were excessive hunger in the low-fat group and intermittent exacerbation of the PD tremor and/or rigidity in the ketogenic group. CONCLUSIONS: It is plausible and safe for PD patients to maintain a low-fat or ketogenic diet for 8 weeks. Both diet groups significantly improved in motor and nonmotor symptoms; however, the ketogenic group showed greater improvements in nonmotor symptoms. © 2018 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society.

Somatotopic heat pain thresholds and intraepidermal nerve fibers in health.

INTRODUCTION: For sequential and somatotopic assessment of small fiber neuropathy, heat pain (HP) tests of hypoalgesia might be used instead of decreased counts of epidermal nerve fibers (ENFs), but then healthy subject reference values of HP thresholds are needed. METHODS: Using the Computer Assisted Sensation Evaluator IVc system, HP thresholds of hypoalgesia were estimated for 10 unilateral sites and counts of ENFs for 4 of them in healthy subjects. RESULTS: In healthy subjects, small but statistically significant differences of both HP thresholds of hypoalgesia and counts of ENFs were observed among tested sites. Significant correlations between HP thresholds and counts of ENFs were not found. DISCUSSION: For the studied somatotopic sites, we provide ≥95th and ≥99th percentile reference limits for HP 0.5 and 5 of 1-10 HP thresholds of hypoalgesia and decreased counts of ENFs at ≤5th and ≤1st percentile levels. Muscle Nerve 58: 509-516, 2018.

Mild acids facilitate functional dentin remineralization under thermo-mechanical stimuli.

PURPOSE: To evaluate if mechanical and thermal cycling promote remineralization at the resin-dentin interface after bonding with three different adhesive approaches. METHODS: Dentin surfaces were subjected to three different treatments: demineralization (1) by 37% phosphoric acid followed by application of an etch-and-rinse dentin adhesive Single Bond (Adper Single Bond) (SB); (2) by 0.5 M ethylenediaminetetraacetic acid (EDTA) followed by SB; (3) application of a self-etch dentin adhesive: Clearfil-SEB (Clearfil SE Bond). Bonded interfaces were stored during 24 hours and then submitted for 3 months to: (1) storage at 37ºC, (2) load cycling, (3) thermocycling, and (4) thermo+load cycling. One section was extracted from each tooth, monthly. Resin-dentin interfaces were analyzed by AFM nano-indentation, Raman spectroscopy/cluster analysis and Masson's trichrome staining at 24 hours, 1, 2 and 3 months, to determine remineralization at the interface. RESULTS: Thermo+load cycling promoted the highest biomimetic remineralization at the hybrid layer formed with EDTA+SB and Clearfil-SEB, at the 1 month time point. A narrow mineral-depleted zone was observed after thermo+load cycling with EDTA+ SB, and at those specimens bonded with Clearfil-SEB. Thermo+load cycling remineralized the dentin interface treated with EDTA+SB and Clearfil-SEB, after 1 month of study period, providing bioactivity and maturity of formed minerals. CLINICAL SIGNIFICANCE: In vitro challenging (thermo+load cycling) favors dentin remineralization at the resin-dentin bonded interfaces promoted with mild conditioning acids.

Human In Situ Study of the effect of Bis(2-Methacryloyloxyethyl) Dimethylammonium Bromide Immobilized in Dental Composite on Controlling Mature Cariogenic Biofilm.

Cariogenic oral biofilms cause recurrent dental caries around composite restorations, resulting in unprosperous oral health and expensive restorative treatment. Quaternary ammonium monomers that can be copolymerized with dental resin systems have been explored for the modulation of dental plaque biofilm growth over dental composite surfaces. Here, for the first time, we investigated the effect of bis(2-methacryloyloxyethyl) dimethylammonium bromide (QADM) on human overlying mature oral biofilms grown intra-orally in human participants for 7⁻14 days. Seventeen volunteers wore palatal devices containing composite specimens containing 10% by mass of QADM or a control composite without QADM. After 7 and 14 days, the adherent biofilms were collected to determine bacterial counts via colony-forming unit (CFU) counts. Biofilm viability, chronological changes, and percentage coverage were also determined through live/dead staining. QADM composites caused a significant inhibition of Streptococcus mutans biofilm formation for up to seven days. No difference in the CFU values were found for the 14-day period. Our findings suggest that: (1) QADM composites were successful in inhibiting 1⁻3-day biofilms in the oral environment in vivo; (2) QADM significantly reduced the portion of the S. mutans group; and (3) stronger antibiofilm activity is required for the control of mature long-term cariogenic biofilms. Contact-killing strategies using dental materials aimed at preventing or at least reducing high numbers of cariogenic bacteria seem to be a promising approach in patients at high risk of the recurrence of dental caries around composites.

Catabolic Defect of Branched-Chain Amino Acids Promotes Heart Failure.

BACKGROUND: Although metabolic reprogramming is critical in the pathogenesis of heart failure, studies to date have focused principally on fatty acid and glucose metabolism. Contribution of amino acid metabolic regulation in the disease remains understudied. METHODS AND RESULTS: Transcriptomic and metabolomic analyses were performed in mouse failing heart induced by pressure overload. Suppression of branched-chain amino acid (BCAA) catabolic gene expression along with concomitant tissue accumulation of branched-chain α-keto acids was identified as a significant signature of metabolic reprogramming in mouse failing hearts and validated to be shared in human cardiomyopathy hearts. Molecular and genetic evidence identified the transcription factor Krüppel-like factor 15 as a key upstream regulator of the BCAA catabolic regulation in the heart. Studies using a genetic mouse model revealed that BCAA catabolic defect promoted heart failure associated with induced oxidative stress and metabolic disturbance in response to mechanical overload. Mechanistically, elevated branched-chain α-keto acids directly suppressed respiration and induced superoxide production in isolated mitochondria. Finally, pharmacological enhancement of branched-chain α-keto acid dehydrogenase activity significantly blunted cardiac dysfunction after pressure overload. CONCLUSIONS: BCAA catabolic defect is a metabolic hallmark of failing heart resulting from Krüppel-like factor 15-mediated transcriptional reprogramming. BCAA catabolic defect imposes a previously unappreciated significant contribution to heart failure.

Nanoscale magnetic ratchets based on shape anisotropy.

Controlling magnetization using piezoelectric strain through the magnetoelectric effect offers several orders of magnitude reduction in energy consumption for spintronic applications. However strain is a uniaxial effect and, unlike directional magnetic field or spin-polarized current, cannot induce a full 180° reorientation of the magnetization vector when acting alone. We have engineered novel 'peanut' and 'cat-eye' shaped nanomagnets on piezoelectric substrates that undergo repeated deterministic 180° magnetization rotations in response to individual electric-field-induced strain pulses by breaking the uniaxial symmetry using shape anisotropy. This behavior can be likened to a magnetic ratchet, advancing magnetization clockwise with each piezostrain trigger. The results were validated using micromagnetics implemented in a multiphysics finite elements code to simulate the engineered spatial and temporal magnetic behavior. The engineering principles start from a target device function and proceed to the identification of shapes that produce the desired function. This approach opens a broad design space for next generation magnetoelectric spintronic devices.

Control of VWF A2 domain stability and ADAMTS13 access to the scissile bond of full-length VWF.

Rheological shear forces in the blood trigger von Willebrand factor (VWF) unfolding which exposes the Y1605-M1606 scissile bond within the VWF A2 domain for cleavage by ADAMTS13. The VWF A2 domain contains 2 structural features that provide it with stability: a vicinal disulphide bond and a Ca(2+)-binding site (CBS). We investigated how these 2 structural features interplay to determine stability and regulate the exposure of the scissile bond in full-length VWF. We have used differential scanning fluorimetry together with site-directed mutagenesis of residues involved in both the vicinal disulphide bond and the CBS to demonstrate that both of these sites contribute to stability against thermal unfolding of the isolated VWF A2 domain. Moreover, we show that the combination of site mutations can result in increased susceptibility of FL-VWF to proteolysis by ADAMTS13, even in the absence of an agent (such as urea) required to induce unfolding. These studies demonstrate that VWF A2 domain stability provided by its 2 structural elements (vicinal disulphide bond and CBS) is a key protective determinant against FL-VWF cleavage by ADAMTS13. They suggest a 2-step mechanism for VWF A2 domain unfolding.

Global deletion of BCATm increases expression of skeletal muscle genes associated with protein turnover.

Consumption of a protein-containing meal by a fasted animal promotes protein accretion in skeletal muscle, in part through leucine stimulation of protein synthesis and indirectly through repression of protein degradation mediated by its metabolite, α-ketoisocaproate. Mice lacking the mitochondrial branched-chain aminotransferase (BCATm/Bcat2), which interconverts leucine and α-ketoisocaproate, exhibit elevated protein turnover. Here, the transcriptomes of gastrocnemius muscle from BCATm knockout (KO) and wild-type mice were compared by next-generation RNA sequencing (RNA-Seq) to identify potential adaptations associated with their persistently altered nutrient signaling. Statistically significant changes in the abundance of 1,486/∼39,010 genes were identified. Bioinformatics analysis of the RNA-Seq data indicated that pathways involved in protein synthesis [eukaryotic initiation factor (eIF)-2, mammalian target of rapamycin, eIF4, and p70S6K pathways including 40S and 60S ribosomal proteins], protein breakdown (e.g., ubiquitin mediated), and muscle degeneration (apoptosis, atrophy, myopathy, and cell death) were upregulated. Also in agreement with our previous observations, the abundance of mRNAs associated with reduced body size, glycemia, plasma insulin, and lipid signaling pathways was altered in BCATm KO mice. Consistently, genes encoding anaerobic and/or oxidative metabolism of carbohydrate, fatty acids, and branched chain amino acids were modestly but systematically reduced. Although there was no indication that muscle fiber type was different between KO and wild-type mice, a difference in the abundance of mRNAs associated with a muscular dystrophy phenotype was observed, consistent with the published exercise intolerance of these mice. The results suggest transcriptional adaptations occur in BCATm KO mice that along with altered nutrient signaling may contribute to their previously reported protein turnover, metabolic and exercise phenotypes.