Kinase-catalyzed crosslinking: A comparison of ATP-crosslinker analogs.

Protein phosphorylation is catalyzed by kinases to regulate cellular events and disease states. Identifying kinase-substrate relationships represents a powerful strategy to understand cell biology and disease yet remains challenging due to the rapid dynamics of phosphorylation. Over the last decade, several γ-phosphoryl modified ATP analogs containing crosslinkers were developed to covalently conjugate kinases, their substrates, and their associated proteins for subsequent characterization. Here, kinetics and crosslinking experiments demonstrated that the UV-activated analogs, ATP-aryl azide and ATP-benzophenone, offered the most robust crosslinking, whereas electrophilic ATP-aryl fluorosulfate promoted the most effective proximity-enabled crosslinking. The data will guide future applications of kinase-catalyzed crosslinking to study normal and disease biology.

Phosphorylation of cell cycle and apoptosis regulatory protein-1 by stress activated protein kinase P38γ is a novel mechanism of apoptosis signaling by genotoxic chemotherapy.

CARP-1, a perinuclear phospho-protein, regulates cell survival and apoptosis signaling induced by genotoxic drugs. However, kinase(s) phosphorylating CARP-1 and down-stream signal transduction events remain unclear. Here we find that CARP-1 Serine (S)626 and Threonine (T)627 substitution to Alanines (AA) inhibits genotoxic drug-induced apoptosis. CARP-1 T627 is followed by a Proline (P), and this TP motif is conserved in vertebrates. Based on these findings, we generated affinity-purified, anti-phospho-CARP-1 T627 rabbit polyclonal antibodies, and utilized them to elucidate chemotherapy-activated, CARP-1-dependent cell growth signaling mechanisms. Our kinase profiling studies revealed that MAPKs/SAPKs phosphorylated CARP-1 T627. We then UV cross-linked protein extracts from Adriamycin-treated HeLa cervical cancer cells with a CARP-1 (614-638) peptide, and conducted liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses of the peptide-bound protein complexes. This experiment revealed SAPK p38γ interaction with CARP-1 (614-638) peptide. Our studies further established that SAPK p38γ, but not other MAPKs, phosphorylates CARP-1 T627 in cancer cells treated with genotoxic drugs. Loss of p38γ abrogates CARP-1 T627 phosphorylation, and results in enhanced survival of breast cancer cells by genotoxic drugs. CARP-1 T627 phosphorylation was also noted in breast tumors from patients treated with radiation or endocrine therapies. We conclude that genotoxic drugs activate p38γ-dependent CARP-1 T627 phosphorylation to inhibit cell growth.

Protein kinase A is a functional component of focal adhesions.

Focal adhesions (FAs) form the junction between extracellular matrix (ECM)-bound integrins and the actin cytoskeleton and also transmit signals that regulate cell adhesion, cytoskeletal dynamics, and cell migration. While many of these signals are rooted in reversible tyrosine phosphorylation, phosphorylation of FA proteins on Ser/Thr residues is far more abundant yet its mechanisms and consequences are far less understood. The cAMP-dependent protein kinase (protein kinase A; PKA) has important roles in cell adhesion and cell migration and is both an effector and regulator of integrin-mediated adhesion to the ECM. Importantly, subcellular localization plays a critically important role in specifying PKA function. Here, we show that PKA is present in isolated FA-cytoskeleton complexes and active within FAs in live cells. Furthermore, using kinase-catalyzed biotinylation of isolated FA-cytoskeleton complexes, we identify 53 high-stringency candidate PKA substrates within FAs. From this list, we validate tensin-3 (Tns3)-a well-established molecular scaffold, regulator of cell migration, and a component of focal and fibrillar adhesions-as a novel direct substrate for PKA. These observations identify a new pathway for phospho-regulation of Tns3 and, importantly, establish a new and important niche for localized PKA signaling and thus provide a foundation for further investigation of the role of PKA in the regulation of FA dynamics and signaling.

Protein Kinase A is a Functional Component of Focal Adhesions.

Focal adhesions (FAs) form the junction between extracellular matrix (ECM)-bound integrins and the actin cytoskeleton and also transmit signals that regulate cell adhesion, cytoskeletal dynamics, and cell migration. While many of these signals are rooted in reversible tyrosine phosphorylation, phosphorylation of FA proteins on Ser/Thr residues is far more abundant yet its mechanisms and consequences are far less understood. The cAMP-dependent protein kinase (protein kinase A; PKA) has important roles in cell adhesion and cell migration and is both an effector and regulator of integrin-mediated adhesion to the ECM. Importantly, subcellular localization plays a critically important role in specifying PKA function. Here, we show that PKA is present in isolated FA-cytoskeleton complexes and active within FAs in live cells. Furthermore, using kinase-catalyzed biotinylation of isolated FA-cytoskeleton complexes, we identify fifty-three high-stringency candidate PKA substrates within FAs. From this list, we validate tensin-3 (Tns3) - a well-established molecular scaffold, regulator of cell migration, and component of focal and fibrillar adhesions - as a novel direct substrate for PKA. These observations identify a new pathway for phospho-regulation of Tns3 and, importantly, establish a new and important niche for localized PKA signaling and thus provide a foundation for further investigation of the role of PKA in the regulation of FA dynamics and signaling.

Kinase-Catalyzed Biotinylation to Identify Phosphatase Substrates (K-BIPS).

Phosphorylation is a reversible post-translational modification that alters the functions of proteins to govern various cellular events, including cell signaling. Kinases catalyze the transfer of a phosphoryl group onto the hydroxyl residue of serine, threonine, and tyrosine, while phosphatases catalyze the removal. Unregulated kinase and phosphatase activity have been observed in various cancers and neurodegenerative diseases. Despite their importance in cell biology, the role of phosphatases in cellular events has yet to be fully characterized, partly due to the lack of tools to identify phosphatase-substrate pairs in a biological context. The method called kinase-catalyzed biotinylation to identify phosphatase substrates (K-BIPS) was developed to remedy the lack of information surrounding phosphatase biology, particularly focused on substrate identification. In the K-BIPS method, the γ-phosphoryl modified adenosine 5'-triphosphate (ATP) analog, ATP-biotin, is used by kinases to biotin-label phosphoproteins. Because phosphatases must initially remove a phosphoryl group for subsequent biotinylation by ATP-biotin, phosphatase substrates are identified in K-BIPS by comparing biotinylated proteins in the presence and absence of active phosphatases. K-BIPS has been used to discover novel substrates of both serine/threonine and tyrosine phosphatases. This chapter describes the K-BIPS method to enable the identification of substrates to any phosphatases of interest, which will augment studies of phosphatase biology.

Cotadutide promotes glycogenolysis in people with overweight or obesity diagnosed with type 2 diabetes.

Cotadutide is a dual glucagon-like peptide 1 and glucagon receptor agonist under development for the treatment of non-alcoholic steatohepatitis and type 2 diabetes mellitus (T2DM) and chronic kidney disease. Non-alcoholic steatohepatitis is a complex disease with no approved pharmacotherapies, arising from an underlying state of systemic metabolic dysfunction in association with T2DM and obesity. Cotadutide has been shown to improve glycaemic control, body weight, lipids, liver fat, inflammation and fibrosis. We conducted a two-part, randomized phase 2a trial in men and women with overweight or obesity diagnosed with T2DM to evaluate the efficacy and safety of cotadutide compared with placebo and liraglutide. The primary endpoints were change from baseline to day 28 of treatment in postprandial hepatic glycogen (part A) and to day 35 of treatment in fasting hepatic glycogen (part B) with cotadutide versus placebo. Secondary endpoints in part B were changes in fasting hepatic glycogen with cotadutide versus the mono glucagon-like peptide 1 receptor agonist, liraglutide, and change in hepatic fat fraction. The trial met its primary endpoint. We showed that cotadutide promotes greater reductions in liver glycogen and fat compared with placebo and liraglutide. Safety and tolerability findings with cotadutide were comparable to those of previous reports. Thus, this work provides evidence of additional benefits of cotadutide that could be attributed to glucagon receptor engagement. Our results suggest that cotadutide acts on the glucagon receptor in the human liver to promote glycogenolysis and improve the metabolic health of the liver. ClinicalTrials.gov registration: NCT03555994 .

Identification of PP1c-PPP1R12A Substrates Using Kinase-Catalyzed Biotinylation to Identify Phosphatase Substrates.

Protein phosphatase 1 regulatory subunit 12A (PPP1R12A) interacts with the catalytic subunit of protein phosphatase 1 (PP1c) to form the myosin phosphatase complex. In addition to a well-documented role in muscle contraction, the PP1c-PPP1R12A complex is associated with cytoskeleton organization, cell migration and adhesion, and insulin signaling. Despite the variety of biological functions, only a few substrates of the PP1c-PPP1R12A complex are characterized, which limit a full understanding of PP1c-PPP1R12A activities in muscle contraction and cytoskeleton regulation. Here, the chemoproteomics method Kinase-catalyzed Biotinylation to Identify Phosphatase Substrates (K-BIPS) was used to identify substrates of the PP1c-PPP1R12A complex in L6 skeletal muscle cells. K-BIPS enriched 136 candidate substrates with 14 high confidence hits. One high confidence hit, AKT1 kinase, was validated as a novel PP1c-PPP1R12A substrate. Given the previously documented role of AKT1 in PPP1R12A phosphorylation and cytoskeleton organization, the data suggest that PP1c-PPP1R12A regulates its own phosphatase activity through an AKT1-dependent feedback mechanism to influence cytoskeletal arrangement in muscle cells.

Valproate regulates inositol synthesis by reducing expression of myo-inositol-3-phosphate synthase.

Inositol depletion is a hypothesized mechanism of action of mood stabilization drugs used in the treatment of bipolar disorder. It was previously reported that the mood stabilizer valproate (VPA) increased phosphorylation of myo-inositol-3-phosphate synthases (MIPS), the rate limiting enzyme of inositol synthesis. Phosphosites were identified and examination of site-directed mutants suggested that phosphorylation leads to decreased enzymatic activity. In this study, we examined the extent of MIPS phosphorylation in response to VPA and used two interaction screens to identify protein kinases that interact with MIPS. Using an epitope tagged MIPS construct, we determined the fraction of phosphorylated MIPS to be very low (less than 2% of total), and we could not detect phosphorylation of untagged MIPS in response to VPA. In vitro analyses of phosphorylation revealed that putative protein kinases, PKC and CKII, have low specificity toward MIPS. These findings suggest that VPA likely depletes inositol via a mechanism other than MIPS phosphorylation. Consistent with this, mRNA levels of the MIPS-encoding gene INO1 and MIPS protein levels were significantly reduced during the mid-log growth phase in response to VPA treatment. These findings suggest that the mechanism whereby VPA causes inositol depletion is by reducing expression of the rate-limiting enzyme MIPS.

Kinase-catalyzed Biotinylation with Inactivated Lysates for Discovery of Substrates (K-BILDS).

Protein phosphorylation is catalyzed by kinases to regulate a large variety of cellular activities, including growth and signal transduction. Methods to identify kinase substrates are crucial to fully understand phosphorylation-mediated cellular events and disease states. Here, we report a set of protocols to identify substrates of a target kinase using Kinase-catalyzed Biotinylation with Inactivated Lysates for Discovery of Substrates (K-BILDS). As described in these protocols, K-BILDS involves inactivation of endogenous kinases in lysates, followed by addition of an active exogenous kinase and the γ-phosphate-modified ATP analog ATP-biotin for kinase-catalyzed biotinylation of cellular substrates. Avidin enrichment isolates biotinylated substrates of the active kinase, which can be monitored by western blot. Substrates of the target kinase can also be discovered using mass spectrometry analysis. Key advantages of K-BILDS include compatibility with any lysate, tissue homogenate, or complex mixture of biological relevance and any active kinase of interest. K-BILDS is a versatile method for studying or discovering substrates of a kinase of interest to characterize biological pathways thoroughly. © 2023 Wiley Periodicals LLC. Basic Protocol 1: FSBA treatment of lysates to inactivate kinases Basic Protocol 2: Kinase-catalyzed Biotinylation with Inactivated Lysates for Discovery of Substrates (K-BILDS).

Affinity-Based Kinase-Catalyzed Crosslinking to Study Kinase-Substrate Pairs.

Phosphorylation of proteins by kinase enzymes is a post-translational modification involved in a myriad of biological events, including cell signaling and disease development. Identifying the interactions between a kinase and its phosphorylated substrate(s) is necessary to characterize phosphorylation-mediated cellular events and encourage development of kinase-targeting drugs. One method for substrate-kinase identification utilizes photocrosslinking γ-phosphate-modified ATP analogues to covalently link kinases to their substrates for subsequent monitoring. Because photocrosslinking ATP analogues require UV light, which could influence cell biology, we report here two ATP analogues, ATP-aryl fluorosulfate (ATP-AFS) and ATP-hexanoyl bromide (ATP-HexBr), that crosslink kinase-substrate pairs via proximity-mediated reactions without the need for UV irradiation. Both ATP-AFS and ATP-HexBr acted as cosubstrates with a variety of kinases for affinity-based crosslinking, with ATP-AFS showing more robust complexes. Importantly, ATP-AFS promoted crosslinking in lysates, which demonstrates compatibility with complex cellular mixtures for future application to kinase-substrate identification.

Feasibility and self-perceived effectiveness of an online training program on dysphagia in residential aged care homes in mainland China.

BACKGROUND: With a rapidly aging population in mainland China, dysphagia has become one of the common geriatric disorders which creates a huge demand on speech and language therapists (SLTs). The major challenge is the shortage of SLTs in China. In addition, frontline practitioners in mainland China may not be well equipped with the knowledge and practical skills in dysphagia management due to lack of systematic training and the work nature. AIMS: This study evaluates the self-perceived effectiveness and feasibility of an online training program that aims to enhance the self-assessed knowledge and skills of SLTs providing dysphagia care in residential aged care homes. METHODS AND PROCEDURES: Sixteen SLTs working in a residential aged care homes in mainland China attended a three-hour pilot online training program which consists of didactic lecture and practical skills activity components. A total of 10 participants completed an online questionnaire one month after the training to evaluate the feasibility and effectiveness of this online training program. OUTCOMES AND RESULTS: The preliminary results demonstrated participants' self-perception of high training effectiveness in theoretical knowledge and practical skills. A majority of the participants perceived that the training enhanced their theoretical knowledge and all of them perceived that they acquired practical skills. All respondents were satisfied with the online training approach. They also highlighted the advantage and challenges of the online training approach. CONCLUSIONS AND IMPLICATIONS: Online training is an effective and feasible approach for theoretical knowledge and practical skills transfer in SLT training and could ultimately benefit the delivery of services for individuals with dysphagia in mainland China. WHAT THIS PAPER ADDS: What is already known on the subject Previous studies have shown that online training approach is as effective as face-to-face training in increasing professional knowledge. Online training programs may be more cost efficient and time efficient when compared with face-to-face training. What this study adds The present study provided preliminary evidence to support the feasibility and effectiveness of using online training on dysphagia for speech and language therapists working in residential aged care homes in mainland China. What are the clinical implications of this work? From the participants' perception, online training approach is effective and feasible in delivering theoretical knowledge and practical skills. It may be a better training approach for mainland China considering the lack of expertise and accessibility to training.

Replacing Foods with a High-Glycemic Index and High in Saturated Fat by Alternatives with a Low Glycemic Index and Low Saturated Fat Reduces Hepatic Fat, Even in Isocaloric and Macronutrient Matched Conditions.

BACKGROUND: Current guidelines aim to limit the dietary glycemic index (GI) and intake of saturated fatty acids (SFA). Several studies have shown favorable effects of low-GI or low-SFA diets on intrahepatic lipid content (IHL), but these studies were performed under overfeeding conditions or extreme differences in GI or SFA to maximize the contrast between diets. By combining changes in GI and SFA, we can mimic how people can improve their diet in a realistic setting. OBJECTIVES: We investigated the effect on liver fat content and substrate metabolism of both reducing GI and replacing SFA with polyunsaturated fat in practically realistic amounts under isocaloric conditions. DESIGN AND METHODS: In a randomized crossover study, thirteen overweight participants consumed two diets, one high in GI and SFA (high GI/SFA) and one low in GI and SFA (low GI/SFA) with identical macronutrient composition, for two weeks each. Diets were equal in caloric content, consisted of habitual food items, and had a macronutrient composition that can be easily achieved in daily life. At the end of each intervention, IHL content/composition and liver glycogen were measured by magnetic resonance spectroscopy. Additionally, fasted and postprandial hepatic de novo lipogenesis and glycemic and metabolic responses were investigated. RESULTS: IHL was significantly lower (-28%) after the two-week low-GI/SFA diet (2.4 ± 0.5% 95% CI [1.4, 3.4]) than after the two-week high-GI/SFA diet (3.3 ± 0.6% 95% CI [1.9, 4.7], p < 0.05). Although hepatic glycogen content, hepatic de novo lipogenesis, hepatic lipid composition, and substrate oxidation during the night were similar between the two diets, the glycemic response to the low-GI/SFA diet was reduced (p < 0.05). CONCLUSIONS: Changes in macronutrient quality can already have drastic effects on liver fat content and postprandial glycemia after two weeks and even when energy content and the percentage of total fat and carbohydrate remains unchanged.

A prolonged fast improves overnight substrate oxidation without modulating hepatic glycogen in adults with and without nonalcoholic fatty liver: A randomized crossover trial.

OBJECTIVE: Increasing overnight fasting time seems a promising strategy to improve metabolic health in individuals with nonalcoholic fatty liver (NAFL). Mechanisms underlying the beneficial effects of fasting may be related to larger fluctuations in hepatic glycogen and higher fat oxidation. This study investigated whether prolonging an overnight fast depletes hepatic glycogen stores and improves substrate metabolism in individuals with NAFL and healthy lean individuals. METHODS: Eleven individuals with NAFL and ten control individuals participated in this randomized crossover trial. After a 9.5-hour or 16-hour fast, hepatic glycogen was measured by using carbon-13 magnetic resonance spectroscopy, and a meal test was performed. Nocturnal substrate oxidation was measured with indirect calorimetry. RESULTS: Extending fasting time led to lower nocturnal carbohydrate oxidation and higher fat oxidation in both groups (intervention × time, p < 0.005 for carbohydrate and fat oxidation). In both arms, the respiratory exchange ratio measured during the night remained higher in the group with NAFL compared with the control group (population p < 0.001). No changes were observed in hepatic glycogen depletion with a prolonged overnight fast in the group with NAFL or the control group. CONCLUSIONS: These results suggest that acutely prolonging the overnight fast can improve overnight substrate oxidation and that these alterations are not mediated by changes in hepatic glycogen depletion.

Proteomics-based trapping with single or multiple inactive mutants reproducibly profiles histone deacetylase 1 substrates.

Histone deacetylase 1 (HDAC1) plays a key role in diverse cellular processes. With the aberrant expression of HDAC1 linked to many diseases, including cancers, HDAC inhibitors have been used successfully as therapeutics. HDAC1 has been predominantly associated with histone deacetylation and gene expression. Recently, non-histone substrates have revealed diverse roles of HDAC1 beyond epigenetics. To augment discovery of non-histone substrates, we introduced "substrate trapping" to enrich HDAC1 substrates using an inactive mutant. Herein, we performed a series of proteomics studies to test the robustness of HDAC1 substrate trapping. Based on our recent results documenting that different HDAC1 mutants preferentially bound different substrates, which suggested that multiple mutants could be used for efficient trapping, trapping with three single point mutants simultaneously identified several potential substrates uniquely compared to a single mutant alone. However, a greater number of biologically interesting hits were observed using only a single mutant, which suggests that the C151A HDAC1 mutant is the optimal trap. Importantly, comparing independent trials with a single mutant performed by different experimentalists and HEK293 cell populations, trapping was robust and reproducible. Based on the reproducible trapping data, carnosine N-methyltransferase 1 (CARNMT1) was validated as an HDAC1 substrate. The data document that mutant trapping is an effective method for discovery of unanticipated HDAC substrates. SIGNIFICANCE: Histone deacetylase (HDAC) proteins are well established epigenetic transcriptional regulators that deacetylate histone substrates to control gene expression. More recently, deacetylation of non-histone substrates has linked HDAC activity to functions outside of epigenetics. Given the use of HDAC inhibitor drugs as anti-cancer therapeutics, understanding the full functions of HDAC proteins in cell biology is essential to future drug design. To discover unanticipated non-histone substrates and further characterize HDAC functions, inactive mutants have been used to "trap" putative substrates, which were identified with mass spectrometry-based proteomics analysis. Here multiple trapping studies were performed to test the robustness of using inactive mutants and proteomics for HDAC substrate discovery. The data confirm the value of trapping mutants as effective tools to discover HDAC substrates and link HDAC activity to unexpected biological functions.

Rational design of metabolically stable HDAC inhibitors: An overhaul of trifluoromethyl ketones.

Epigenetic regulation of gene expression using histone deacetylase (HDAC) inhibitors is a promising strategy for developing new anticancer agents. The most common HDAC inhibitors are hydroxamates, which, though highly potent, have limitations due to their poor pharmacokinetic properties and lack of isoform selectivity. Trifluoromethylketones (TFMK) developed as alternatives to hydroxamates are rapidly metabolized to inactive trifluoromethyl alcohols in vivo, which prevented their further development as potential drug candidates. In order to overcome this limitation, we designed trifluoropyruvamides (TFPAs) as TFMK surrogates. The presence of an additional electron withdrawing group next to the ketone carbonyl group made the hydrate form of the ketone more stable, thus preventing its metabolic reduction to alcohol in vivo. In addition, this structural modification reduces the potential of the TFMK group to act as a covalent warhead to eliminate off-target effects. Additional structural changes in the cap group of the inhibitors gave analogues with IC50 values ranging from upper nanomolar to low micromolar in the cytotoxicity assay, and they were more selective for cancer cells over normal cells. Some of the most active analogues inhibited HDAC enzymes with low nanomolar IC50 values and were found to be more selective for HDAC8 over other isoforms. These molecules provide a new class of HDAC inhibitors with a metabolically stable metal-binding group that could be used to develop selective HDAC inhibitors by further structural modification.

Kinase-Catalyzed Crosslinking and Immunoprecipitation (K-CLIP) to Explore Kinase-Substrate Pairs.

Kinases are responsible for phosphorylation of proteins and are involved in many biological processes, including cell signaling. Identifying the kinases that phosphorylate specific phosphoproteins is critical to augment the current understanding of cellular events. Herein, we report a general protocol to study the kinases of a target substrate phosphoprotein using kinase-catalyzed crosslinking and immunoprecipitation (K-CLIP). K-CLIP uses a photocrosslinking γ-phosphoryl-modified ATP analog, such as ATP-arylazide, to covalently crosslink substrates to kinases with UV irradiation. Crosslinked kinase-substrate complexes can then be enriched by immunoprecipitating the target substrate phosphoprotein, with bound kinase(s) identified using Western blot or mass spectrometry analysis. K-CLIP is an adaptable chemical tool to investigate and discover kinase-substrate pairs, which will promote characterization of complex phosphorylation-mediated cell biology. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Kinase-catalyzed crosslinking of lysates Basic Protocol 2: Kinase-catalyzed crosslinking and immunoprecipitation (K-CLIP).

Three weeks of time-restricted eating improves glucose homeostasis in adults with type 2 diabetes but does not improve insulin sensitivity: a randomised crossover trial.

AIMS/HYPOTHESIS: Time-restricted eating (TRE) is suggested to improve metabolic health by limiting food intake to a defined time window, thereby prolonging the overnight fast. This prolonged fast is expected to lead to a more pronounced depletion of hepatic glycogen stores overnight and might improve insulin sensitivity due to an increased need to replenish nutrient storage. Previous studies showed beneficial metabolic effects of 6-8 h TRE regimens in healthy, overweight adults under controlled conditions. However, the effects of TRE on glucose homeostasis in individuals with type 2 diabetes are unclear. Here, we extensively investigated the effects of TRE on hepatic glycogen levels and insulin sensitivity in individuals with type 2 diabetes. METHODS: Fourteen adults with type 2 diabetes (BMI 30.5±4.2 kg/m2, HbA1c 46.1±7.2 mmol/mol [6.4±0.7%]) participated in a 3 week TRE (daily food intake within 10 h) vs control (spreading food intake over ≥14 h) regimen in a randomised, crossover trial design. The study was performed at Maastricht University, the Netherlands. Eligibility criteria included diagnosis of type 2 diabetes, intermediate chronotype and absence of medical conditions that could interfere with the study execution and/or outcome. Randomisation was performed by a study-independent investigator, ensuring that an equal amount of participants started with TRE and CON. Due to the nature of the study, neither volunteers nor investigators were blinded to the study interventions. The quality of the data was checked without knowledge on intervention allocation. Hepatic glycogen levels were assessed with 13C-MRS and insulin sensitivity was assessed using a hyperinsulinaemic-euglycaemic two-step clamp. Furthermore, glucose homeostasis was assessed with 24 h continuous glucose monitoring devices. Secondary outcomes included 24 h energy expenditure and substrate oxidation, hepatic lipid content and skeletal muscle mitochondrial capacity. RESULTS: Results are depicted as mean ± SEM. Hepatic glycogen content was similar between TRE and control condition (0.15±0.01 vs 0.15±0.01 AU, p=0.88). M value was not significantly affected by TRE (19.6±1.8 vs 17.7±1.8 µmol kg-1 min-1 in TRE vs control, respectively, p=0.10). Hepatic and peripheral insulin sensitivity also remained unaffected by TRE (p=0.67 and p=0.25, respectively). Yet, insulin-induced non-oxidative glucose disposal was increased with TRE (non-oxidative glucose disposal 4.3±1.1 vs 1.5±1.7 µmol kg-1 min-1, p=0.04). TRE increased the time spent in the normoglycaemic range (15.1±0.8 vs 12.2±1.1 h per day, p=0.01), and decreased fasting glucose (7.6±0.4 vs 8.6±0.4 mmol/l, p=0.03) and 24 h glucose levels (6.8±0.2 vs 7.6±0.3 mmol/l, p<0.01). Energy expenditure over 24 h was unaffected; nevertheless, TRE decreased 24 h glucose oxidation (260.2±7.6 vs 277.8±10.7 g/day, p=0.04). No adverse events were reported that were related to the interventions. CONCLUSIONS/INTERPRETATION: We show that a 10 h TRE regimen is a feasible, safe and effective means to improve 24 h glucose homeostasis in free-living adults with type 2 diabetes. However, these changes were not accompanied by changes in insulin sensitivity or hepatic glycogen. TRIAL REGISTRATION: ClinicalTrials.gov NCT03992248 FUNDING: ZonMW, 459001013.

Evidence that HDAC7 acts as an epigenetic "reader" of AR acetylation through NCoR-HDAC3 dissociation.

Histone deacetylase (HDAC) proteins are epigenetic regulators that govern a wide variety of cellular events. With a role in cancer formation, HDAC inhibitors have emerged as anti-cancer therapeutics. Among the eleven metal-dependent class I, II, and IV HDAC proteins targeted by inhibitor drugs, class IIa HDAC4, -5, -7, and -9 harbor low deacetylase activity and are hypothesized to be "reader" proteins, which bind to post-translationally acetylated lysine. However, evidence linking acetyllysine binding to a downstream functional event is lacking. Here, we report for the first time that HDAC4, -5, and -7 dissociated from corepressor NCoR in the presence of an acetyllysine-containing peptide, consistent with reader function. Documenting the biological consequences of this possible reader function, mutation of a critical acetylation site regulated androgen receptor (AR) transcriptional activation function through HDAC7-NCoR-HDAC3 dissociation. The data document the first evidence consistent with epigenetic-reader functions of class IIa HDAC proteins.

Evaluating the contribution of a wildlife health capacity building program on orangutan conservation.

One Health is increasingly being used as a tool in ecosystem protection. The Orangutan Veterinary Advisory Group (OVAG) is working to address One Health concerns in Pongo spp. (orangutan) welfare and conservation. Orangutans are vital contributors to the ecosystem health of their range areas. Strengthening national capacity is crucial to make a lasting difference in the currently bleak outlook for orangutan species survival. OVAG is a capacity strengthening and expertise network that brings together all those working with orangutans, in- and ex-situ, to share knowledge, skills, and to collectively learn. Using the One Health paradigm embedded to enhance professional development, the OVAG network is successfully supporting conservation outcomes and impact. As part of our adaptive management approach, and to assess individual and organizational change attributable to the capacity strengthening work of OVAG, we evaluated technical skill test data, program satisfaction data, and asked participants to complete a self-reflective survey. This pilot study of our work demonstrates statistically significant improvements in conservation medicine (t = 5.481, p < 0.0001) and wildlife clinical skills knowledge (t = 3.923, p < 0.001) for those in the OVAG program. Most consider OVAG participation to be either critical or very useful in their conservation medicine decision-making process, with a perceived positive impact on their skills at handling multiple situations. Additionally, participant feedback shows a sense of being able to drive positive change locally and nationally (within their own countries) as a consequence of OVAG participation. The authors hope the OVAG model including its associated capacity support mechanisms and pedagogical approaches can be used as a template for other One Health efforts.

Early Development of Neural Speech Encoding Depends on Age but Not Native Language Status: Evidence From Lexical Tone.

We investigated the development of early-latency and long-latency brain responses to native and non-native speech to shed light on the neurophysiological underpinnings of perceptual narrowing and early language development. Specifically, we postulated a two-level process to explain the decrease in sensitivity to non-native phonemes toward the end of infancy. Neurons at the earlier stages of the ascending auditory pathway mature rapidly during infancy facilitating the encoding of both native and non-native sounds. This growth enables neurons at the later stages of the auditory pathway to assign phonological status to speech according to the infant's native language environment. To test this hypothesis, we collected early-latency and long-latency neural responses to native and non-native lexical tones from 85 Cantonese-learning children aged between 23 days and 24 months, 16 days. As expected, a broad range of presumably subcortical early-latency neural encoding measures grew rapidly and substantially during the first two years for both native and non-native tones. By contrast, long-latency cortical electrophysiological changes occurred on a much slower scale and showed sensitivity to nativeness at around six months. Our study provided a comprehensive understanding of early language development by revealing the complementary roles of earlier and later stages of speech processing in the developing brain.