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The article focuses on a study which showed the use of metabolic profiling of synovial tissue in accurately diagnosing rheumatoid arthritis RA). Topics discussed include the altered glucose and choline metabolism in the synovial tissue of RA samples, the relative quantification of metabolites for evaluating the metabolic differences between the tested groups, and the similarity of the observed metabolic pattern in the synovial fluid (SF) of RA and osteoarthritis patients.

Background: There is limited evidence of potential associations between body mass index (BMI) and risk of vulvar and vaginal cancer. We explored these associations in a large cohort of Norwegian women., Methods: The analytical dataset included 889,441 women aged 16-75 years at baseline in 1963-1975. Multivariable Cox regression analyses were used to estimate hazard ratios (HRs) and 95% confidence intervals (CIs) for the associations between BMI and vulvar and vaginal cancer incidence., Results: During 30.1 million person-years of follow-up, 1748 incident vulvar and 408 incident vaginal cancer cases occurred. The HRs (95% CIs) for vulvar cancer for a BMI of 15- < 18.5, 18.5- < 25, 25- < 30, 30- < 35, ≥ 35 were 0.62 (0.38-1.01), 1.00 (reference), 1.23 (1.10-1.40), 1.43 (1.23-1.66) and 1.72 (1.35-2.20, p trend  < 0.001), and per 5 kg/m 2 increment was 1.20 (1.13-1.26). The corresponding HRs (95% CIs) for vaginal cancer were 1.05 (0.52-2.15), 1.00, 0.89 (0.71-1.12), 0.95 (0.68-1.34), and 2.01 (1.29-3.13, p trend  < 0.001), respectively, and per 5 kg/m 2 was 1.11 (0.99-1.25). The HR (95% CI) per 5 kg/m 2 increase in BMI at ages 16-29 was 1.28 (1.07-1.54, n = 250 cases) for vulvar and 1.53 (1.11-2.11, n = 66 cases) for vaginal cancers. The HR (95% CI) per 5 kg/m 2 for early-onset (< 50 years age at diagnosis) vulvar cancer was 0.92 (0.66-1.28, n = 87 cases) and 1.70 (1.05-2.76, n = 21 cases) for vaginal cancer., Conclusion: These results further support the associations between higher BMI and increased risk of vulvar and vaginal cancers, with suggestive stronger associations between BMI in early adulthood for both cancers and for early-onset vaginal cancer. Further studies are needed to elucidate these findings and investigate the underlying mechanisms., (© 2024. The Author(s).)

Ablation of the immune-specific catalytic subunit Cβ2 of protein kinase A is associated with a proinflammatory phenotype and increased sensitivity to autoimmunity in mice. Here we show that tumour growth of the adenocarcinoma cell line EO771 in the breast and in the lung after injection into the mammary fat pad and tail vein, respectively, was significantly reduced in mice ablated for Cβ2 compared to wild-type mice. In both cases, the breast and lung tumours showed increased infiltration of immune cells in the mice lacking Cβ2 compared to wild-type mice. Despite this, it appeared that solid tissue- versus intravenously injected EO771 cells evoked different immune responses. This was reflected by significantly increased levels of splenic proinflammatory immune cells and circulating cytokines in Cβ2 ablated mice carrying breast- but not the lung tumours. Moreover, Cβ2 ablated mice injected with EO771 cells showed increased overall survival compared to wild-type mice. Taken together, our results suggest for a role for immune cell-specific Cβ2 in protecting against tumour growth induced by EO771 cells in mice that is reflected in improved overall survival., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Periampullary cancers, including pancreatic ductal adenocarcinoma, ampullary-, cholangio-, and duodenal carcinoma, are frequently diagnosed in an advanced stage and are associated with poor overall survival. They are difficult to differentiate from each other and challenging to distinguish from benign periampullary disease preoperatively. To improve the preoperative diagnostics of periampullary neoplasms, clinical or biological markers are warranted.In this study, 28 blood plasma amino acids and derivatives from preoperative patients with benign (N = 45) and malignant (N = 72) periampullary disease were analyzed by LC-MS/MS.Principal component analysis and consensus clustering both separated the patients with cancer and the patients with benign disease. Glutamic acid had significantly higher plasma expression and 15 other metabolites significantly lower plasma expression in patients with malignant disease compared with patients having benign disease. Phenylalanine was the only metabolite associated with improved overall survival (HR = 0.50, CI 0.30-0.83, P < 0.01).Taken together, plasma metabolite profiles from patients with malignant and benign periampullary disease were significantly different and have the potential to distinguish malignant from benign disease preoperatively., (© 2024. The Author(s).)

Surviving long periods without food has shaped human evolution. In ancient and modern societies, prolonged fasting was/is practiced by billions of people globally for religious purposes, used to treat diseases such as epilepsy, and recently gained popularity as weight loss intervention, but we still have a very limited understanding of the systemic adaptions in humans to extreme caloric restriction of different durations. Here we show that a 7-day water-only fast leads to an average weight loss of 5.7 kg (±0.8 kg) among 12 volunteers (5 women, 7 men). We demonstrate nine distinct proteomic response profiles, with systemic changes evident only after 3 days of complete calorie restriction based on in-depth characterization of the temporal trajectories of ~3,000 plasma proteins measured before, daily during, and after fasting. The multi-organ response to complete caloric restriction shows distinct effects of fasting duration and weight loss and is remarkably conserved across volunteers with >1,000 significantly responding proteins. The fasting signature is strongly enriched for extracellular matrix proteins from various body sites, demonstrating profound non-metabolic adaptions, including extreme changes in the brain-specific extracellular matrix protein tenascin-R. Using proteogenomic approaches, we estimate the health consequences for 212 proteins that change during fasting across ~500 outcomes and identified putative beneficial (SWAP70 and rheumatoid arthritis or HYOU1 and heart disease), as well as adverse effects. Our results advance our understanding of prolonged fasting in humans beyond a merely energy-centric adaptions towards a systemic response that can inform targeted therapeutic modulation., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Introduction: Carbohydrates and fats are the primary energy substrates during exercise, but proteins can also contribute. When proteins are degraded in the body, the amino groups are mainly converted to urea and excreted. Therefore, nitrogen excretion has been used as a marker of protein degradation, but a clear conclusion has yet to be reached on the effect of exercise on nitrogen excretion. Thus, we tested whether exercise increases nitrogen excretion., Methods: Fifteen young, healthy, moderate-to-well-trained participants (4 females, 11 males, VO 2max 54.4 ± 1.7 mL·kg -1 ·min -1 ; mean ± SEM) participated in a randomized, balanced cross-over design investigation consisting of 1 day with 5 h of exercise (exercise day, EX) and 1 day with no exercise (control day, CON). The participants recorded their dietary intake the day before from 16:00 and throughout the intervention day. They then repeated these dietary intakes on the second trial day. A standardized lunch was provided on both days. In addition, participants were allowed to consume almost protein-free snacks in EX to ensure the same energy balance during both trial days. Urine was collected throughout the whole testing period, and urinary 3-methylhistidine (3-MH) excretion was measured to examine muscular catabolism. The sweat rate was calculated during the exercise period., Results and Discussion: The urinary nitrogen and 3-MH excretions did not differ significantly between EX and CON ( p  = 0.764 and p  = 0.953). The sweat rate was 2.55 ± 0.25 L in EX and 0.14 ± 0.15 L in CON ( p  < 0.001), and by estimating sweat nitrogen excretion, total nitrogen excretion was shown to differ with exercise. Our results showed that 5 hours of mixed exercise did not significantly impact urinary nitrogen and 3-MH excretions in healthy moderate-to-well-trained young adults., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Clauss, Burkhardt, Wöber, Skålhegg and Jensen.)

The gut hormone ghrelin drives food motivation and increases food intake, but it is also involved in the anticipation of and response to rewards other than food. This pre-registered study investigated how naturally varying ghrelin concentrations affect the processing of touch as a social reward in humans. Sixty-seven volunteers received slow caressing touch (so-called CT-targeted touch) as a social reward and control touch on their shins during 3T functional imaging on two test days. On one occasion, participants were fasted, and on another, they received a meal. On each occasion, plasma ghrelin was measured at three time points. All touch was rated as more pleasant after the meal, but there was no association between ghrelin concentrations and pleasantness. CT-targeted touch was rated as the most pleasant and activated somatosensory and reward networks (whole brain). A region-of-interest in the right medial orbitofrontal cortex (mOFC) showed lower activation during all touches, the higher the ghrelin concentrations were. During CT-targeted touch, a larger satiety response (ghrelin decrease after the meal) was associated with higher mOFC activation, and this mOFC activation was associated with higher experienced pleasantness. Overall, higher ghrelin concentrations appear to be related to a lower reward value for touch. Ghrelin may reduce the value of social stimuli, such as touch, to promote food search and intake in a state of low energy. This suggests that the role of ghrelin goes beyond assigning value to food reward., (© 2023 The Authors. Psychophysiology published by Wiley Periodicals LLC on behalf of Society for Psychophysiological Research.)

The main purpose of this study was to investigate the effect of a novel alginate-encapsulated carbohydrate-protein (CHO-PRO ratio 2:1) supplement (ALG) on cycling performance. The ALG, designed to control the release of nutrients, was compared to an isocaloric carbohydrate-only control (CON). Alginate encapsulation of CHOs has the potential to reduce the risk of carious lesions., Methods: In a randomised cross-over clinical trial, 14 men completed a preliminary test over 2 experimental days separated by ~6 days. An experimental day consisted of an exercise bout (EX1) of cycling until exhaustion at W ~73% , followed by 5 h of recovery and a subsequent time-to-exhaustion (TTE) performance test at W ~65% . Subjects ingested either ALG (0.8 g CHO/kg/hr + 0.4 g PRO/kg/hr) or CON (1.2 g CHO/kg/hr) during the first 2 h of recovery., Results: Participants cycled on average 75.2 ± 5.9 min during EX1. Levels of plasma branched-chain amino acids decreased significantly after EX1, and increased significantly with the intake of ALG during the recovery period. During recovery, a significantly higher plasma insulin and glucose response was observed after intake of CON compared to ALG. Intake of ALG increased plasma glucagon, free fatty acids, and glycerol significantly. No differences were found in the TTE between the supplements ( p = 0.13) nor in the pH of the subjects' saliva., Conclusions: During the ALG supplement, plasma amino acids remained elevated during the recovery. Despite the 1/3 less CHO intake with ALG compared to CON, the TTE performance was similar after intake of either supplement.

Introduction: Exercise with low carbohydrate availability increases protein degradation, which may reduce subsequent performance considerably. The present study aimed to investigate the effect of carbohydrate ingestion during standardized exercise with and without exhaustion on protein degradation and next-day performance., Methods: Seven trained male cyclists (V̇O 2max 66.8 ± 1.9 mL·kg -1 ·min -1 ; mean ± SEM) cycled to exhaustion (~2.5 h) at a power output eliciting 68% of V̇O 2max (W 68% ). This was followed by repeating 1-min work/1-min recovery intervals at 90% of V̇O 2max (W 90% ) until exhaustion. During W 68% , cyclists consumed a placebo water drink (PLA) the first time and a carbohydrate drink (CHO), 1 g carbohydrate·kg -1 ·h -1 , the second time. The participants performed the same amount of work under the two conditions, separated by at least 1 wk. A standardized diet was provided to the participants so that the two conditions were isoenergetic. To test the impact of carbohydrates on recovery, participants completed a time trial (TT) the next day., Results: Carbohydrate ingestion maintained carbohydrate availability during W 68% and W 90% : total carbohydrate oxidation was significantly higher in CHO ( P = 0.022), and plasma glucose concentration was maintained compared with PLA ( P = 0.025). Next-day performance during TT was better after CHO ingestion (CHO, 41:49 ± 1:38 min; PLA, 42:50 ± 1:46 min; P = 0.020; effect size d = 0.23, small), as was gross efficiency (CHO, 18.6% ± 0.3%; PLA, 17.9% ± 0.3%; P = 0.019). Urinary nitrogen excretion ( P = 0.897) and urinary 3-methylhistidine excretion ( P = 0.673) did not significantly differ during the study period. Finally, tyrosine and phenylalanine plasma concentrations increased in PLA but not in CHO ( P = 0.018)., Conclusions: Carbohydrate ingestion during exhaustive exercise reduced deterioration in next-day performance through reduced metabolic stress and development of fatigue. In addition, some parameters point toward less protein degradation, which would preserve muscle function., (Copyright © 2023 by the American College of Sports Medicine.)

Background: The aim of this study was to explore the associations between BMI and cancer of the liver, bile ducts, and gallbladder., Methods: A registry-based cohort study was performed by linking data from several national registries in Norway., Results: The cohort comprised 1 723 692 individuals including 4768 hepatobiliary cancer cases during 55 743 509 person-years of follow-up. In men, we found increased risk of cancer per 5 kg/m 2 BMI increase for hepatocellular carcinoma and extrahepatic cholangiocarcinoma. In women there was increased risk of extrahepatic cholangiocarcinoma and gallbladder cancer. Women with high BMI in early adulthood had increased risk of intrahepatic cholangiocarcinoma. Reduced cancer-specific survival was found for all hepatobiliary malignancies in women with overweight and obesity. In men, reduced survival was observed in individuals with obesity for all hepatobiliary cancers, except gallbladder cancer. Increased risk of cancer-death per 5 kg/m 2 BMI increase was found for hepatocellular carcinoma, intra-, and extrahepatic cholangiocarcinoma in women. For men, 5 kg/m 2 BMI increase was positively associated with cancer-death from intrahepatic cholangiocarcinoma., Discussion: This study supports the notion of an increased risk of hepatobiliary cancers with increasing BMI, with sex and age variations. The findings also suggest a higher risk of cancer-death with increasing BMI., Competing Interests: Declarations of competing interest None to declare., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Aim: The aim of the present study was to compare performance 5 h after a 90-min endurance training session when either carbohydrate only or carbohydrate with added whey hydrolysate or whey isolate was ingested during the first 2 h of the recovery period., Methods: Thirteen highly trained competitive male cyclists completed three exercise and diet interventions (double-blinded, randomized, crossover design) separated by 1 week. The 90-min morning session (EX1) included a 60 min time-trial (TT 60 ). Immediately and 1 h after exercise, participants ingested either (1) 1.2 g carbohydrate∙kg -1 ∙h -1 (CHO), (2) 0.8 g carbohydrate∙kg -1 ∙h -1  + 0.4 g isolate whey protein∙kg -1 ∙h -1 (ISO) or (3) 0.8 g carbohydrate∙kg -1 ∙h -1  + 0.4 g hydrolysate whey protein∙kg -1 ∙h -1 (HYD). Additional intakes were identical between interventions. After 5 h of recovery, participants completed a time-trial performance (TT P ) during which a specific amount of work was performed. Blood and urine were collected throughout the day., Results: TT P did not differ significantly between dietary interventions (CHO: 43:54 ± 1:36, ISO: 46:55 ± 2:32, HYD: 44:31 ± 2:01 min). Nitrogen balance during CHO was lower than ISO (p < 0.0001) and HYD (p < 0.0001), with no difference between ISO and HYD (p = 0.317). In recovery, the area under the curve for blood glucose was higher in CHO compared to ISO and HYD. HR, VO 2 , RER, glucose, and lactate during EX2 were similar between interventions., Conclusion: Performance did not differ after 5 h of recovery whether carbohydrate only or isocaloric carbohydrate plus protein was ingested during the first 2 h. Correspondingly, participants were not in negative nitrogen balance in any dietary intervention., (© 2023 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Antibody-based blocking of vascular endothelial growth factor (VEGF) reduces choroidal neovascularization (CNV) and retinal edema, rescuing vision in patients with neovascular age-related macular degeneration (nAMD). However, poor response and resistance to anti-VEGF treatment occurs. We report that targeting the Notch ligand Jagged1 by a monoclonal antibody reduces neovascular lesion size, number of activated phagocytes and inflammatory markers and vascular leakage in an experimental CNV mouse model. Additionally, we demonstrate that Jagged1 is expressed in mouse and human eyes, and that Jagged1 expression is independent of VEGF signaling in human endothelial cells. When anti-Jagged1 was combined with anti-VEGF in mice, the decrease in lesion size exceeded that of either antibody alone. The therapeutic effect was solely dependent on blocking, as engineering antibodies to abolish effector functions did not impair the therapeutic effect. Targeting of Jagged1 alone or in combination with anti-VEGF may thus be an attractive strategy to attenuate CNV-bearing diseases., (© 2023. The Author(s).)

Aim: The aim was to explore potential associations between the body mass index (BMI) and the risk of colorectal cancer (CRC), including subsites of the colon, and cancer-specific death., Methods: A registry-based cohort study was conducted with baseline data gathered from the Norwegian Tuberculosis Screening Programme, collected between 1963 and 1975, and linked to follow-up data from the Cancer Registry of Norway and the Norwegian Cause of Death Registry. Cox regression models were used to explore associations between BMI and CRC risk and cancer-specific death., Results: Of 1 723 692 included individuals, 76 616 developed CRC during 55 370 707 person-years of follow-up. In men, a 5 kg/m 2 increase in BMI was associated with an increased risk of colon cancer, including both right and left subsites, and rectal cancer. Allowing for nonlinearities, we found a U-shaped association for the right colon and an inverse U-shape for the left colon and rectum cancer. In women, a 5 kg/m 2 increase in BMI in early adulthood was associated with increased risk of colon cancer, including both subsites. In women, an increased risk of CRC death with increasing BMI was found for colon cancer., Conclusions: Men of all ages have an increased risk of CRC with increasing BMI, with the highest risk for right-sided colon cancer. An increased risk for colon cancer was also found in women with high BMI in early adulthood. Furthermore, women of all age groups appeared to have an increased risk of CRC death with higher BMI., (© 2022 The Authors. Colorectal Disease published by John Wiley & Sons Ltd on behalf of Association of Coloproctology of Great Britain and Ireland.)

Background and Objective: An association between body mass index (BMI) and pancreatic cancer is suggested in observational studies. However, further studies are required to substantiate available evidence. The aim of this study was to explore the association between BMI and pancreatic ductal adenocarcinoma (PDAC) risk, treatment, and mortality., Methods: A registry-based cohort study was performed by combining data from four registries in Norway. Baseline data were collected between 1963 and 1975 with follow-up data collected until 2018. Kaplan-Meier curves and multivariable Cox regressions were estimated. Chi-square tests were used to analyze differences between groups., Results: The study cohort consisted of 1,723,692 individuals. A total of 8973 PDAC cases were identified during 55,744,749 person-years of follow-up. A 5 kg/m 2 increase in BMI was associated with an increased risk of PDAC if high BMI at young age (16-29 years) (hazard ratio (HR): 1.21, 95% confidence interval (CI): 1.13-1.31), both for men (HR: 1.30, 95% CI: 1.15-1.46) and women (HR: 1.16, 95% CI: 1.05-1.28). In men, there was a 52% increase in risk of early-onset PDAC (2 increase in BMI. A total of 2645 individuals were diagnosed with stage 1-3 disease, of whom 1131 underwent curative surgery. In all, 49% of normal weight, 38% overweight, and 30% obese individuals with stage 1-3 PDAC underwent surgery with curative intent (p < 0.001). Cancer survival was lower in overweight and obese individuals for both sexes. Analysis adjusted for sex, age, and period of diagnosis confirmed increased risk of cancer death in obese individuals., Conclusion: This study suggests that increased BMI in young adults may increase the risk of PDAC, and higher BMI in men is associated with an increased risk of early onset PDAC (

Methotrexate is one of the cornerstones of rheumatoid arthritis (RA) therapy. Genetic factors or single nucleotide polymorphisms (SNPs) are responsible for 15%-30% of the variation in drug response. Identification of clinically effective SNP biomarkers for predicting methotrexate (MTX) sensitivity has been a challenge. The aim of this study was to explore the association between the disease related outcome of MTX treatment and 23 SNPs in 8 genes of the MTX pathway, as well as one pro-inflammatory related gene in RA patients naïve to MTX. Categorical outcomes such as Disease Activity Score (DAS)-based European Alliance of Associations for Rheumatology (EULAR) non-response at 4 months, The American College of Rheumatology and EULAR (ACR/EULAR) non-remission at 6 months, and failure to sustain MTX monotherapy from 12 to 24 months were assessed, together with continuous outcomes of disease activity, joint pain and fatigue. We found that the SNPs rs1801394 in the MTRR gene, rs408626 in DHFR gene, and rs2259571 in AIF-1 gene were significantly associated with disease activity relevant continuous outcomes. Additionally, SNP rs1801133 in the MTHFR gene was identified to be associated with improved fatigue. Moreover, associations with p values at uncorrected significance level were found in SNPs and different categorical outcomes: 1) rs1476413 in the MTHFR gene and rs3784864 in ABCC1 gene are associated with ACR/EULAR non-remission; 2) rs1801133 in the MTHFR gene is associated with EULAR response; 3) rs246240 in the ABCC1 gene, rs2259571 in the AIF-1 gene, rs2274808 in the SLC19A1 gene and rs1476413 in the MTHFR gene are associated with failure to MTX monotherapy after 12-24 months. The results suggest that SNPs in genes associated with MTX activity may be used to predict MTX relevant-clinical outcomes in patients with RA., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Kolan, Li, Grimolizzi, Sexton, Goll, Kvien, Sundlisæter, Zucknick, Lillegraven, Haavardsholm and Skålhegg.)

To meet the demand for energy and biomass, T lymphocytes (T cells) activated to proliferation and clonal expansion, require uptake and metabolism of glucose (Gluc) and the amino acid (AA) glutamine (Gln). Whereas exogenous Gln is converted to glutamate (Glu) by glutaminase (GLS), Gln is also synthesized from the endogenous pool of AA through Glu and activity of glutamine synthase (GS). Most of this knowledge comes from studies on cell cultures under ambient oxygen conditions (normoxia, 21% O2). However, in vivo, antigen induced T-cell activation often occurs under moderately hypoxic (1-4% O2) conditions and at various levels of exogenous nutrients. Here, CD4+ T cells were stimulated for 72 h with antibodies targeting the CD3 and CD28 markers at normoxia and hypoxia (1% O2). This was done in the presence and absence of the GLS and GS inhibitors, Bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl) ethyl sulfide (BPTES) and methionine sulfoximine (MSO) and at various combinations of exogenous Gluc, Gln and pyruvate (Pyr) for the last 12 h of stimulation. We found that T-cell proliferation, viability and levels of endogenous AA were significantly influenced by the availability of exogenous Gln, Gluc and Pyr as well as inhibition of GLS and GS. Moreover, inhibition of GLS and GS and levels of oxygen differentially influenced oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). Finally, BPTES-dependent down-regulation of ECAR was associated with reduced hexokinase (HK) activity at both normoxia and hypoxia. Our results demonstrate that Gln availability and metabolism is rate-limiting for CD4+ T-cell activity., (© 2022 The Author(s).)

Pancreatic cancer remains a disease with unmet clinical needs and inadequate diagnostic, prognostic, and predictive biomarkers. In-depth characterization of the disease proteome is limited. This study thus aims to define and describe protein networks underlying pancreatic cancer and identify protein centric subtypes with clinical relevance. Mass spectrometry-based proteomics was used to identify and quantify the proteome in tumor tissue, tumor-adjacent tissue, and patient-derived xenografts (PDX)-derived cell lines from patients with pancreatic cancer, and tissues from patients with chronic pancreatitis. We identified, quantified, and characterized 11,634 proteins from 72 pancreatic tissue samples. Network focused analysis of the proteomics data led to identification of a tumor epithelium-specific module and an extracellular matrix (ECM)-associated module that discriminated pancreatic tumor tissue from both tumor adjacent tissue and pancreatitis tissue. On the basis of the ECM module, we defined an ECM-high and an ECM-low subgroup, where the ECM-high subgroup was associated with poor prognosis (median survival months: 15.3 vs . 22.9 months; log-rank test, P = 0.02). The ECM-high tumors were characterized by elevated epithelial-mesenchymal transition and glycolytic activities, and low oxidative phosphorylation, E2F, and DNA repair pathway activities. This study offers novel insights into the protein network underlying pancreatic cancer opening up for proteome precision medicine development., Significance: Pancreatic cancer lacks reliable biomarkers for prognostication and treatment of patients. We analyzed the proteome of pancreatic tumors, nonmalignant tissues of the pancreas and PDX-derived cell lines, and identified proteins that discriminate between patients with good and poor survival. The proteomics data also unraveled potential novel drug targets., Competing Interests: J. Lehtio reports other from Fenomark Diagnostics outside the submitted work; and J. Lehtio is involved in Cancer Core Europe BoB trial financed by Roche (not related to this work). No other disclosures were reported., (© 2022 The Authors; Published by the American Association for Cancer Research.)

Protein kinase A (PKA) is a holoenzyme consisting of a regulatory (R)-subunit dimer and two catalytic (C)-subunits. There are two major families of C-subunits, C α and C β , and four functionally nonredundant R-subunits (RI α , RI β , RII α , RII β ). In addition to binding to and being regulated by the R-subunits, the C-subunits are regulated by two tail regions that each wrap around the N- and C-lobes of the kinase core. Although the C-terminal (Ct-) tail is classified as an intrinsically disordered region (IDR), the N-terminal (Nt-) tail is dominated by a strong helix that is flanked by short IDRs. In contrast to the Ct-tail, which is a conserved and highly regulated feature of all PKA, PKG, and protein kinase C protein kinase group (AGC) kinases, the Nt-tail has evolved more recently and is highly variable in vertebrates. Surprisingly and in contrast to the kinase core and the Ct-tail, the entire Nt-tail is not conserved in nonmammalian PKAs. In particular, in humans, C β actually represents a large family of C-subunits that are highly variable in their Nt-tail and also expressed in a highly tissue-specific manner. Although we know so much about the C α 1-subunit, we know almost nothing about these C β isoforms wherein C β 2 is highly expressed in lymphocytes, and C β 3 and C β 4 isoforms account for ∼50% of PKA signaling in brain. Based on recent disease mutations, the C β proteins appear to be functionally important and nonredundant with the C α isoforms. Imaging in retina also supports nonredundant roles for C β as well as isoform-specific localization to mitochondria. This represents a new frontier in PKA signaling. SIGNIFICANCE STATEMENT: How tails and adjacent domains regulate each protein kinase is a fundamental challenge for the biological community. Here we highlight how the N- and C-terminal tails of PKA (Nt-tails/Ct-tails) affect the structure and regulate the function of the kinase core and show the combinatorial variations that are introduced into the Nt-tail of the C α - and C β -subunits in contrast to the Ct-tail, which is conserved across the entire AGC subfamily of protein kinases., (Copyright © 2022 by The American Society for Pharmacology and Experimental Therapeutics.)

T lymphocytes (T cells) are divided into two functionally different subgroups the CD4+ T helper cells (Th) and the CD8+ cytotoxic T lymphocytes (CTL). Adequate CD4 and CD8 T cell activation to proliferation, clonal expansion and effector function is crucial for efficient clearance of infection by pathogens. Failure to do so may lead to T cell exhaustion. Upon activation by antigen presenting cells, T cells undergo metabolic reprograming that support effector functions. In this review we will discuss how metabolic reprograming dictates functionality during viral infections using severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and human immunodeficiency virus (HIV) as examples. Moreover, we will briefly discuss T cell metabolic programs during bacterial infections exemplified by Mycobacterium tuberculosis (MT) infection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Wik and Skålhegg.)

DNA-damaging therapy is the basis for treatment of most cancers, including B-cell precursor acute lymphoblastic leukemia (BCP-ALL, hereafter ALL). We have previously shown that cAMP-activating factors present in the bone marrow render ALL cells less sensitive to DNA damage-induced apoptosis, by enhancing autophagy and suppressing p53. To sensitize ALL cells to DNA-damaging therapy, we have searched for novel targets that may counteract the effects induced by cAMP signaling. In the current study, we have identified PARP1 as a potential target. We show that the PARP1 inhibitors olaparib or PJ34 inhibit cAMP-mediated autophagy and thereby potentiate the DNA-damaging treatment. Furthermore, we reveal that cAMP-mediated PARP1 activation is preceded by induction of reactive oxygen species (ROS) and results in depletion of nicotinamide adenine dinucleotide (NAD), both of which are autophagy-promoting events. Accordingly, we demonstrate that scavenging ROS by N-acetylcysteine and repleting NAD independently reduce DNA damage-induced autophagy. In addition, olaparib augmented the effect of DNA-damaging treatment in a human xenograft model of ALL in NOD-scidIL2Rgammanull mice. On the basis of the current findings, we suggest that PARP1 inhibitors may enhance the efficiency of conventional genotoxic therapies and thereby provide a novel treatment strategy for pediatric patients with ALL., Implications: PARP1 inhibitors augment the DNA damage-induced killing of ALL cells by limiting the opposing effects of cAMP-mediated autophagy, which involves ROS-induced PARP1 activation and depletion of cellular NAD levels., (©2021 American Association for Cancer Research.)

In humans, there are two forms of glutaminase (GLS), designated GLS1 and GLS2. These enzymes catalyse the conversion of glutamine to glutamate. GLS1 exists as two isozymes: kidney glutaminase (KGA) and glutaminase C (GAC). Several GLS inhibitors have been identified, of which DON (6-diazo-5-oxonorleucine), BPTES (bis-2-(5-phenylacetamido-1, 3, 4-thiadiazol-2-yl) ethyl sulphide), 968 (5-(3-Bromo-4-(dimethylamino)phenyl)-2,2-dimethyl-2,3,5,6-tetrahydrobenzo[a]phenanthridin-4(1H)-one) and CB839 (Telaglenastat) are the most widely used. However, these inhibitors have variable efficacy, specificity and bioavailability in research and clinical settings, implying the need for novel and improved GLS inhibitors. Based on this need, a diverse library of 28,000 compounds from Enamine was screened for inhibition of recombinant, purified GAC. From this library, one inhibitor designated compound 19 (C19) was identified with kinetic features revealing allosteric inhibition of GAC in the µm range. Moreover, C19 inhibits anti-CD3/CD28-induced CD4+ T-cell proliferation and cytokine production with similar or greater potency as compared to BPTES. Taken together, our data suggest that C19 has the potential to modulate GLS1 activity and alter metabolic activity of T cells., (© 2021 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Delayed-type hypersensitivity arthritis (DTHA) is a recently established experimental model of rheumatoid arthritis (RA) in mice with pharmacological values. Despite an indispensable role of CD4+ T cells in inducing DTHA, a potential role for CD4+ T cell subsets is lacking. Here we have quantified CD4+ subsets during DTHA development and found that levels of activated, pro-inflammatory Th1, Th17, and memory CD4+ T cells in draining lymph nodes were increased with differential dynamic patterns after DTHA induction. Moreover, according to B-cell depletion experiments, it has been suggested that this cell type is not involved in DTHA. We show that DTHA is associated with increased levels of B cells in draining lymph nodes accompanied by increased levels of circulating IgG. Finally, using the anti-rheumatoid agents, methotrexate (MTX) and the anti-inflammatory drug dexamethasone (DEX), we show that MTX and DEX differentially suppressed DTHA-induced paw swelling and inflammation. The effects of MTX and DEX coincided with differential regulation of levels of Th1, Th17, and memory T cells as well as B cells. Our results implicate Th1, Th17, and memory T cells, together with activated B cells, to be involved and required for DTHA-induced paw swelling and inflammation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Li, Kolan, Guo, Marciniak, Kolan, Malachin, Grimolizzi, Haraldsen and Skålhegg.)

3',5'-cyclic adenosine monophosphate (cAMP) dependent protein kinase or protein kinase A (PKA) has served as a prototype for the large family of protein kinases that are crucially important for signal transduction in eukaryotic cells. The PKA catalytic subunits are encoded by the two major genes PRKACA and PRKACB, respectively. The PRKACA gene encodes two known splice variants, the ubiquitously expressed Cα1 and the sperm-specifically expressed Cα2. In contrast, the PRKACB gene encodes several splice variants expressed in a highly cell and tissue-specific manner. The Cβ proteins are called Cβ1, Cβ2, Cβ3, Cβ4 and so-called abc variants of Cβ3 and Cβ4. Whereas Cβ1 is ubiquitously expressed, Cβ2 is enriched in immune cells and the Cβ3, Cβ4 and their abc variants are solely expressed in neuronal cells. All Cα and Cβ splice variants share a kinase-conserved catalytic core and a C-terminal tail encoded by exons 2 through 10 in the PRKACA and PRKACB genes, respectively. All Cα and Cβ splice variants with the exception of Cα1 and Cβ1 are hyper-variable at the N-terminus. Here, we will discuss how the PRKACA and PRKACB genes have developed as paralogs that encode distinct and functionally non-redundant proteins. The fact that Cα and Cβ splice variant mutations are associated with numerous diseases further opens new windows for PKA-induced disease pathologies., (© 2021 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Endothelial cell function and metabolism are closely linked to differential use of energy substrate sources and combustion. While endothelial cell migration is promoted by 2-phosphofructokinase-6/fructose-2,6-bisphosphatase (PFKFB3)-driven glycolysis, proliferation also depends on fatty acid oxidation for dNTP synthesis. We show that inflammatory activation of human umbilical vein endothelial cells (HUVECs) by interleukin-1β (IL-1β), despite inhibiting proliferation, promotes a shift toward more metabolically active phenotype. This was reflected in increased cellular glucose uptake and consumption, which was preceded by an increase in PFKFB3 mRNA and protein expression. However, despite a modest increase in extracellular acidification rates, the increase in glycolysis did not correlate with extracellular lactate accumulation. Accordingly, IL-1β stimulation also increased oxygen consumption rate, but without a concomitant rise in fatty acid oxidation. Together, this suggests that the IL-1β-stimulated energy shift is driven by shunting of glucose-derived pyruvate into mitochondria to maintain elevated oxygen consumption in HUVECs. We also revealed a marked donor-dependent variation in the amplitude of the metabolic response to IL-1β and postulate that the donor-specific response should be taken into account when considering targeting dysregulated endothelial cell metabolism., (© 2021 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Background: Current nutritional management of infants born very preterm results in significant deficiency of the essential fatty acids (FAs) arachidonic acid (ARA) and docosahexaenoic acid (DHA). The impact of this deficit on brain maturation and inflammation mediated neonatal morbidities are unknown. The aim of this study is to determine whether early supply of ARA and DHA improves brain maturation and neonatal outcomes in infants born before 29 weeks of gestation., Methods: Infants born at Oslo University Hospital are eligible to participate in this double-blind randomized controlled trial. Study participants are randomized to receive an enteral FA supplement of either 0.4 ml/kg MCT-oil™ (medium chain triglycerides) or 0.4 ml/kg Formulaid™ (100 mg/kg of ARA and 50 mg/kg of DHA). The FA supplement is given from the second day of life to 36 weeks' postmenstrual age (PMA). The primary outcome is brain maturation assessed by Magnetic Resonance Imaging (MRI) at term equivalent age. Secondary outcomes include quality of growth, incidence of neonatal morbidities, cardiovascular health and neuro-development. Target sample size is 120 infants (60 per group), this will provide 80% power to detect a 0.04 difference in mean diffusivity (MD, mm 2 /sec) in major white matter tracts on MRI., Discussion: Supplementation of ARA and DHA has the potential to improve brain maturation and reduce inflammation related diseases. This study is expected to provide valuable information for future nutritional guidelines for preterm infants., Trial Registration: Clinicaltrials.gov ID: NCT03555019 . Registered 4 October 2018- Retrospectively registered.

PRKACA and PRKACB code for two catalytic subunits (Cα and Cβ) of cAMP-dependent protein kinase (PKA), a pleiotropic holoenzyme that regulates numerous fundamental biological processes such as metabolism, development, memory, and immune response. We report seven unrelated individuals presenting with a multiple congenital malformation syndrome in whom we identified heterozygous germline or mosaic missense variants in PRKACA or PRKACB. Three affected individuals were found with the same PRKACA variant, and the other four had different PRKACB mutations. In most cases, the mutations arose de novo, and two individuals had offspring with the same condition. Nearly all affected individuals and their affected offspring shared an atrioventricular septal defect or a common atrium along with postaxial polydactyly. Additional features included skeletal abnormalities and ectodermal defects of variable severity in five individuals, cognitive deficit in two individuals, and various unusual tumors in one individual. We investigated the structural and functional consequences of the variants identified in PRKACA and PRKACB through the use of several computational and experimental approaches, and we found that they lead to PKA holoenzymes which are more sensitive to activation by cAMP than are the wild-type proteins. Furthermore, expression of PRKACA or PRKACB variants detected in the affected individuals inhibited hedgehog signaling in NIH 3T3 fibroblasts, thereby providing an underlying mechanism for the developmental defects observed in these cases. Our findings highlight the importance of both Cα and Cβ subunits of PKA during human development., (Copyright © 2020 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

In a healthy person, metabolically quiescent T lymphocytes (T cells) circulate between lymph nodes and peripheral tissues in search of antigens. Upon infection, some T cells will encounter cognate antigens followed by proliferation and clonal expansion in a context-dependent manner, to become effector T cells. These events are accompanied by changes in cellular metabolism, known as metabolic reprogramming. The magnitude and variation of metabolic reprogramming are, in addition to antigens, dependent on factors such as nutrients and oxygen to ensure host survival during various diseases. Herein, we describe how metabolic programmes define T cell subset identity and effector functions. In addition, we will discuss how metabolic programs can be modulated and affect T cell activity in health and disease using cancer and autoimmunity as examples., (© 2020 The Authors. Scandinavian Journal of Immunology published by John Wiley & Sons Ltd on behalf of The Scandinavian Foundation for Immunology.)

The present study compared the effects of postexercise carbohydrate plus protein (CHO+PROT) and carbohydrate (CHO)-only supplementation on muscle glycogen metabolism, anabolic cell signaling, and subsequent exercise performance. Nine endurance-trained males cycled twice to exhaustion (muscle glycogen decreased from ~495 to ~125 mmol/kg dry wt) and received either CHO only (1.2 g·kg -1 ·h -1 ) or CHO+PROT (0.8/0.4 g·kg -1 ·h -1 ) during the first 90 min of recovery. Glycogen content was similar before the performance test after 5 h of recovery. Glycogen synthase (GS) fractional activity increased after exhaustive exercise and remained activated 5 h after, despite substantial glycogen synthesis (176.1 ± 19.1 and 204.6 ± 27.0 mmol/kg dry wt in CHO and CHO+PROT, respectively; P = 0.15). Phosphorylation of GS at site 3 and site 2+2a remained low during recovery. After the 5-h recovery, cycling time to exhaustion was improved by CHO+PROT supplementation compared with CHO supplementation (54.6 ± 11.0 vs. 46.1 ± 9.8 min; P = 0.009). After the performance test, muscle glycogen was equally reduced in CHO+PROT and CHO. Akt Ser 473 and p70s6k Thr 389 phosphorylation was elevated after 5 h of recovery. There were no differences in Akt Ser 473 , p70s6k Thr 389 , or TSC2 Thr 1462 phosphorylation between treatments. Nitrogen balance was positive in CHO+PROT (19.6 ± 7.6 mg nitrogen/kg; P = 0.04) and higher than CHO (-10.7 ± 6.3 mg nitrogen/kg; P = 0.009). CHO+PROT supplementation during exercise recovery improved subsequent endurance performance relative to consuming CHO only. This improved performance after CHO+PROT supplementation could not be accounted for by differences in glycogen metabolism or anabolic cell signaling, but may have been related to differences in nitrogen balance. NEW & NOTEWORTHY Endurance athletes competing consecutive days need optimal dietary intake during the recovery period. We report that coingestion of protein and carbohydrate soon after exhaustive exercise, compared with carbohydrate only, resulted in better performance the following day. The better performance after coingestion of protein and carbohydrate was not associated with a higher rate of glycogen synthesis or activation of anabolic signaling compared with carbohydrate only. Importantly, nitrogen balance was positive after coingestion of protein and carbohydrate, which was not the case after intake of carbohydrate only, suggesting that protein synthesis contributes to the better performance the following day.

Inhibition of the key glycolytic activator 6-phosphofructokinase 2/fructose-2,6-bisphosphatase-3 (PFKFB3) by 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO) strongly attenuates pathological angiogenesis in cancer and inflammation. In addition to modulating endothelial proliferation and migration, 3PO also dampens proinflammatory activation of endothelial cells and experimental inflammation in vivo, suggesting a potential for 3PO in the treatment of chronic inflammation. The aim of our study was to explore if the anti-inflammatory action of 3PO in human endothelial cells was mediated by inhibition of PFKFB3 and glycolysis and assess if other means of PFKFB3 inhibition reduced inflammatory activation in a similar manner. We found that 3PO caused a rapid and transient reduction in IL-1β- and TNF-induced phosphorylation of both IKKα/β and JNK, thus inhibiting signaling through the NFκB and the stress-activated kinase pathways. However, in contrast to 3PO-treatment, neither shRNA-mediated silencing of PFKFB3 nor treatment with the alternative PFKFB3 inhibitor 7,8-dihydroxy-3-(4-hydroxy-phenyl)-chromen-4-one (YN1) prevented cytokine-induced NFκB signaling and upregulation of the adhesion molecules VCAM-1 and E-selectin, implying off target effects of 3PO. Collectively, our results suggest that the anti-inflammatory action of 3PO in human endothelial cells is not limited to inhibition of PFKFB3 and cellular glycolysis., Competing Interests: The authors have declared that no competing interests exist.

The aim of the present study was to investigate the effect of protein and carbohydrate ingestion during early recovery from exhaustive exercise on performance after 18-h recovery. Eight elite cyclists (V̇o 2max : 74.0 ± 1.6 ml·kg -1 ·min -1 ) completed two exercise and diet interventions in a double-blinded, randomized, crossover design. Participants cycled first at 73% of V̇o 2max (W 73% ) followed by 1-min intervals at 90% of V̇o 2max until exhaustion. During the first 2 h of recovery, participants ingested either 1.2 g carbohydrate·kg -1 ·h -1 (CHO) or 0.8 g carbohydrate + 0.4 g protein·kg -1 ·h -1 (CHO + PROT). The diet during the remaining recovery period was similar for both interventions and adjusted to body weight. After an 18-h recovery, cycling performance was assessed with a 10-s sprint test, 30 min of cycling at W 73% , and a cycling time trial (TT). The TT was 8.5% faster (41:53 ± 1:51 vs. 45:26 ± 1:32 min; P < 0.03) after CHO + PROT compared with CHO. Mean power output during the sprints was 3.7% higher in CHO + PROT compared with CHO (1,063 ± 54 vs. 1,026 ± 53 W; P = 0.01). Nitrogen balance in the recovery period was negative in CHO and neutral in CHO + PROT (-82.4 ± 11.5 vs. 7.0 ± 15.4 mg/kg; P  < 0.01). In conclusion, TT and sprint performances were improved 18 h after exhaustive cycling by CHO + PROT supplementation during the first 2 h of recovery compared with isoenergetic CHO supplementation. Our results indicate that intake of carbohydrate plus protein after exhaustive endurance exercise more rapidly converts the body from a catabolic to an anabolic state than carbohydrate alone, thus speeding recovery and improving subsequent cycling performance. NEW & NOTEWORTHY Prolonged high intensity endurance exercise depends on glycogen utilization and high oxidative capacity. Still, exhaustion develops and effective recovery strategies are required to compete in multiday stage races. We show that coingestion of protein and carbohydrate during the first 2 h of recovery is superior to isoenergetic intake of carbohydrate to stimulate recovery, and improves both endurance time-trial and 10-s sprint performance the following day in elite cyclists.

Assembly of multi enzyme complexes at subcellular localizations by anchoring- and scaffolding proteins represents a pivotal mechanism for achieving spatiotemporal regulation of cellular signaling after hormone receptor targeting [for review, see (1)]. In the 3' 5'-cyclic adenosine monophosphate (cAMP) dependent protein kinase (PKA) signaling pathway it is generally accepted that specificity is secured at several levels. This includes at the first level stimulation of receptors coupled to heterotrimeric G proteins which through stimulation of adenylyl cyclase (AC) forms the second messenger cAMP. Cyclic AMP has several receptors including PKA. PKA is a tetrameric holoenzyme consisting of a regulatory (R) subunit dimer and two catalytic (C) subunits. The R subunit is the receptor for cAMP and compartmentalizes cAMP signals through binding to cell and tissue-specifically expressed A kinase anchoring proteins (AKAPs). The current dogma tells that in the presence of cAMP, PKA dissociates into an R subunit dimer and two C subunits which are free to phosphorylate relevant substrates in the cytosol and nucleus. The release of the C subunit has raised the question how specificity of the cAMP and PKA signaling pathway is maintained when the C subunit no longer is attached to the R subunit-AKAP complex. An increasing body of evidence points toward a regulatory role of the cAMP and PKA signaling pathway by targeting the C subunits to various C subunit binding proteins in the cytosol and nucleus. Moreover, recent identification of isoform specific amino acid sequences, motifs and three dimensional structures have together provided new insight into how PKA at the level of the C subunit may act in a highly isoform-specific fashion. Here we discuss recent understanding of specificity of the cAMP and PKA signaling pathway based on C subunit subcellular targeting as well as evolution of the C subunit structure that may contribute to the dynamic regulation of C subunit activity.

Protein kinase A (PKA) is a holoenzyme composed of a regulatory subunit dimer and two catalytic subunits and regulates numerous cellular functions including immune cell activity. There are two major catalytic subunit genes, PRKACA and PRKACB encoding the catalytic subunits Cα and Cβ. The PRKACB gene encodes several splice variants including Cβ2, which is enriched in T-, B- and natural killer cells. Cβ2 is significantly larger (46 kDa) than any other C splice variant. In this study we characterized mice ablated for the Cβ2 protein demonstrating a significantly reduced cAMP-induced catalytic activity of PKA in the spleenocytes, lymphocytes and thymocytes. We also observed a significantly increased number of CD62L-expressing CD4 + and CD8 + T cells in LNs, accompanied by increased susceptibility to systemic inflammation by the Cβ2 ablated mice. The latter was reflected in an elevated sensitivity to collagen-induced arthritis (CIA), as well as higher concentration of TNF-α and lower concentration of IL-10 in response to LPS challenges. We suggest a role of Cβ2 in regulating innate as well as adaptive immune sensitivity in vivo., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Ameloblastin (AMBN), an important component of the self-assembled enamel extra cellular matrix, contains several in silico predicted phosphorylation sites. However, to what extent these sites actually are phosphorylated and the possible effects of such post-translational modifications are still largely unknown. Here we report on in vitro experiments aimed at investigating what sites in AMBN are phosphorylated by casein kinase 2 (CK2) and protein kinase A (PKA) and the impact such phosphorylation has on self-assembly and calcium binding. All predicted sites in AMBN can be phosphorylated by CK2 and/or PKA. The experiments show that phosphorylation, especially in the exon 5 derived part of the molecule, is inversely correlated with AMBN self-assembly. These results support earlier findings suggesting that AMBN self-assembly is mostly dependent on the exon 5 encoded region of the AMBN gene. Phosphorylation was significantly more efficient when the AMBN molecules were in solution and not present as supramolecular assemblies, suggesting that post-translational modification of AMBN must take place before the enamel matrix molecules self-assemble inside the ameloblast cell. Moreover, phosphorylation of exon 5, and the consequent reduction in self-assembly, seem to reduce the calcium binding capacity of AMBN suggesting that post-translational modification of AMBN also can be involved in control of free Ca 2+ during enamel extra cellular matrix biomineralization. Finally, it is speculated that phosphorylation can provide a functional crossroad for AMBN either to be phosphorylated and act as monomeric signal molecule during early odontogenesis and bone formation, or escape phosphorylation to be subsequently secreted as supramolecular assemblies that partake in enamel matrix structure and mineralization.

The 3',5'-cyclic adenosine monophosphate (cAMP)-dependent protein kinase, or protein kinase A (PKA), pathway is one of the most versatile and best studied signaling pathways in eukaryotic cells. The two paralogous PKA catalytic subunits Cα and Cβ, encoded by the genes PRKACA and PRKACB, respectively, are among the best understood model kinases in signal transduction research. In this work, we explore and elucidate the evolution of the alternative 5' exons and the splicing pattern giving rise to the numerous PKA catalytic subunit isoforms. In addition to the universally conserved Cα1/Cβ1 isoforms, we find kinase variants with short N-termini in all main vertebrate classes, including the sperm-specific Cα2 isoform found to be conserved in all mammals. We also describe, for the first time, a PKA Cα isoform with a long N-terminus, paralogous to the PKA Cβ2 N-terminus. An analysis of isoform-specific variation highlights residues and motifs that are likely to be of functional importance.

The IL-17-producing CD4 + T helper cell (Th17) differentiation is affected by stimulation of the aryl hydrocarbon receptor (AhR) pathway and by hypoxia-inducible factor 1 alpha (HIF-1α). In some cases, Th17 become non-pathogenic and produce IL-10. However, the initiating events triggering this phenotype are yet to be fully understood. Here, we show that such cells may be differentiated at low oxygen and regardless of AhR ligand treatment such as cigarette smoke extract. Hypoxia led to marked alterations of the transcriptome of IL-10-producing Th17 cells affecting genes involved in metabolic, anti-apoptotic, cell cycle, and T cell functional pathways. Moreover, we show that oxygen regulates the expression of CD52, which is a cell surface protein that has been shown to suppress the activation of other T cells upon release. Taken together, these findings suggest a novel ability for Th17 cells to regulate immune responses in vivo in an oxygen-dependent fashion.

Background: Today overtreatment of indolent prostate cancers and undertreatment of aggressive prostate cancer are a major concern for patients, their families, and the health care system. New biomarkers distinguishing indolent and aggressive prostate cancer are needed to improve precision medicine. In prostate cancer, protein kinase A (PKA) is known to activate the androgen receptor and published data indicate that PKA subunits can act as predictive markers for response to radiation and chemotherapy. We have previously shown that the catalytic subunit, Cβ2, of PKA is up-regulated in prostate cancer and we would in this study investigate the potential of Cβ2 to become a prognostic biomarker in prostate cancer., Methods: Data were sampled from a total of 241 patients from 3 independent cohorts. We measured and compared Cβ2 messenger RNA (mRNA) levels in prostate tumor and nontumor samples (n = 22), and exon levels in a cohort of 50 tumor samples, as well as acquiring mRNA data from the publicly available database The cancer genome atlas (n = 169)., Results: Cβ2 mRNA was up-regulated in prostate cancer in all 3 cohorts, measured by 3 different methods. Furthermore, the relative Cβ2 mRNA expression levels were lower in prostate cancer samples with Gleason score 8 to 10 compared with samples with Gleason score<8 (P = 0.004). Finally, low expression of Cβ2 mRNA in prostate cancer biopsies correlated with poor survival (hazard ratio = 0.20; 95% CI: 0.048-0.86; P = 0.031), adjusted for risk group and age., Conclusions: We suggest that Cβ2 mRNA expression may be used as a biomarker together with established prognostic markers to more precisely predict aggressiveness in patients diagnosed with prostate cancer., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Introduction: Aging is associated with increased levels of circulating inflammatory markers and reduced muscle mass and strength., Objective: We investigated whether intake of protein-enriched milk for 12 weeks would influence markers of inflammation among adults ≥70years of age with reduced physical strength., Methods: In a double-blind randomized controlled intervention study, subjects were randomly allocated into two groups, receiving a protein-enriched milk (2×20g protein/d, n=14, mean (±SD) age 76.9±4.9 yrs) or an isocaloric carbohydrate drink (n=17, age 77.7±4.8 yrs) for 12 weeks. We measured serum and mRNA expression levels of inflammatory markers in PBMCs., Results: Significant differences in the mRNA expression of nuclear receptor subfamily, group H, member 3 (NR1H3, encoding the LXRα transcription factor) and interferon gamma (INFG) were observed between groups. The mRNA level of TNFRSF1A was significantly reduced, while the mRNA level of dipeptidyl-peptidase 4 (DPP4) was significantly increased, in the control group. The serum level of TNFα increased significantly in the control group, while sTNFRSF1A increased significantly in both groups, but with no significant differences between groups., Conclusion: Consumption of a low-fat, protein-enriched milk for 12 weeks had minor effects on inflammatory related markers in older adults compared to an isocaloric carbohydrate drink., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Immune responses often take place where nutrients and O2 availability are limited. This has an impact on T cell metabolism and influences activation and effector functions. T cell proliferation and expansion are associated with increased consumption of glutamine which is needed in a number of metabolic pathways and regulate various physiological processes. The first step in endogenous glutamine metabolism is reversible and is regulated by glutaminase (GLS1 and GLS2) and glutamine synthase (GLUL). There are two isoforms of GLS1, Kidney type glutaminase (KGA) and Glutaminase C (GAC). The aim of this study is to investigate the expression, localization and role of GLS1 and GLUL in naïve and activated human CD4+ T cells stimulated through the CD3 and CD28 receptors under normoxia and hypoxia. In proliferating cells, GAC was upregulated and KGA was downregulated, and both enzymes were located to the mitochondria irrespective of O2 levels. By contrast GLUL is localized to the cytoplasm and was upregulated under hypoxia. Proliferation was dependent on glutamine consumption, as glutamine deprivation and GLS1 inhibition decreased proliferation and expression of CD25 and CD226, regardless of O2 availability. Again irrespective of O2, GLS1 inhibition decreased the proportion of CCR6 and CXCR3 expressing CD4+ T cells as well as cytokine production. We propose that systemic Th cell activation and expansion might be dependent on glutamine but not O2 availability.

Study Question: Do the known capacitating agents HCO(3)(-) and serum albumin regulate the generation of ATP required for sperm motility and capacitation?, Summary Answer: Serum albumin and HCO(3)(-) seem to regulate two separate pools of ATP by different mechanisms in human spermatozoa., What Is Known Already: Sperm capacitation is a maturation process that naturally occurs in the female reproductive tract preparing the sperm cell for fertilization. It is a highly energy-depending process as it involves hyperactive motility and substantial levels of protein phosphorylation., Study Design, Size, Duration: Human sperm cells from four (motility experiments) and three (all other experiments) healthy donors were used. Untreated cells were compared with cells treated with HCO(3)(-) and serum albumin for up to 4 h., Participants/materials, Setting, Methods: Changes in glycolysis and mitochondrial respiration rates upon treatment with serum albumin and HCO(3)(-) were analysed by metabolic tracing of (13)C-labelled substrates and respirometry studies, respectively. Levels of hyperactive spermatozoa and ATP content were measured during 4 h of incubation under capacitating conditions., Main Results and the Role of Chance: We found that HCO(3)(-) significantly (P < 0.05) increased glycolytic flux by >3-folds via a cAMP/PKA sensitive pathway. This was accompanied by an increase in hyperactive motility. In contrast, serum albumin significantly increased endogenous ATP levels by 50% without stimulating hyperactive motility or glycolysis, indicating that this pool of ATP is separately located from the HCO(3)(-)-induced ATP. The increase in ATP induced by albumin could be mimicked by treatment with the cholesterol acceptors 2-hydroxypropyl- and methyl-β-cyclodextrin and counteracted by co-incubation with cholesterol sulphate to the level of the non-treated control (P < 0.05), pointing to cholesterol extraction from the sperm cell membrane as the main mechanism. However, the concentration of cyclodextrins needed to directly detect cholesterol extraction from the sperm cells was not compatible with maintenance of sperm viability. The increase in ATP seemed not to be dependent on the sperm-specific Ca(2+) channel CatSper. Finally, we demonstrated that neither HCO(3)(-) nor serum albumin stimulated mitochondrial respiration rates. However, serum albumin increased the respiratory capacity of mitochondria by >50%, an effect that was counteracted by HCO(3)(-)., Limitations, Reasons for Caution: Great variation in motility and capacitation is observed between sperm cells from different species. Hence, caution should be taken when extrapolating the findings in this work on human spermatozoa to sperm from other species., Wider Implications of the Findings: It is already established that an efficient energy-generation is required to support sperm motility and capacitation. However, the mechanisms explaining how ATP production is regulated in spermatozoa are not fully understood. Our findings indicate that HCO(3)(-) stimulates hyperactive motility by increasing glycolytic flux and ATP production in a cAMP/PKA sensitive fashion. On the other hand, serum albumin seems to increase ATP concentration at a different location and by a mechanism different from glycolysis that involves extraction of cholesterol from the sperm cell membrane. These new insights into sperm metabolism may pave the way for both the development of new and improved male contraceptives and optimized assisted reproduction techniques., Study Funding: The work was funded by Spermatech AS, The University of Oslo and the Research Council of Norway., Competing Interest(s): T.H.H. and K.R.R. are employees at Spermatech. B.S.S is a shareholder in Spermatech.

3',5'-cyclic adenosine monophosphate (cAMP) dependent protein kinase or protein kinase A (PKA) has served as a prototype for the large family of protein kinases that are crucially important for signal transduction in eukaryotic cells. The PKA catalytic subunits Cα and Cβ, encoded by the two genes PRKACA and PRKACB, respectively, are among the best understood and characterized human kinases. Here we have studied the evolution of this gene family in chordates, arthropods, mollusks and other animals employing probabilistic methods and show that Cα and Cβ arose by duplication of an ancestral PKA catalytic subunit in a common ancestor of vertebrates. The two genes have subsequently been duplicated in teleost fishes. The evolution of the PRKACG retroposon in simians was also investigated. Although the degree of sequence conservation in the PKA Cα/Cβ kinase family is exceptionally high, a small set of signature residues defining Cα and Cβ subfamilies were identified. These conserved residues might be important for functions that are unique to the Cα or Cβ clades. This study also provides a good example of a seemingly simple phylogenetic problem which, due to a very high degree of sequence conservation and corresponding weak phylogenetic signals, combined with problematic nonphylogenetic signals, is nontrivial for state-of-the-art probabilistic phylogenetic methods.

Protein kinase A (PKA) exists as several tissue-specific isoforms that through phosphorylation of serine and threonine residues of substrate proteins act as key regulators of a number of cellular processes. We here demonstrate that the human sperm-specific isoform of PKA named Cα2 is important for sperm motility and thus male fertility. Furthermore, we report on the first three-dimensional crystal structure of human apo Cα2 to 2.1 Å. Apo Cα2 displays an open conformation similar to the well-characterized apo structure of murine Cα1. The asymmetric unit contains two molecules and the core of the small lobe is rotated by almost 13° in the A molecule relative to the B molecule. In addition, a salt bridge between Lys72 and Glu91 was observed for Cα2 in the apo-form, a conformation previously found only in dimeric or ternary complexes of Cα1. Human Cα2 and Cα1 share primary structure with the exception of the amino acids at the N-terminus coded for by an alternative exon 1. The N-terminal glycine of Cα1 is myristoylated and this aliphatic chain anchors the N-terminus to an intramolecular hydrophobic pocket. Cα2 cannot be myristoylated and the crystal structure revealed that the equivalent hydrophobic pocket is unoccupied and exposed. Nuclear magnetic resonance (NMR) spectroscopy further demonstrated that detergents with hydrophobic moieties of different lengths can bind deep into this uncovered pocket. Our findings indicate that Cα2 through the hydrophobic pocket has the ability to bind intracellular targets in the sperm cell, which may modulate protein stability, activity and/or cellular localization., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The genes PRKACA and PRKACB encode the principal catalytic (C) subunits of protein kinase A (PKA) Cα and Cβ, respectively. Cα is expressed in all eukaryotic tissues examined and studies of Cα knockout mice demonstrate a crucial role for Cα in normal physiology. We have sequenced exon 2 through 10 of PRKACA from the genome of 498 Norwegian donors and extracted information about PRKACA mutations from public databases. We identified four interesting nonsynonymous point mutations, Arg45Gln, Ser109Pro, Gly186Val, and Ser263Cys, in the Cα1 splice variant of the kinase. Cα variants harboring the different amino acid mutations were analyzed for kinase activity and regulatory (R) subunit binding. Whereas mutation of residues 45 and 263 did not alter catalytic activity or R subunit binding, mutation of Ser(109) significantly reduced kinase activity while R subunit binding was unaltered. Mutation of Cα Gly(186) completely abrogated kinase activity and PKA type I but not type II holoenzyme formation. Gly(186) is located in the highly conserved DFG motif of Cα and mutation of this residue to Val was predicted to result in loss of binding of ATP and Mg(2+), which may explain the kinetic inactivity. We hypothesize that individuals born with mutations of Ser(109) or Gly(186) may be faced with abnormal development and possibly severe disease.

Accumulation of the complex set of alternatively processed mRNA from the adenovirus major late transcription unit (MLTU) is subjected to a temporal regulation involving both changes in poly (A) site choice and alternative 3' splice site usage. We have previously shown that the adenovirus L4-33K protein functions as an alternative splicing factor involved in activating the shift from L1-52,55K to L1-IIIa mRNA. Here we show that L4-33K specifically associates with the catalytic subunit of the DNA-dependent protein kinase (DNA-PK) in uninfected and adenovirus-infected nuclear extracts. Further, we show that L4-33K is highly phosphorylated by DNA-PK in vitro in a double stranded DNA-independent manner. Importantly, DNA-PK deficient cells show an enhanced production of the L1-IIIa mRNA suggesting an inhibitory role of DNA-PK on the temporal switch in L1 alternative RNA splicing. Moreover, we show that L4-33K also is phosphorylated by protein kinase A (PKA), and that PKA has an enhancer effect on L4-33K-stimulated L1-IIIa splicing. Hence, we demonstrate that these kinases have opposite effects on L4-33K function; DNA-PK as an inhibitor and PKA as an activator of L1-IIIa mRNA splicing. Taken together, this is the first report identifying protein kinases that phosphorylate L4-33K and to suggest novel regulatory roles for DNA-PK and PKA in adenovirus alternative RNA splicing.

Background: There has been an ongoing debate in the reproductive field about whether mammalian spermatozoa rely on glycolysis, oxidative phosphorylation or both for their energy production. Recent studies have proposed that human spermatozoa depend mainly on glucose for motility and fertilization but the mechanism behind an efficient glycolysis in human spermatozoa is not well understood. Here, we demonstrate how human spermatozoa utilize exogenous pyruvate to enhance glycolytic ATP production, motility, hyperactivation and capacitation, events that are crucial for male fertility., Methods: Purified human spermatozoa from healthy donors were incubated under capacitating conditions (including albumin, bicarbonate and glucose) and tested for changes in ATP levels, motility, hyperactivation and tyrosine phosphorylation after treatment with pyruvate. The experiments were repeated in the presence of sodium cyanide in order to assess the contribution from mitochondrial respiration. The metabolism of (13)C labeled glucose and pyruvate was traced by a combination of liquid chromatography and mass spectrometry., Results: The treatment of human spermatozoa with exogenous pyruvate increased intracellular ATP levels, progressive motility and hyperactivation by 56, 21 and 130%, respectively. In addition, added pyruvate induced a significant increase in tyrosine phosphorylation levels. Blocking of the electron transport chain did not markedly affect the results, indicating that the mechanism is independent of oxidative phosphorylation. However, the observed effects could be counteracted by oxamate, an inhibitor of lactate dehydrogenase (LDH). Metabolic tracing experiments revealed that the observed rise in ATP concentration resulted from an enhanced glycolytic flux, which was increased by more than 50% in the presence of exogenous pyruvate. Moreover, all consumed (13)C labeled pyruvate added was converted to lactate rather than oxidized in the tricarboxylic acid cycle., Conclusions: Human spermatozoa seem to rely mainly, if not entirely, on glycolysis as the source of ATP fueling the energy-demanding processes of motility and capacitation. The efficient glycolysis is dependent on exogenous pyruvate, which indirectly feeds the accelerated glycolysis with NAD(+) through the LDH-mediated conversion of pyruvate to lactate. Pyruvate is present in the human female reproductive tract at concentrations in accordance with our results. As seen in other mammals, the motility and fertility of human spermatozoa seem to be dictated by the available energy substrates present in the conspecific female.

Background: Post-transcriptional processing of pre-mRNA takes place in several steps and requires involvement of a number of RNA-binding proteins. How pre-mRNA processing is regulated is in large enigmatic. The catalytic (C) subunit of protein kinase A (PKA) is a serine/threonine kinase, which regulates numerous cellular processes including pre-mRNA splicing. Despite that a significant fraction of the C subunit is found in splicing factor compartments in the nucleus, there are no indications of a direct interaction between RNA and PKA. Based on this we speculate if the specificity of the C subunit in regulating pre-mRNA splicing may be mediated indirectly through other nuclear proteins., Results: Using yeast two-hybrid screening with the PKA C subunit Cbeta2 as bait, we identified the G-patch domain and KOW motifs-containing protein (GPKOW), also known as the T54 protein or MOS2 homolog, as an interaction partner for Cbeta2. We demonstrate that GPKOW, which contains one G-patch domain and two KOW motifs, is a nuclear RNA-binding protein conserved between species. GPKOW contains two sites that are phosphorylated by PKA in vitro. By RNA immunoprecipitation and site directed mutagenesis of the PKA phosphorylation sites we revealed that GPKOW binds RNA in vivo in a PKA sensitive fashion., Conclusion: GPKOW is a RNA-binding protein that binds RNA in a PKA regulated fashion. Together with our previous results demonstrating that PKA regulates pre-mRNA splicing, our results suggest that PKA phosphorylation is involved in regulating RNA processing at several steps.

Background: The two variants of the α-form of the catalytic (C) subunit of protein kinase A (PKA), designated Cα1 and Cα2, are encoded by the PRKACA gene. Whereas Cα1 is ubiquitous, Cα2 expression is restricted to the sperm cell. Cα1 and Cα2 are encoded with different N-terminal domains. In Cα1 but not Cα2 the N-terminal end introduces three sites for posttranslational modifications which include myristylation at Gly1, Asp-specific deamidation at Asn2 and autophosphorylation at Ser10. Previous reports have implicated specific biological features correlating with these modifications on Cα1. Since Cα2 is not modified in the same way as Cα1 we tested if they have distinct biochemical activities that may be reflected in different biological properties., Results: We show that Cα2 interacts with the two major forms of the regulatory subunit (R) of PKA, RI and RII, to form cAMP-sensitive PKAI and PKAII holoenzymes both in vitro and in vivo as is also the case with Cα1. Moreover, using Surface Plasmon Resonance (SPR), we show that the interaction patterns of the physiological inhibitors RI, RII and PKI were comparable for Cα2 and Cα1. This is also the case for their potency to inhibit catalytic activities of Cα2 and Cα1., Conclusion: We conclude that the regulatory complexes formed with either Cα1 or Cα2, respectively, are indistinguishable.