Validation of the Structured Assessment of Gastrointestinal Symptoms Scale to Support Standardized Evaluation and Follow-up.

OBJECTIVES: Patient-reported outcome measures facilitate evaluation of patients and allow to better assess treatment effects. Validated tools are lacking for pediatric gastroenterological patients. We thus aimed to adapt and validate for pediatric populations a self-administered Structured Assessment of Gastrointestinal Symptoms (SAGIS) tool that previously has been validated in adult cohorts. METHODS: Each item of the original SAGIS instrument was thoroughly reviewed for its relevance in the pediatric population. The resulting pediatric (p)SAGIS was utilized over a 35 months' period in consecutive patients in a pediatric outpatient GI clinic. Principal component analysis (PCA) followed by varimax rotation and confirmatory factor analysis (CFA) was performed in derivation and validation samples. Responsiveness to change was assessed in 32 children with inflammatory bowel disease (IBD) after 12 months of therapy. RESULTS: The final pediatric SAGIS (pSAGIS) consisted of 21 GI-related Likert-type questions, 8 dichotomous questions assessing extra-intestinal symptoms, and 2 most bothersome symptoms; 1153 children/adolescents completed a total of 2647 questionnaires. Cronbach alpha was 0.89, indicating good internal consistency. PCA supported a 5-factor model (symptom groups: abdominal pain, dyspepsia, diarrhea, constipation, dysphagia/nausea) and CFA showed good model fit (comparative fit index: 0.96, root-mean-square error of approximation: 0.075). The initial mean total GI symptom score in IBD patients (8.7 ± 10.3) decreased to 3.6 ± 7.7 after 1 year of therapy ( P < 0.01), and 4 of 5 symptom group scores decreased significantly upon treatment ( P < 0.05). CONCLUSIONS: The pSAGIS is a novel, easy to use, self-administered instrument for GI-symptom assessment in children/adolescents with excellent psychometric properties. It may standardize GI-symptom assessment and may enable uniform clinical analysis of treatment outcomes.

Significance of validated symptom assessment versus breath testing for malabsorption after lactose load in children.

BACKGROUND AND OBJECTIVES: Lactose malabsorption and lactose-induced symptoms are poorly correlated, as shown by breath tests and various symptom assessment methods. Validated assessment is the key to overcome the limitations of biased symptom measurements. We characterized lactose-induced symptoms with the population-specific, validated paediatric carbohydrate perception questionnaire (pCPQ) and their correlation with the history of symptoms (HoS). METHODS: A total of 130 patients with functional gastrointestinal symptoms underwent a lactose hydrogen breath and tolerance test (LBTT) allowing for a diagnosis of malabsorption (M+) and lactose sensitivity (S+). HoS indicative of lactose-induced symptoms (abdominal pain, nausea, bloating, flatulence, diarrhoea) in the 4 weeks preceding the test was determined using a validated questionnaire. The pCPQ was used to score lactose-induced symptoms. MAIN RESULTS: The LBTT revealed 41 children (31.5%) with lactose malabsorption (M+), 56 (43.1%) with lactose sensitivity (S+) and 24 (18.5%) were M+/S+. Sensitivity correlated with HoS (P < 0.001), regardless of whether malabsorption was detectable. Malabsorption status did not correlate with HoS (NS). The odds of lactose sensitivity significantly increased when abdominal pain [odds ratio (OR) 3.5, confidence interval (CI) 1.6-7.8], nausea (OR 2.3, CI, 1.1-4.9) and flatulence (OR 3.1, CI 1.4-6.8) were reported in the 4 weeks preceding the LBTT. Symptoms after the lactose load were similar for M+/S+ and M-/S+, except for flatulence, which was more frequent in malabsorbers (P < 0.01). CONCLUSION: Our findings fit well with the emerging view of the important role of a validated symptom assessment after a lactose load. The determination of symptoms may be more relevant than malabsorption for the clinical outcomes of paediatric patients with lactose-related gastrointestinal symptoms.

Characterization of the genetic switch from phage ɸ13 important for Staphylococcus aureus colonization in humans.

Temperate phages are bacterial viruses that after infection either reside integrated into a bacterial genome as prophages forming lysogens or multiply in a lytic lifecycle. The decision between lifestyles is determined by a switch involving a phage-encoded repressor, CI, and a promoter region from which lytic and lysogenic genes are divergently transcribed. Here, we investigate the switch of phage ɸ13 from the human pathogen Staphylococcus aureus. ɸ13 encodes several virulence factors and is prevalent in S. aureus strains colonizing humans. We show that the ɸ13 switch harbors a cI gene, a predicted mor (modulator of repression) gene, and three high-affinity operator sites binding CI. To quantify the decision between lytic and lysogenic lifestyle, we introduced reporter plasmids that carry the 1.3 kb switch region from ɸ13 with the lytic promoter fused to lacZ into S. aureus and Bacillus subtilis. Analysis of ß-galactosidase expression indicated that decision frequency is independent of host factors. The white "lysogenic" phenotype, which relies on the expression of cI, could be switched to a stable blue "lytic" phenotype by DNA damaging agents. We have characterized lifestyle decisions of phage ɸ13, and our approach may be applied to other temperate phages encoding virulence factors in S. aureus.

Beneficial effect of voluntary physical exercise in Plakophilin2 transgenic mice.

Arrhythmogenic right ventricular cardiomyopathy is a hereditary, rare disease with an increased risk for sudden cardiac death. The disease-causing mutations are located within the desmosomal complex and the highest incidence is found in plakophilin2. However, there are other factors playing a role for the disease progression unrelated to the genotype such as inflammation or exercise. Competitive sports have been identified as risk factor, but the type and extend of physical activity as cofactor for arrhythmogenesis remains under debate. We thus studied the effect of light voluntary exercise on cardiac health in a mouse model. Mice with a heterozygous PKP2 loss-of-function mutation were given the option to exercise in a running wheel which was monitored 24 h/d. We analyzed structural and functional development in vivo by echocardiography which revealed that neither the genotype nor the exercise caused any significant structural changes. Ejection fraction and fractional shortening were not influenced by the genotype itself, but exercise did cause a drop in both parameters after 8 weeks, which returned to normal after 16 weeks of training. The electrophysiological analysis revealed that the arrhythmogenic potential was slightly higher in heterozygous animals (50% vs 18% in wt littermates) and that an additional stressor (isoprenaline) did not lead to an increase of arrhythmogenic events pre run or after 8 weeks of running but the vulnerability was increased after 16 weeks. Exercise-induced alterations in Ca handling and contractility of isolated myocytes were mostly abolished in heterozygous animals. No fibrofatty replacements or rearrangement of gap junctions could be observed. Taken together we could show that light voluntary exercise can cause a transient aggravation of the mutation-induced phenotype which is abolished after long term exercise indicating a beneficial effect of long term light exercise.

Relevance of Methane and Carbon Dioxide Evaluation in Breath Tests for Carbohydrate Malabsorption in a Paediatric Cohort.

OBJECTIVES: The relevance of methane measurement in breath tests for the detection of carbohydrate malabsorption in children is controversial. The need for correction for poor sample collection is disputed. We evaluated the relevance of methane/CO2 measurements for the diagnosis of paediatric carbohydrate malabsorption. METHODS: A total of 132 breath tests (fructose: n = 54; lactose: n = 78) were performed in 91 children/adolescents with functional abdominal complaints. Breath samples were collected and analysed for hydrogen, methane, and CO2. Malabsorption was defined by a net increase over baseline of ≥20 parts per million (ppm) for hydrogen, ≥5 to ≥12 ppm for methane, and ≥10 to ≥15 ppm for hydrogen-plus-methane. The diagnosis was made before and after the use of a CO2-based correction factor (5.5% as the numerator). Hydrogen-based test results were compared with results obtained with other cut-off values. RESULTS: Fifty-eight positive tests were obtained by hydrogen measurement (without CO2 correction). The addition of methane measurements did not significantly influence the test results (P > 0.05). Only under the use of extraordinary cut-offs (combined hydrogen-plus-methane smaller than ≥18 ppm) did the rate of malabsorbers increase significantly (P < 0.05). After CO2 correction, hydrogen ≥20 ppm was detected in 4 additional patients, but 1 patient lost the hydrogen-based diagnosis of malabsorption (Cohen kappa = 0.92). CONCLUSIONS: Methane measurement did not significantly affect the detection rate of carbohydrate malabsorbers in children/adolescents with functional abdominal complaints when established cut-offs are used. The use of CO2 correction altered the diagnosis of malabsorption in a minority of patients but did not significantly alter overall test results.

Hypertonicity counteracts MCL-1 and renders BCL-XL a synthetic lethal target in head and neck cancer.

Head and neck squamous cell carcinoma (HNSCC) is an aggressive and difficult-to-treat cancer entity. Current therapies ultimately aim to activate the mitochondria-controlled (intrinsic) apoptosis pathway, but complex alterations in intracellular signaling cascades and the extracellular microenvironment hamper treatment response. On the one hand, proteins of the BCL-2 family set the threshold for cell death induction and prevent accidental cellular suicide. On the other hand, controlling a cell's readiness to die also determines whether malignant cells are sensitive or resistant to anticancer treatments. Here, we show that HNSCC cells upregulate the proapoptotic BH3-only protein NOXA in response to hyperosmotic stress. Induction of NOXA is sufficient to counteract the antiapoptotic properties of MCL-1 and switches HNSCC cells from dual BCL-XL/MCL-1 protection to exclusive BCL-XL addiction. Hypertonicity-induced functional loss of MCL-1 renders BCL-XL a synthetically lethal target in HNSCC, and inhibition of BCL-XL efficiently kills HNSCC cells that poorly respond to conventional therapies. We identify hypertonicity-induced upregulation of NOXA as link between osmotic pressure in the tumor environment and mitochondrial priming, which could perspectively be exploited to boost efficacy of anticancer drugs.

Long-term effects of empagliflozin on excitation-contraction-coupling in human induced pluripotent stem cell cardiomyocytes.

The SGLT2 inhibitor empagliflozin improved cardiovascular outcomes in patients with diabetes. As the cardiac mechanisms remain elusive, we investigated the long-term effects (up to 2 months) of empagliflozin on excitation-contraction (EC)-coupling in human cardiomyocytes derived from induced pluripotent stem cells (iPSC-CM) in a blinded manner. IPSC from 3 donors, differentiated into pure iPSC-CM (4 differentiations), were treated with a clinically relevant concentration of empagliflozin (0.5 µmol/l) or vehicle control. Treatment, data acquisition, and analysis were conducted externally blinded. Epifluorescence microscopy measurements in iPSC-CM showed that empagliflozin has neutral effects on Ca2+ transient amplitude, diastolic Ca2+ levels, Ca2+ transient kinetics, or sarcoplasmic Ca2+ load after 2 weeks or 8 weeks of treatment. Confocal microscopy determining possible effects on proarrhythmogenic diastolic Ca2+ release events showed that in iPSC-CM, Ca2+ spark frequency and leak was not altered after chronic treatment with empagliflozin. Finally, in patch-clamp experiments, empagliflozin did not change action potential duration, amplitude, or resting membrane potential compared with vehicle control after long-term treatment. Next-generation RNA sequencing (NGS) and mapped transcriptome profiles of iPSC-CMs untreated and treated with empagliflozin for 8 weeks showed no differentially expressed EC-coupling genes. In line with NGS data, Western blots indicate that empagliflozin has negligible effects on key EC-coupling proteins. In this blinded study, direct treatment of iPSC-CM with empagliflozin for a clinically relevant duration of 2 months did not influence cardiomyocyte EC-coupling and electrophysiology. Therefore, it is likely that other mechanisms independent of cardiomyocyte EC-coupling are responsible for the beneficial treatment effect of empagliflozin. KEY MESSAGES: This blinded study investigated the clinically relevant long-term effects (up to 2 months) of empagliflozin on cardiomyocyte excitation-contraction (EC)-coupling. Human cardiomyocytes derived from induced pluripotent stem cells (iPSC-CM) were used to study a human model including a high repetition number of experiments. Empagliflozin has neutral effects on cardiomyocyte Ca2+ transients, sarcoplasmic Ca2+ load, and diastolic sarcoplasmic Ca2+ leak. In patch-clamp experiments, empagliflozin did not change the action potential. Next-generation RNA sequencing, mapped transcriptome profiles, and Western blots of iPSC-CM untreated and treated with empagliflozin showed no differentially expressed EC-coupling candidates.

Revealing the mechanism of repressor inactivation during switching of a temperate bacteriophage.

Temperate bacteriophages can enter one of two life cycles following infection of a sensitive host: the lysogenic or the lytic life cycle. The choice between the two alternative life cycles is dependent upon a tight regulation of promoters and their cognate regulatory proteins within the phage genome. We investigated the genetic switch of TP901-1, a bacteriophage of Lactococcus lactis, controlled by the CI repressor and the modulator of repression (MOR) antirepressor and their interactions with DNA. We determined the solution structure of MOR, and we solved the crystal structure of MOR in complex with the N-terminal domain of CI, revealing the structural basis of MOR inhibition of CI binding to the DNA operator sites. 15N NMR Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion and rotating frame R1ρ measurements demonstrate that MOR displays molecular recognition dynamics on two different time scales involving a repacking of aromatic residues at the interface with CI. Mutations in the CI:MOR binding interface impair complex formation in vitro, and when introduced in vivo, the bacteriophage switch is unable to choose the lytic life cycle showing that the CI:MOR complex is essential for proper functioning of the genetic switch. On the basis of sequence alignments, we show that the structural features of the MOR:CI complex are likely conserved among a larger family of bacteriophages from human pathogens implicated in transfer of antibiotic resistance.

Development and validation of the paediatric Carbohydrate Perception Questionnaire (pCPQ), an instrument for the assessment of carbohydrate-induced gastrointestinal symptoms in the paediatric population.

BACKGROUND: There is an unmet need for a validated, test-specific symptom questionnaire to evaluate carbohydrate perception during breath tests. Our aim was to develop and validate a questionnaire for the assessment of symptoms after a provocative carbohydrate load. METHODS: After a literature search and initial focus group-style interviews, five relevant complaints were identified. Responses were given on a Likert-type faces scale with a language children use and understand. Reliability, validity and responsiveness to change were established by the implementation of the questionnaire during breath tests in 215 pediatric subjects. Correlation between the questionnaire and a medical interview by a pediatrician who was blinded to the results of the questionnaire (n = 19) was determined. KEY RESULTS: The questionnaire had good face and content validity (Lawshe ratio = 1). Intraclass correlation coefficients for test-retest reliability (n = 116) demonstrated good repeatability (P < .001), and effect sizes were small (Cohen's d < 0.15 for all symptoms). Convergent validity and discriminant validity were supported according to the multitrait-multimethod matrix method. The results obtained by the questionnaire correlated highly with the result of the medical interview (P < .001; Fisher's exact test). Cronbach's alpha was 0.81. Responsiveness was verified for the whole patient group and subgroups with medium to high effect sizes. CONCLUSIONS AND INFERENCES: The paediatric Carbohydrate Perception Questionnaire (pCPQ) is a simple, test-specific questionnaire for a pediatric population. It is a valid instrument with excellent psychometric properties to assess gastrointestinal symptoms after carbohydrate ingestion. The pCPQ can replace non-validated symptom assessment during carbohydrate breath tests and allows a standardized diagnosis of carbohydrate intolerance.

NCX1 represents an ionic Na+ sensing mechanism in macrophages.

Inflammation and infection can trigger local tissue Na+ accumulation. This Na+-rich environment boosts proinflammatory activation of monocyte/macrophage-like cells (MΦs) and their antimicrobial activity. Enhanced Na+-driven MΦ function requires the osmoprotective transcription factor nuclear factor of activated T cells 5 (NFAT5), which augments nitric oxide (NO) production and contributes to increased autophagy. However, the mechanism of Na+ sensing in MΦs remained unclear. High extracellular Na+ levels (high salt [HS]) trigger a substantial Na+ influx and Ca2+ loss. Here, we show that the Na+/Ca2+ exchanger 1 (NCX1, also known as solute carrier family 8 member A1 [SLC8A1]) plays a critical role in HS-triggered Na+ influx, concomitant Ca2+ efflux, and subsequent augmented NFAT5 accumulation. Moreover, interfering with NCX1 activity impairs HS-boosted inflammatory signaling, infection-triggered autolysosome formation, and subsequent antibacterial activity. Taken together, this demonstrates that NCX1 is able to sense Na+ and is required for amplifying inflammatory and antimicrobial MΦ responses upon HS exposure. Manipulating NCX1 offers a new strategy to regulate MΦ function.

Repression of the lysogenic PR promoter in bacteriophage TP901-1 through binding of a CI-MOR complex to a composite OM-OR operator.

A functional genetic switch from the lactococcal bacteriophage TP901-1, deciding which of two divergently transcribing promoters becomes most active and allows this bi-stable decision to be inherited in future generations requires a DNA region of less than 1 kb. The fragment encodes two repressors, CI and MOR, transcribed from the PR and PL promoters respectively. CI can repress the transcription of the mor gene at three operator sites (OR, OL, and OD), leading to the immune state. Repression of the cI gene, leading to the lytic (anti-immune) state, requires interaction between CI and MOR by an unknown mechanism, but involving a CI:MOR complex. A consensus for putative MOR binding sites (OM sites), and a common topology of three OM sites adjacent to the OR motif was here identified in diverse phage switches that encode CI and MOR homologs, in a search for DNA sequences similar to the TP901-1 switch. The OR site and all putative OM sites are important for establishment of the anti-immune repression of PR, and a putative DNA binding motif in MOR is needed for establishment of the anti-immune state. Direct evidence for binding between CI and MOR is here shown by pull-down experiments, chemical crosslinking, and size exclusion chromatography. The results are consistent with two possible models for establishment of the anti-immune repression of cI expression at the PR promoter.

Valid Assessment of Carbohydrate Intolerance and the Need for a Distinction to Carbohydrate Malabsorption. Comment on "Roles of Lactose and Fructose Malabsorption and Dietary Outcomes in Children Presenting with Chronic Abdominal Pain.", Nutrients, 2019, 11, 3063.

We have read with interest the recent paper by Posovszky and Roesler et al [1] that reports, based on the doctoral thesis by Dr. Roesler [2] [...].

Ca2+/calmodulin-dependent protein kinase II is essential in hyperacute pressure overload.

BACKGROUND: Activation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) is established as a central intracellular trigger for various cardiac pathologies such as hypertrophy, heart failure or arrhythmias in animals and humans suggesting CaMKII as a promising target protein for future medical treatments. However, the physiological role of CaMKII is scarcely well defined. AIM & METHODS: To investigate the role of CaMKII in hyperacute pressure overload, we evaluated the effects of pressure overload induced by transverse aortic constriction (TAC) on survival, cardiac function, protein expression and excitation-contraction coupling (ECC) in female WT littermate vs. AC3-I mice 2 days after TAC (2d post TAC). AC3-I mice express the CaMKII inhibitor autocamtide-3 related inhibitory peptide (AiP) under the control of the α-myosin heavy chain promotor in the heart. RESULTS: CaMKII activation is significantly increased in WT TAC vs. sham mice 2d post TAC. Interestingly, survival is significantly reduced in AC3-I animals within the first five days after TAC compared to WT TAC littermates, while systolic cardiac function is markedly reduced in AC3-I TAC vs. AC3-I sham mice, but preserved in WT TAC vs. WT sham mice. Proteins regulating ECC such as ryanodine receptors (RyR2) and phospholamban (PLB) are hypophosphorylated at their CaMKII phosphorylation site in AC3-I TAC mice, but hyperphosphorylated in WT TAC mice compared to controls. In isolated cardiomyocytes fractional shortening is significantly impaired in AC3-I compared to WT mice 2d post TAC, and CaMKII incubation with AiP mimics the AC3-I phenotype in cardiomyocytes from WT TAC mice in vitro. In summary, this suggests cardiac dysfunction due to CaMKII inhibition as a potential cause of increased mortality in AC3-I TAC mice. However, proarrhythmic spontaneous Ca2+ release events (SCR) appear less frequent in cardiomyocytes from AC3-I TAC mice than in WT TAC mice. CONCLUSIONS: Our data indicate that excessive CaMKII inhibition as present in AC3-I transgenic mice leads to an impaired adaptation of ECC to hyperacute pressure overload resulting in diminished cardiac contractility and increased death. Thus, our data suggest that in pressure overload the activation of CaMKII is a pivotal, but previously unknown part of hyperacute stress physiology in the heart, while CaMKII inhibition, albeit potentially antiarrhythmic, can be detrimental. This should be taken into account for future studies with CaMKII inhibitors as therapeutic agents.

Genetics of Lactococci.

Lactococcus lactis is the best characterized species among the lactococci, and among the most consumed food-fermenting bacteria worldwide. Thanks to their importance in industrialized food production, lactococci are among the lead bacteria understood for fundamental metabolic pathways that dictate growth and survival properties. Interestingly, lactococci belong to the Streptococcaceae family, which includes food, commensal and virulent species. As basic metabolic pathways (e.g., respiration, metal homeostasis, nucleotide metabolism) are now understood to underlie virulence, processes elucidated in lactococci could be important for understanding pathogen fitness and synergy between bacteria. This chapter highlights major findings in lactococci and related bacteria, and covers five themes: distinguishing features of lactococci, metabolic capacities including the less known respiration metabolism in Streptococcaceae, factors and pathways modulating stress response and fitness, interbacterial dialogue via metabolites, and novel applications in health and biotechnology.

HIF1A and NFAT5 coordinate Na+-boosted antibacterial defense via enhanced autophagy and autolysosomal targeting.

Infection and inflammation are able to induce diet-independent Na+-accumulation without commensurate water retention in afflicted tissues, which favors the pro-inflammatory activation of mouse macrophages and augments their antibacterial and antiparasitic activity. While Na+-boosted host defense against the protozoan parasite Leishmania major is mediated by increased expression of the leishmanicidal NOS2 (nitric oxide synthase 2, inducible), the molecular mechanisms underpinning this enhanced antibacterial defense of mouse macrophages with high Na+ (HS) exposure are unknown. Here, we provide evidence that HS-increased antibacterial activity against E. coli was neither dependent on NOS2 nor on the phagocyte oxidase. In contrast, HS-augmented antibacterial defense hinged on HIF1A (hypoxia inducible factor 1, alpha subunit)-dependent increased autophagy, and NFAT5 (nuclear factor of activated T cells 5)-dependent targeting of intracellular E. coli to acidic autolysosomal compartments. Overall, these findings suggest that the autolysosomal compartment is a novel target of Na+-modulated cell autonomous innate immunity. Abbreviations: ACT: actins; AKT: AKT serine/threonine kinase 1; ATG2A: autophagy related 2A; ATG4C: autophagy related 4C, cysteine peptidase; ATG7: autophagy related 7; ATG12: autophagy related 12; BECN1: beclin 1; BMDM: bone marrow-derived macrophages; BNIP3: BCL2/adenovirus E1B interacting protein 3; CFU: colony forming units; CM-H2DCFDA: 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester; CTSB: cathepsin B; CYBB: cytochrome b-245 beta chain; DAPI: 4,6-diamidino-2-phenylindole; DMOG: dimethyloxallyl glycine; DPI: diphenyleneiodonium chloride; E. coli: Escherichia coli; FDR: false discovery rate; GFP: green fluorescent protein; GSEA: gene set enrichment analysis; GO: gene ontology; HIF1A: hypoxia inducible factor 1, alpha subunit; HUGO: human genome organization; HS: high salt (+ 40 mM of NaCl to standard cell culture conditions); HSP90: heat shock 90 kDa proteins; LDH: lactate dehydrogenase; LPS: lipopolysaccharide; Lyz2/LysM: lysozyme 2; NFAT5/TonEBP: nuclear factor of activated T cells 5; MΦ: macrophages; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MFI: mean fluorescence intensity; MIC: minimum inhibitory concentration; MOI: multiplicity of infection; MTOR: mechanistic target of rapamycin kinase; NaCl: sodium chloride; NES: normalized enrichment score; n.s.: not significant; NO: nitric oxide; NOS2/iNOS: nitric oxide synthase 2, inducible; NS: normal salt; PCR: polymerase chain reaction; PGK1: phosphoglycerate kinase 1; PHOX: phagocyte oxidase; RFP: red fluorescent protein; RNA: ribonucleic acid; ROS: reactive oxygen species; sCFP3A: super cyan fluorescent protein 3A; SBFI: sodium-binding benzofuran isophthalate; SLC2A1/GLUT1: solute carrier family 2 (facilitated glucose transporter), member 1; SQSTM1/p62: sequestosome 1; ULK1: unc-51 like kinase 1; v-ATPase: vacuolar-type H+-ATPase; WT: wild type.

Empagliflozin reduces Ca/calmodulin-dependent kinase II activity in isolated ventricular cardiomyocytes.

AIMS: The EMPA-REG OUTCOME study showed reduced mortality and hospitalization due to heart failure (HF) in diabetic patients treated with empagliflozin. Overexpression and Ca2+ -dependent activation of Ca2+ /calmodulin-dependent kinase II (CaMKII) are hallmarks of HF, leading to contractile dysfunction and arrhythmias. We tested whether empagliflozin reduces CaMKII- activity and improves Ca2+ -handling in human and murine ventricular myocytes. METHODS AND RESULTS: Myocytes from wild-type mice, mice with transverse aortic constriction (TAC) as a model of HF, and human failing ventricular myocytes were exposed to empagliflozin (1 µmol/L) or vehicle. CaMKII activity was assessed by CaMKII-histone deacetylase pulldown assay. Ca2+ spark frequency (CaSpF) as a measure of sarcoplasmic reticulum (SR) Ca2+ leak was investigated by confocal microscopy. [Na+ ]i was measured using Na+ /Ca2+ -exchanger (NCX) currents (whole-cell patch clamp). Compared with vehicle, 24 h empagliflozin exposure of murine myocytes reduced CaMKII activity (1.6 ± 0.7 vs. 4.2 ± 0.9, P < 0.05, n = 10 mice), and also CaMKII-dependent ryanodine receptor phosphorylation (0.8 ± 0.1 vs. 1.0 ± 0.1, P < 0.05, n = 11 mice), with similar results upon TAC. In murine myocytes, empagliflozin reduced CaSpF (TAC: 1.7 ± 0.3 vs. 2.5 ± 0.4 1/100 µm-1  s-1 , P < 0.05, n = 4 mice) but increased SR Ca2+ load and Ca2+ transient amplitude. Importantly, empagliflozin also significantly reduced CaSpF in human failing ventricular myocytes (1 ± 0.2 vs. 3.3 ± 0.9, P < 0.05, n = 4 patients), while Ca2+ transient amplitude was increased (F/F0 : 0.53 ± 0.05 vs. 0.36 ± 0.02, P < 0.05, n = 3 patients). In contrast, 30 min exposure with empagliflozin did not affect CaMKII activity nor Ca2+ -handling but significantly reduced [Na+ ]i . CONCLUSIONS: We show for the first time that empagliflozin reduces CaMKII activity and CaMKII-dependent SR Ca2+ leak. Reduced Ca2+ leak and improved Ca2+ transients may contribute to the beneficial effects of empagliflozin in HF.

Correction to: Relationship Between Abdominal Symptoms and Fructose Ingestion in Children with Chronic Abdominal Pain.

The original version of this article unfortunately contained an error in a couple of reference citation in Discussion Section, paragraph 6. The reference citation number should be changed from [6] to [9] in the below sentences so that it reads.

Relationship Between Abdominal Symptoms and Fructose Ingestion in Children with Chronic Abdominal Pain.

BACKGROUND: Limited valid data are available regarding the association of fructose-induced symptoms, fructose malabsorption, and clinical symptoms. AIM: To develop a questionnaire for valid symptom assessment before and during a carbohydrate breath test and to correlate symptoms with fructose breath test results in children/adolescents with functional abdominal pain. METHODS: A Likert-type questionnaire assessing symptoms considered relevant for hydrogen breath test in children was developed and underwent initial validation. Fructose malabsorption was determined by increased breath hydrogen in 82 pediatric patients with functional abdominal pain disorders; fructose-induced symptoms were quantified by symptom score ≥2 and relevant symptom increase over baseline. The results were correlated with clinical symptoms. The time course of symptoms during the breath test was assessed. RESULTS: The questionnaire exhibited good psychometric properties in a standardized assessment of the severity of carbohydrate-related symptoms. A total of 40 % (n = 33) had malabsorption; symptoms were induced in 38 % (n = 31), but only 46 % (n = 15) with malabsorption were symptomatic. There was no significant correlation between fructose malabsorption and fructose-induced symptoms. Clinical symptoms correlated with symptoms evoked during the breath test (p < 0.001, r2 = 0.21) but not with malabsorption (NS). Malabsorbers did not differ from non-malabsorbers in terms of symptoms during breath test. Symptomatic patients had significantly higher pain and flatulence scores over the 9-h observation period (p < 0.01) than did nonsymptomatic patients; the meteorism score was higher after 90 min. CONCLUSIONS: Fructose-induced symptoms but not fructose malabsorption are related to increased abdominal symptoms and have distinct timing patterns.

Ribosomal dimerization factor YfiA is the major protein synthesized after abrupt glucose depletion in Lactococcus lactis.

We analysed the response of the model bacterium Lactococcus lactis to abrupt depletion of glucose after several generations of exponential growth. Glucose depletion resulted in a drastic drop in the energy charge accompanied by an extremely low GTP level and an almost total arrest of protein synthesis. Strikingly, the cell prioritized the continued synthesis of a few proteins, of which the ribosomal dimerization factor YfiA was the most highly expressed. Transcriptome analysis showed no immediate decrease in total mRNA levels despite the lowered nucleotide pools and only marginally increased levels of the yfiA transcript. Severe up-regulation of genes in the FruR, CcpA, ArgR and AhrC regulons were consistent with a downshift in carbon and energy source. Based upon the results, we suggest that transcription proceeded long enough to record the transcriptome changes from activation of the FruR, CcpA, ArgR and AhrC regulons, while protein synthesis stopped due to an extremely low GTP concentration emerging a few minutes after glucose depletion. The yfiA deletion mutant exhibited a longer lag phase upon replenishment of glucose and a faster death rate after prolonged starvation supporting that YfiA-mediated ribosomal dimerization is important for keeping long-term starved cells viable and competent for growth initiation.