Mutations that prevent caspase cleavage of RIPK1 cause autoinflammatory disease.

RIPK1 is a key regulator of innate immune signalling pathways. To ensure an optimal inflammatory response, RIPK1 is regulated post-translationally by well-characterized ubiquitylation and phosphorylation events, as well as by caspase-8-mediated cleavage1-7. The physiological relevance of this cleavage event remains unclear, although it is thought to inhibit activation of RIPK3 and necroptosis8. Here we show that the heterozygous missense mutations D324N, D324H and D324Y prevent caspase cleavage of RIPK1 in humans and result in an early-onset periodic fever syndrome and severe intermittent lymphadenopathy-a condition we term 'cleavage-resistant RIPK1-induced autoinflammatory syndrome'. To define the mechanism for this disease, we generated a cleavage-resistant Ripk1D325A mutant mouse strain. Whereas Ripk1-/- mice died postnatally from systemic inflammation, Ripk1D325A/D325A mice died during embryogenesis. Embryonic lethality was completely prevented by the combined loss of Casp8 and Ripk3, but not by loss of Ripk3 or Mlkl alone. Loss of RIPK1 kinase activity also prevented Ripk1D325A/D325A embryonic lethality, although the mice died before weaning from multi-organ inflammation in a RIPK3-dependent manner. Consistently, Ripk1D325A/D325A and Ripk1D325A/+ cells were hypersensitive to RIPK3-dependent TNF-induced apoptosis and necroptosis. Heterozygous Ripk1D325A/+ mice were viable and grossly normal, but were hyper-responsive to inflammatory stimuli in vivo. Our results demonstrate the importance of caspase-mediated RIPK1 cleavage during embryonic development and show that caspase cleavage of RIPK1 not only inhibits necroptosis but also maintains inflammatory homeostasis throughout life.

Mast cell activation and degranulation in acute artery injury: A target for post-operative therapy.

The increasing incidence of cardiovascular disease (CVD) has led to a significant ongoing need to address this surgically through coronary artery bypass grafting (CABG) and percutaneous coronary interventions (PCI). From this, there continues to be a substantial burden of mortality and morbidity due to complications arising from endothelial damage, resulting in restenosis. Whilst mast cells (MC) have been shown to have a causative role in atherosclerosis and other vascular diseases, including restenosis due to vein engraftment; here, we demonstrate their rapid response to arterial wire injury, recapitulating the endothelial damage seen in PCI procedures. Using wild-type mice, we demonstrate accumulation of MC in the femoral artery post-acute wire injury, with rapid activation and degranulation, resulting in neointimal hyperplasia, which was not observed in MC-deficient KitW-sh/W-sh mice. Furthermore, neutrophils, macrophages, and T cells were abundant in the wild-type mice area of injury but reduced in the KitW-sh/W-sh mice. Following bone-marrow-derived MC (BMMC) transplantation into KitW-sh/W-sh mice, not only was the neointimal hyperplasia induced, but the neutrophil, macrophage, and T-cell populations were also present in these transplanted mice. To demonstrate the utility of MC as a target for therapy, we administered the MC stabilizing drug, disodium cromoglycate (DSCG) immediately following arterial injury and were able to show a reduction in neointimal hyperplasia in wild-type mice. These studies suggest a critical role for MC in inducing the conditions and coordinating the detrimental inflammatory response seen post-endothelial injury in arteries undergoing revascularization procedures, and by targeting the rapid MC degranulation immediately post-surgery with DSCG, this restenosis may become a preventable clinical complication.

Profiles of disordered eating behaviour in type 1 diabetes using the DEPS-R and behaviour and glycaemic outcomes in a real-life setting.

AIMS: The Diabetes Eating Problems Survey - Revised (DEPS-R) is commonly used to assess disordered eating behaviour (DEB) in individuals with type 1 diabetes and has advantages compared to other measures not specifically tailored to diabetes. A score ≥20 on the DEPS-R is used to indicate clinically significant DEB; however, it does not distinguish between eating disorder (ED) phenotypes necessary to guide treatment decisions, limiting clinical utility. METHODS: The current study used latent class analysis to identify distinct person-centred profiles of DEB in adults with type 1 diabetes using the DEPS-R. Analysis of Variance with Games Howell post-hoc comparisons was then conducted to examine the correspondence between the profiles and binge eating, insulin restriction and glycaemic control (HbA1c, mean blood glucose, and percent time spent in hyperglycaemia) during 3 days of assessment in a real-life setting. RESULTS: Latent class analysis indicated a 4-class solution, with patterns of item endorsement suggesting the following profiles: Bulimia, Binge Eating, Overeating and Low Pathology. Differences in binge eating, insulin restriction and glycaemic control were observed between profiles during 3 days of at-home assessment. The Bulimia profile was associated with highest HbA1c and 3-day mean blood glucose. CONCLUSIONS: There are common patterns of responses on the DEPS-R that appear to reflect different ED phenotypes. Profiles based on the DEPS-R corresponded with behaviour in the real-life setting as expected and were associated with different glycaemic outcomes. Results may have implications for the use of the DEPS-R in research and clinical settings.

Exploration of model misspecification in latent class methods for longitudinal data: Correlation structure matters.

Modeling longitudinal trajectories and identifying latent classes of trajectories is of great interest in biomedical research, and software to identify latent classes of such is readily available for latent class trajectory analysis (LCTA), growth mixture modeling (GMM) and covariance pattern mixture models (CPMM). In biomedical applications, the level of within-person correlation is often non-negligible, which can impact the model choice and interpretation. LCTA does not incorporate this correlation. GMM does so through random effects, while CPMM specifies a model for within-class marginal covariance matrix. Previous work has investigated the impact of constraining covariance structures, both within and across classes, in GMMs-an approach often used to solve convergence problems. Using simulation, we focused specifically on how misspecification of the temporal correlation structure and strength, but correct variances, impacts class enumeration and parameter estimation under LCTA and CPMM. We found (1) even in the presence of weak correlation, LCTA often does not reproduce original classes, (2) CPMM performs well in class enumeration when the correct correlation structure is selected, and (3) regardless of misspecification of the correlation structure, both LCTA and CPMM give unbiased estimates of the class trajectory parameters when the within-individual correlation is weak and the number of classes is correctly specified. However, the bias increases markedly when the correlation is moderate for LCTA and when the incorrect correlation structure is used for CPMM. This work highlights the importance of correlation alone in obtaining appropriate model interpretations and provides insight into model choice.

Solvent effect on the Spectra of Methylene Green and Methylene Blue.

This investigation focuses on dyes that differ only in the nitro substituent. The NO2 group leads to a strong hardening of the fluorescence at 298 K. In methylene green (MG) the excitation energy migrates to the system of triplet states. This non-radiative process causes the MG fuorescence absent or too low compared to methylene blue (MB). Moreover, laser-induced fluorescence is completely absent for MG in the investigated solvents. However, at liquid nitrogen temperature, we recorded fluorescence for MG in ethanol 250 times higher than at room temperature and phosphorescence too. The intensity of the MB fluorescence band in ethanol at 77 K is 6 times higher than at room temperature. According to the results of this study, the lifetime in the excited state decreases in the following order: isopropanol > acetonitrile≈ethanol≈dimethyl sulfoxide > > water for MG and chloroform > acetonitrile≈ethanol≈dimethyl sulfoxide > > water≈isopropanol for MB. In addition, MG has phosphorescence in ethanol at 780 nm (12,800 cm-1) and in chloroform at 810 nm (12,300 cm-1).

Middle age serum sodium levels in the upper part of normal range and risk of heart failure.

AIMS: With increasing prevalence of heart failure (HF) owing to the ageing population, identification of modifiable risk factors is important. In a mouse model, chronic hypohydration induced by lifelong water restriction promotes cardiac fibrosis. Hypohydration elevates serum sodium. Here, we evaluate the association of serum sodium at middle age as a measure of hydration habits with risk to develop HF. METHODS AND RESULTS: We analysed data from Atherosclerosis Risk in Communities study with middle age enrolment (45-66 years) and 25 years of follow-up. Participants without water balance dysregulation were selected: serum sodium within normal range (135-146 mmol/L), not diabetic, not obese and free of HF at baseline (N = 11 814). In time-to-event analysis, HF risk was increased by 39% if middle age serum sodium exceeded 143 mmol/L corresponding to 1% body weight water deficit [hazard ratio 1.39, 95% confidence interval (CI) 1.14-1.70]. In a retrospective case-control analysis performed on 70- to 90-year-old attendees of Visit 5 (N = 4961), serum sodium of 142.5-143 mmol/L was associated with 62% increase in odds of left ventricular hypertrophy (LVH) diagnosis [odds ratio (OR) 1.62, 95% CI 1.03-2.55]. Serum sodium above 143 mmol/L was associated with 107% increase in odds of LVH (OR 2.07, 95% CI 1.30-3.28) and 54% increase in odds of HF (OR 1.54, 95% CI 1.06-2.23). As a result, prevalence of HF and LVH was increased among 70- to 90-year-old participants with higher middle age serum sodium. CONCLUSION: Middle age serum sodium above 142 mmol is a risk factor for LVH and HF. Maintaining good hydration throughout life may slow down decline in cardiac function and decrease prevalence of HF.

Efficacy and Safety of Massage for Osteoarthritis of the Knee: a Randomized Clinical Trial.

BACKGROUND: Current treatment options for knee osteoarthritis have limited effectiveness and potentially adverse side effects. Massage may offer a safe and effective complement to the management of knee osteoarthritis. OBJECTIVE: Examine effects of whole-body massage on knee osteoarthritis, compared to active control (light-touch) and usual care. DESIGN: Multisite RCT assessing the efficacy of massage compared to light-touch and usual care in adults with knee osteoarthritis, with assessments at baseline and weeks 8, 16, 24, 36, and 52. Subjects in massage or light-touch groups received eight weekly treatments, then were randomized to biweekly intervention or usual care to week 52. The original usual care group continued to week 24. Analysis was performed on an intention-to-treat basis. PARTICIPANTS: Five hundred fifty-one screened for eligibility, 222 adults with knee osteoarthritis enrolled, 200 completed 8-week assessments, and 175 completed 52-week assessments. INTERVENTION: Sixty minutes of protocolized full-body massage or light-touch. MAIN MEASURES: Primary: Western Ontario and McMaster Universities Arthritis Index. Secondary: visual analog pain scale, PROMIS Pain Interference, knee range of motion, and timed 50-ft walk. KEY RESULTS: At 8 weeks, massage significantly improved WOMAC Global scores compared to light-touch (- 8.16, 95% CI = - 13.50 to - 2.81) and usual care (- 9.55, 95% CI = - 14.66 to - 4.45). Additionally, massage improved pain, stiffness, and physical function WOMAC subscale scores compared to light-touch (p < 0.001; p = 0.04; p = 0.02, respectively) and usual care (p < 0.001; p = 0.002; p = 0.002; respectively). At 52 weeks, the omnibus test of any group difference in the change in WOMAC Global from baseline to 52 weeks was not significant (p = 0.707, df = 3), indicating no significant difference in change across groups. Adverse events were minimal. CONCLUSIONS: Efficacy of symptom relief and safety of weekly massage make it an attractive short-term treatment option for knee osteoarthritis. Longer-term biweekly dose maintained improvement, but did not provide additional benefit beyond usual care post 8-week treatment. TRIAL REGISTRATION: clinicaltrials.gov NCT01537484.

Health literacy, sociodemographic factors, and cognitive training in the active study of older adults.

OBJECTIVE: Health literacy is critical for understanding information from health-care providers and correct use of medications and includes the capacity to filter other information in navigating health care systems. Older adults with low health literacy exhibit more chronic health conditions, worse physical functioning, and poorer mental health. This study examined the relationship between sociodemographic variables and health literacy, and the impact of cognitive training on change in health literacy over 10 years in older adults. METHODS: Participants (N = 2,802) aged 65 years and older completed assessments, including reading and numeracy health literacy items, as part of the Advanced Cognitive Training for Independent and Vital Elderly (ACTIVE) study. We evaluated baseline sociodemographic variables and change in health literacy over a 10-year period in individuals exposed to cognitive training in reasoning, processing speed, memory, or a no-contact control condition. RESULTS: Age, sex, race, education level, and general cognitive functioning at baseline were all associated with baseline health literacy in older adults. Predictors of change in health literacy over the 10-year follow-up were age, race, education level, general cognitive functioning, and neighborhood income; disparities in health literacy because of race attenuated over time, while the effect of age increased over time. Health literacy was generally stable across the ACTIVE intervention groups over 10 years. CONCLUSIONS: The present study showed important disparities in health literacy level and change over 10 years. Cognitive training did not significantly impact health literacy, suggesting that alternative approaches are needed to reduce the disparities.

Efficacy of a Goal Setting and Implementation Planning Intervention on Firefighters' Cardiorespiratory Fitness.

Bycura, DK, Dmitrieva, NO, Santos, AC, Waugh, KL, and Ritchey, KM. Efficacy of a Goal Setting and Implementation Planning Intervention on Firefighters' Cardiorespiratory Fitness. J Strength Cond Res 33(11): 3151-3161, 2019-The purpose of this study was to assess the effectiveness of a goal setting and implementation planning (GSIP) intervention on cardiorespiratory fitness among firefighters. Male career firefighters (N = 20) from a large municipal fire department were assigned to an intervention arm (n = 12) or passive control arm (n = 8) of a 14-week study involving cardiorespiratory exercise. The intervention consisted of GSIP coaching at baseline and support throughout the study. Cardiorespiratory fitness was assessed through a standardized physical protocol for firefighters (Candidate Physical Ability Test) at baseline, at 6 weeks, and at 14 weeks time points, using the Cosmed K4b portable metabolic system to measure physiological variables. Using intention-to-treat principles, we employed a 2-level multilevel model to examine the effect of intervention group on intercept and change over time on primary outcomes of interest: oxygen transport and utilization (VO2), heart rate (HR), and respiratory exchange ratio (R). Results demonstrated that the intervention and control groups both exhibited a significant increase in VO2 (B = 0.143, SE = 0.025, p < 0.001) and a decline in R (B = -0.003. SE = 0.001, p < 0.01) over the course of the study. Although both groups exhibited improvements on 2 of the 3 cardiorespiratory outcomes, subjects assigned to the intervention did not exhibit superior outcomes when compared to the control group. Given the improvements we observed in 2 out of 3 outcomes across both conditions, future research may consider testing effectiveness of interventions consisting of functional training exercises that closely translate to firefighting tasks over time periods sufficient to elicit aerobic metabolic adaptations.

Time of Day When Type 1 Diabetes Patients With Eating Disorder Symptoms Most Commonly Restrict Insulin.

OBJECTIVE: Restricting insulin to lose weight is a significant problem in the clinical management of type 1 diabetes (T1D). Little is known about this behavior or how to effectively intervene. Identifying when insulin restriction occurs could allow clinicians to target typical high-risk times or formulate hypotheses regarding factors that influence this behavior. The current study investigated the frequency of insulin restriction by time of day. METHODS: Fifty-nine adults with T1D and eating disorder symptoms completed 72 hours of real-time reporting of eating and insulin dosing with continuous glucose monitoring. We used a generalized estimating equation model to test the global hypothesis that frequency of insulin restriction (defined as not taking enough insulin to cover food consumed) varied by time of day, and examined frequency of insulin restriction by hour. We also examined whether patterns of insulin restriction for 72 hours corresponded with patients' interview reports of insulin restriction for the past 28 days. RESULTS: Frequency of insulin restriction varied as a function of time (p = .016). Insulin restriction was the least likely in the morning hours (6:00-8:59 AM), averaging 6% of the meals/snacks consumed. Insulin restriction was more common in the late afternoon (3:00-5:59 PM), peaking at 29%. Insulin was restricted for 32% of the meals/snacks eaten overnight (excluding for hypoglycemia); however, overnight eating was rare. Insulin restriction was associated with higher 120-minute postprandial blood glucose (difference = 44.4 mg/dL, 95% confidence interval = 22.7-68.5, p < .001) and overall poorer metabolic control (r = 0.43-0.62, p's < .01). Patients reported restricting insulin for a greater percentage of meals and snacks for the past 28 days than during the 72 hour real-time assessment; however, the reports were correlated (Spearman's ρ = 0.46, p < .001) and accounted for similar variance in HbA1c (34% versus 35%, respectively). CONCLUSIONS: Findings suggest that insulin restriction may be less likely in the morning, and that late afternoon is a potentially important time for additional therapeutic support. Results also suggest that systematic clinical assessment and treatment of overnight eating might improve T1D management.

Cross-Sectional Positive Association of Serum Lipids and Blood Pressure With Serum Sodium Within the Normal Reference Range of 135-145 mmol/L.

OBJECTIVE: Serum sodium concentration is maintained by osmoregulation within normal range of 135 to 145 mmol/L. Previous analysis of data from the ARIC study (Atherosclerosis Risk in Communities) showed association of serum sodium with the 10-year risk scores of coronary heart disease and stroke. Current study evaluated the association of within-normal-range serum sodium with cardiovascular risk factors. APPROACH AND RESULTS: Only participants who did not take cholesterol or blood pressure medications and had sodium within normal 135 to 145 mmol/L range were included (n=8615), and the cohort was stratified based on race, sex, and smoking status. Multiple linear regression analysis of data from ARIC study was performed, with adjustment for age, blood glucose, insulin, glomerular filtration rate, body mass index, waist to hip ratio, and calorie intake. The analysis showed positive associations with sodium of total cholesterol, low-density lipoprotein cholesterol, and total cholesterol to high-density lipoprotein cholesterol ratio; apolipoprotein B; and systolic and diastolic blood pressure. Increases in lipids and blood pressure associated with 10 mmol/L increase in sodium are similar to the increases associated with 7 to 10 years of aging. Analysis of sodium measurements made 3 years apart demonstrated that it is stable within 2 to 3 mmol/L, explaining its association with long-term health outcomes. Furthermore, elevated sodium promoted lipid accumulation in cultured adipocytes, suggesting direct causative effects on lipid metabolism. CONCLUSIONS: Serum sodium concentration is a cardiovascular risk factor even within the normal reference range. Thus, decreasing sodium to the lower end of the normal range by modification of water and salt intake is a personalizable strategy for decreasing cardiovascular risks.

Secretion of von Willebrand factor by endothelial cells links sodium to hypercoagulability and thrombosis.

Hypercoagulability increases risk of thrombi that cause cardiovascular events. Here we identify plasma sodium concentration as a factor that modulates blood coagulability by affecting the production of von Willebrand factor (vWF), a key initiator of the clotting cascade. We find that elevation of salt over a range from the lower end of what is normal in blood to the level of severe hypernatremia reversibly increases vWF mRNA in endothelial cells in culture and the rate of vWF secretion from them. The high NaCl increases expression of tonicity-regulated transcription factor NFAT5 and its binding to promoter of vWF gene, suggesting involvement of hypertonic signaling in vWF up-regulation. To elevate NaCl in vivo, we modeled mild dehydration, subjecting mice to water restriction (WR) by feeding them with gel food containing 30% water. Such WR elevates blood sodium from 145.1 ± 0.5 to 150.2 ± 1.3 mmol/L and activates hypertonic signaling, evidenced from increased expression of NFAT5 in tissues. WR increases vWF mRNA in liver and lung and raises vWF protein in blood. Immunostaining of liver revealed increased production of vWF protein by endothelium and increased number of microthrombi inside capillaries. WR also increases blood level of D-dimer, indicative of ongoing coagulation and thrombolysis. Multivariate regression analysis of clinical data from the Atherosclerosis Risk in Communities Study demonstrated that serum sodium significantly contributes to prediction of plasma vWF and risk of stroke. The results indicate that elevation of extracellular sodium within the physiological range raises vWF sufficiently to increase coagulability and risk of thrombosis.

Demographic characteristics do not decrease the utility of depressive symptoms assessments: examining the practical impact of item bias in four heterogeneous samples of older adults.

OBJECTIVE: Previous studies have identified differential item function (DIF) in depressive symptoms measures, but the impact of DIF has been rarely reported. Given the critical importance of depressive symptoms assessment among older adults, we examined whether DIF due to demographic characteristics resulted in salient score changes in commonly used measures. METHODS: Four longitudinal studies of cognitive aging provided a sample size of 3754 older adults and included individuals both with and without a clinical diagnosis of major depression. Each study administered at least one of the following measures: the Center for Epidemiologic Studies Depression scale (20-item ordinal response or 10-item dichotomous response versions), the Geriatric Depression Scale, and the Montgomery-Åsberg Depression Rating Scale. Hybrid logistic regression-item response theory methods were used to examine the presence and impact of DIF due to age, sex, race/ethnicity, and years of education on the depressive symptoms items. RESULTS: Although statistically significant DIF due to demographic factors was present on several items, its cumulative impact on depressive symptoms scores was practically negligible. CONCLUSIONS: The findings support substantive meaningfulness of previously reported demographic differences in depressive symptoms among older adults, showing that these individual differences were unlikely to have resulted from item bias attributable to demographic characteristics we examined.

Differential item functioning due to cognitive status does not impact depressive symptom measures in four heterogeneous samples of older adults.

OBJECTIVE: The objective of this study is to determine whether differential item functioning (DIF) due to cognitive status impacted three depressive symptoms measures commonly used with older adults. METHODS: Differential item functioning in depressive symptoms was assessed among participants (N = 3558) taking part in four longitudinal studies of cognitive aging, using the Geriatric Depression Scale, the Montgomery-Åsberg Depression Rating Scale, and the Center for Epidemiologic Studies Depression Scale. Participants were grouped by cognitive status using a general cognitive performance score derived from each study's neuropsychological battery and linked to a national average using a population-based survey representative of the US population. The Clinical Dementia Rating score was used as an alternate grouping variable in three of the studies. RESULTS: Although statistically significant DIF based on cognitive status was found for some depressive symptom items (e.g., items related to memory complaints, appetite loss, lack of energy, and mood), the effect of item bias on the total score for each scale was negligible. CONCLUSIONS: The depressive symptoms scales in these four studies measured depression in the same way, regardless of cognitive status. This may reduce concerns about using these depression measures in cognitive aging research, as relationships between depression and cognitive decline are unlikely to have been due to item bias, at least in the ways that were measured in the datasets we considered.

Evaluation of antibiotic susceptibility of Bacteroides, Prevotella and Fusobacterium species isolated from patients of the N. N. Blokhin Cancer Research Center, Moscow, Russia.

In total 122 non-duplicate Bacteroides, Prevotella and Fusobacterium spp isolated from cancer patients between 2004 and 2014 were involved in this study. Most of the strains belonged to the B. fragilis group (55%), followed by Prevotella strains (34.4%) and Fusobacterium spp (10.6%). The species identification was carried out by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), and they were identified on species level with a log (score) >2.0. The most common isolates were B. fragilis, B. thetaiotaomicron, B. ovatus and B. vulgatus. Among Prevotella species, the most frequently isolated species were P. buccae, P. buccalis, P. oris, P. denticola and P. nigrescens, and most of the Fusobacterium spp. were F. nucleatum. Susceptibilities of the strains were determined by the E-test methodology. The percentage of the susceptibility of B. fragilis group isolates were: metronidazole (MIC ≤4 µg/ml), 97%; imipenem (MIC ≤2 µg/ml), 95.5%; amoxicillin/clavulanate (MIC ≤4 µg/ml), 95.5% and clindamycin (MIC ≤4 µg/ml), 77.6%. Three B. fragilis isolates proved to be multidrug-resistant (parallel resistance to imipenem, amoxicillin/clavulanate and metronidazole or clindamycin was observed). All Prevotella strains tested were susceptible to imipenem and amoxicillin/clavulanate, whereas 78.6% of the pigmented Prevotella species and 46.4% of the non-pigmented species were resistant to penicillin (MIC >0.5 µg/ml). The susceptibility to metronidazole and clindamycin were 93% and 88%, respectively. All Fusobacterium strains were sensitive to all tested antibiotics, including penicillin.

Leisure-time physical activity moderates the longitudinal associations between work-family spillover and physical health.

Previous research has documented cross-sectional associations between negative and positive work-family spillover and physical health. Using an effort-recovery model, the study tested the hypothesis that engagement in greater leisure-time physical activity would facilitate recovery processes that buffer the negative health effects of increasing work-family spillover. Employed adults (N = 1,354) completed two waves of the National Survey of Midlife Development in the United States (MIDUS). Results indicated that an increase in negative work-family spillover across nine years was associated with decreased physical health and increased number of chronic conditions at Time 2. Moreover, more time spent on moderate leisure-time physical activity buffered many of the associations between increasing negative spillover and declining health. Implications of the findings are discussed.

Global discovery of high-NaCl-induced changes of protein phosphorylation.

High extracellular NaCl, such as in the renal medulla, can perturb and even kill cells, but cells mount protective responses that enable them to survive and function. Many high-NaCl-induced perturbations and protective responses are known, but the signaling pathways involved are less clear. Change in protein phosphorylation is a common mode of cell signaling, but there was no unbiased survey of protein phosphorylation in response to high NaCl. We used stable isotopic labeling of amino acids in cell culture coupled to mass spectrometry to identify changes in protein phosphorylation in human embryonic kidney (HEK 293) cells exposed to high NaCl. We reproducibly identify >8,000 unique phosphopeptides in 4 biological replicate samples with a 1% false discovery rate. High NaCl significantly changed phosphorylation of 253 proteins. Western analysis and targeted ion selection mass spectrometry confirm a representative sample of the phosphorylation events. We analyze the affected proteins by functional category to infer how altered protein phosphorylation might signal cellular responses to high NaCl, including alteration of cell cycle, cyto/nucleoskeletal organization, DNA double-strand breaks, transcription, proteostasis, metabolism of mRNA, and cell death.

Disinhibited eating and weight-related insulin mismanagement among individuals with type 1 diabetes.

OBJECTIVE: Withholding insulin for weight control is a dangerous practice among individuals with type 1 diabetes; yet little is known about the factors associated with this behavior. Studies of nondiabetic individuals with weight concerns suggest that eating in a disinhibited manner (e.g., binge eating) predicts the use of maladaptive compensatory strategies (e.g., self-induced vomiting). The purpose of this study was to test whether individuals with type 1 diabetes are less restrained in their eating when they think their blood glucose (BG) is low and whether this contributes to insulin omission for weight control purposes and subsequently higher hemoglobin A1c (HbA1c). METHODS: Two-hundred and seventy-six individuals with type 1 diabetes completed an online survey of eating behaviors, insulin dosing and most recent HbA1c. We used structural equation modeling to test the hypothesis that disinhibited eating when blood sugar is thought to be low predicts weight-related insulin mismanagement, and this, in turn, predicts higher HbA1c. RESULTS: The majority of participants endorsed some degree of disinhibition when they think their blood glucose is low (e.g., eating foods they do not typically allow) and corresponding negative affect (e.g., guilt/shame). The frequency of disinhibited eating was positively associated with weight-related insulin mismanagement. Controlling for age, sex, education, and insulin pump use, the model explained 31.3% of the variance in weight-related insulin mismanagement and 16.8% of the variance in HbA1c. CONCLUSION: Addressing antecedents to disinhibited eating that are unique to type 1 diabetes (e.g., perceived BG level) and associated guilt or shame may reduce weight-related insulin omission.

DNA double-strand breaks induced by high NaCl occur predominantly in gene deserts.

High concentration of NaCl increases DNA breaks both in cell culture and in vivo. The breaks remain elevated as long as NaCl concentration remains high and are rapidly repaired when the concentration is lowered. The exact nature of the breaks, and their location, has not been entirely clear, and it has not been evident how cells survive, replicate, and maintain genome integrity in environments like the renal inner medulla in which cells are constantly exposed to high NaCl concentration. Repair of the breaks after NaCl is reduced is accompanied by formation of foci containing phosphorylated H2AX (γH2AX), which occurs around DNA double-strand breaks and contributes to their repair. Here, we confirm by specific comet assay and pulsed-field electrophoresis that cells adapted to high NaCl have increased levels of double-strand breaks. Importantly, γH2AX foci that occur during repair of the breaks are nonrandomly distributed in the mouse genome. By chromatin immunoprecipitation using anti-γH2AX antibody, followed by massive parallel sequencing (ChIP-Seq), we find that during repair of double-strand breaks induced by high NaCl, γH2AX is predominantly localized to regions of the genome devoid of genes ("gene deserts"), indicating that the high NaCl-induced double-strand breaks are located there. Localization to gene deserts helps explain why the DNA breaks are less harmful than are the random breaks induced by genotoxic agents such as UV radiation, ionizing radiation, and oxidants. We propose that the universal presence of NaCl around animal cells has directly influenced the evolution of the structure of their genomes.