Combining Molecular Dynamics Simulations and Biophysical Characterization to Investigate Protein-Specific Excipient Effects on Reteplase during Freeze Drying.

We performed molecular dynamics simulations of Reteplase in the presence of different excipients to study the stabilizing mechanisms and to identify the role of excipients during freeze drying. To simulate the freeze-drying process, we divided the process into five distinct steps: (i) protein-excipient formulations at room temperature, (ii) the ice-growth process, (iii)-(iv) the partially solvated and fully dried formulations, and (v) the reconstitution. Furthermore, coarse-grained (CG) simulations were employed to explore the protein-aggregation process in the presence of arginine. By using a coarse-grained representation, we could observe the collective behavior and interactions between protein molecules during the aggregation process. The CG simulations revealed that the presence of arginine prevented intermolecular interactions of the catalytic domain of Reteplase, thus reducing the aggregation propensity. This suggests that arginine played a stabilizing role by interacting with protein-specific regions. From the freeze-drying simulations, we could identify several protein-specific events: (i) collapse of the domain structure, (ii) recovery of the drying-induced damages during reconstitution, and (iii) stabilization of the local aggregation-prone region via direct interactions with excipients. Complementary to the simulations, we employed nanoDSF, size-exclusion chromatography, and CD spectroscopy to investigate the effect of the freeze-drying process on the protein structure and stability.

Sculpting conducting nanopore size and shape through de novo protein design.

Transmembrane ß-barrels (TMBs) are widely used for single molecule DNA and RNA sequencing and have considerable potential for a broad range of sensing and sequencing applications. Current engineering approaches for nanopore sensors are limited to naturally occurring channels such as CsgG, which have evolved to carry out functions very different from sensing, and hence provide sub-optimal starting points. In contrast, de novo protein design can in principle create an unlimited number of new nanopores with any desired properties. Here we describe a general approach to the design of transmembrane ß-barrel pores with different diameter and pore geometry. NMR and crystallographic characterization shows that the designs are stably folded with structures close to the design models. We report the first examples of de novo designed TMBs with 10, 12 and 14 stranded ß-barrels. The designs have distinct conductances that correlate with their pore diameter, ranging from 110 pS (~0.5 nm pore diameter) to 430 pS (~1.1 nm pore diameter), and can be converted into sensitive small-molecule sensors with high signal to noise ratio. The capability to generate on demand ß-barrel pores of defined geometry opens up fundamentally new opportunities for custom engineering of sequencing and sensing technologies.

Investigation of the pH-dependent aggregation mechanisms of GCSF using low resolution protein characterization techniques and advanced molecular dynamics simulations.

Granulocyte-colony stimulating factor (GCSF) is a widely used therapeutic protein to treat neutropenia. GCSF has an increased propensity to aggregate if the pH is increased above 5.0. Although GCSF is very well experimentally characterized, the exact pH-dependent aggregation mechanism of GCSF is still under debate. This study aimed to model the complex pH-dependent aggregation behavior of GCSF using state-of-the-art simulation techniques. The conformational stability of GCSF was investigated by performing metadynamics simulations, while the protein-protein interactions were investigated using coarse-grained (CG) simulations of multiple GCSF monomers. The CG simulations were directly compared with small-angle X-ray (SAXS) data. The metadynamics simulations demonstrated that the orientations of Trp residues in GCSF are dependent on pH. The conformational change of Trp residues is due to the loss of Trp-His interactions at the physiological pH, which in turn may increase protein flexibility. The helical structure of GCSF was not affected by the pH conditions of the simulations. Our CG simulations indicate that at pH 4.0, the colloidal stability may be more important than the conformational stability of GCSF. The electrostatic potential surface and CG simulations suggested that the basic residues are mainly responsible for colloidal stability as deprotonation of these residues causes a reduction of the highly positively charged electrostatic barrier close to the aggregation-prone long loop regions.

Parathyroid Hormone Concentrations in Maintenance Hemodialysis: Longitudinal Evaluation of Intact and Biointact Assays.

RATIONALE & OBJECTIVE: Management of chronic kidney disease mineral and bone disorder requires parathyroid hormone (PTH) concentrations. "Biointact" PTH immunoassays detect "whole" PTH (wPTH), whereas "intact" immunoassays measure PTH plus PTH fragments (iPTH). We aimed to determine whether longitudinal changes in PTH concentrations can be evaluated using biointact and intact immunoassays alike. STUDY DESIGN: Open noninterventional longitudinal cohort study. SETTING & PARTICIPANTS: PTH concentrations were measured quarterly up to 5 times in 102 hemodialysis patients. PREDICTORS & TESTS COMPARED: Age, sex, phosphate levels, and others as clinical predictors for PTH trend. Tests compared were iPTH immunoassays from Siemens and Roche and wPTH immunoassays from Roche and DiaSorin. OUTCOMES: PTH concentration trend; regression equations; test bias. ANALYTICAL APPROACH: Predictive regression-to-the-mean model for PTH slope; Bland-Altman plots, Passing-Bablok regression, and reference change values for test comparisons. RESULTS: wPTH concentrations were similar with both immunoassays (wPTH-Roche = 11.7 + 0.97 × wPTH-DiaSorin, r = 0.99; mean ± 1.96 SD bias, 8.2 ± 43.3 pg/mL [17.5% ± 40.9%], by Bland-Altman plots). iPTH-Siemens concentrations were higher than iPTH-Roche concentrations (iPTH-Siemens = -5.4 + 1.33 × iPTH-Roche, r = 0.99; mean ± 1.96 SD bias, 84.0 ± 180.2 pg/mL [21.1% ± 29.8%], by Bland-Altman plots). iPTH-Roche and iPTH-Siemens concentrations were 2- and 2.5-fold higher than wPTH concentrations, respectively. Full agreement among all 4 immunoassays in detecting both significant and insignificant changes in PTH concentrations, upward or downward from one quarter to the next, was reached in 87% of consecutive measurements. In a predictive model, baseline PTH concentrations > 199 pg/mL (wPTH-Roche), 204 pg/mL (wPTH-DiaSorin), 386 pg/mL (iPTH-Roche), and 417 pg/mL (iPTH-Siemens) correctly predicted declining PTH concentration trend in 62% to 68% of patients, but age, sex, hemodialysis vintage, and calcium and phosphate levels were no significant predictors. LIMITATIONS: Limited number of immunoassays, only 59 patients attended all quarterly samplings. CONCLUSIONS: wPTH-Roche and wPTH-DiaSorin concentrations were similar, while iPTH was higher than wPTH concentrations. The iPTH-Siemens immunoassay is either higher calibrated or detects more fragments than iPTH-Roche. However, longitudinal PTH concentration changes largely coincided with all tested immunoassays.

Combining Unfolding Reversibility Studies and Molecular Dynamics Simulations to Select Aggregation-Resistant Antibodies.

The efficient development of new therapeutic antibodies relies on developability assessment with biophysical and computational methods to find molecules with drug-like properties such as resistance to aggregation. Despite the many novel approaches to select well-behaved proteins, antibody aggregation during storage is still challenging to predict. For this reason, there is a high demand for methods that can identify aggregation-resistant antibodies. Here, we show that three straightforward techniques can select the aggregation-resistant antibodies from a dataset with 13 molecules. The ReFOLD assay provided information about the ability of the antibodies to refold to monomers after unfolding with chemical denaturants. Modulated scanning fluorimetry (MSF) yielded the temperatures that start causing irreversible unfolding of the proteins. Aggregation was the main reason for poor unfolding reversibility in both ReFOLD and MSF experiments. We therefore performed temperature ramps in molecular dynamics (MD) simulations to obtain partially unfolded antibody domains in silico and used CamSol to assess their aggregation potential. We compared the information from ReFOLD, MSF, and MD to size-exclusion chromatography (SEC) data that shows whether the antibodies aggregated during storage at 4, 25, and 40 °C. Contrary to the aggregation-prone molecules, the antibodies that were resistant to aggregation during storage at 40 °C shared three common features: (i) higher tendency to refold to monomers after unfolding with chemical denaturants, (ii) higher onset temperature of nonreversible unfolding, and (iii) unfolding of regions containing aggregation-prone sequences at higher temperatures in MD simulations.

Biophysical Characterization of Binary Therapeutic Monoclonal Antibody Mixtures.

Coformulations containing two therapeutic monoclonal antibodies (mAbs) could offer various benefits like enhanced therapeutic efficacy and better patient compliance. However, there are very few published studies on coformulations and binary mixtures of mAbs. It remains unclear to what extent mAbs with different physicochemical properties can be combined in solution without detrimental effects on protein stability. Here, we present a study including six model mAbs of the IgG1 subclass that are commercially available. In silico and biophysical characterization shows that the proteins have different physicochemical properties. Thus, their combinations represent various scenarios for coformulation development. We prepared all possible binary mixtures of the six mAbs and determined several biophysical parameters that are assessed during early-stage protein drug product development. The measured biophysical parameters are indicative of the conformational protein stability (inflection points of the thermal protein unfolding transitions) and the colloidal protein stability (aggregation onset temperatures and interaction parameter kD from dynamic light scattering). Remarkably, all 15 binary mAb mixtures do not exhibit biophysical parameters that indicate inferior conformational or colloidal stability compared to the least stable mAb in the mixture. Our findings suggest that the coformulation of some therapeutic monoclonal antibodies of the IgG1 subclass could be possible in a straightforward way as severe detrimental effects on the stability of these proteins in binary mixtures were not observed.

Bioimpedance spectroscopy for fluid status assessment in patients with decompensated liver cirrhosis: Implications for peritoneal dialysis.

Bioimpedance spectroscopy (BIS) is routinely used in peritoneal dialysis patients and might aid fluid status assessment in patients with liver cirrhosis, but the effect of ascites volume removal on BIS-readings is unknown. Here we determined changes in BIS-derived parameters and clinical signs of fluid overload from before to after abdominal paracentesis. Per our pre-specified sample size calculation, we studied 31 cirrhotic patients, analyzing demographics, labs and clinical parameters along with BIS results. Mean volume of the abdominal paracentesis was 7.8 ± 2.6 L. From pre-to post-paracentesis, extracellular volume (ECV) decreased (20.2 ± 5.2 L to 19.0 ± 4.8 L), total body volume decreased (39.8 ± 9.8 L to 37.8 ± 8.5 L) and adipose tissue mass decreased (38.4 ± 16.0 kg to 29.9 ± 12.9 kg; all p < 0.002). Correlation of BIS-derived parameters from pre to post-paracentesis ranged from R² = 0.26 for body cell mass to R² = 0.99 for ECV. Edema did not correlate with BIS-derived fluid overload (FO ≥ 15% ECV), which occurred in 16 patients (51.6%). In conclusion, BIS-derived information on fluid status did not coincide with clinical judgement. The changes in adipose tissue mass support the BIS-model assumption that fluid in the peritoneal cavity is not detectable, suggesting that ascites (or peritoneal dialysis fluid) mass should be subtracted from adipose tissue if BIS is used in patients with a full peritoneal cavity.

Medication Adherence and Coping Strategies in Patients with Rheumatoid Arthritis: A Cross-Sectional Study.

OBJECTIVES: The aim of this study was to determine if strategies for coping with illnesses, demographic factors, and clinical factors were associated with medication adherence among patients with rheumatoid arthritis (RA). METHODS: This cross-sectional study was conducted at a Viennese rheumatology outpatient clinic on RA patients. Medication adherence was assessed using the Medication Adherence Report Scale. Strategies for coping with illness were assessed using the Freiburg Questionnaire for Coping with Illness. RESULTS: Half (N=63, 52.5%) of the 120 patients included in the study were considered completely medication adherent. Female sex (odds ratio [OR]: 4.57, 95% confidence interval [CI]: 1.14 - 18.42), older age (54-65 yr vs. <45 yr OR: 9.2, CI:2.0-40.70; >65 yr vs. <45 yr OR 6.93, CI:1,17 - 40.87), middle average income (middle average income vs. lowest income class OR= 0.06, CI= 0.01-0.43), and shorter disease duration (5-10 yr vs. >10 yr OR= 3.53, CI= 1.04-11.95; 1-4 yr vs. >10 yr OR=3.71, CI= 1.02-13.52) were associated with higher medication adherence. Levels of active coping (15.57 vs. 13.47, p=0.01) or diversion and self-encouragement (16.10 vs. 14.37, p=0.04) were significantly higher among adherent as opposed to less adherent participants. However, in multivariate regression models, coping strategies were not significantly associated with adherence. CONCLUSIONS: Age, sex, monthly net income, and disease duration were found to be associated with an increased risk for medication nonadherence among patients with RA. Coping strategies such as active coping, diversion, and self-encouragement were associated with adherence in univariate models, but not when adjusted for demographic and clinical factors.

Sex-specific analysis of haemodialysis prevalence, practices and mortality over time: the Austrian Dialysis Registry from 1965 to 2014.

BACKGROUND: Despite a higher prevalence of chronic kidney disease among women, more men than women start renal replacement therapy (RRT). We hypothesized that gender differences in health care access exist and therefore aimed at determining whether characteristics and outcomes of haemodialysis patients over time differ by sex. METHODS: We studied all 28 323 adults who began haemodialysis during 1965-2014 in the Austrian Dialysis Registry, analysing trends in patient characteristics by sex and decade with mortality (via Cox regression), which was compared with the mortality of the Austrian general population. RESULTS: More men than women started haemodialysis (60.1% men versus 39.9% women overall), with minor differences among decades and age groups. The male:female mortality rate ratio in the general population ranged from 1.2 to 2.4 for age groups >18 years and in haemodialysis patients ranged from 0.80 to 1.3 (closer to 1 than in the general population, but consistently >1 in Decades 3-5). In recent decades, diabetes and hypertension replaced glomerulonephritis as the primary cause of end-stage renal disease in both men and women. Interaction analyses showed the mortality risk associated with haemodialysis access (only recorded in Decade 5) was significantly lower for men than for women. CONCLUSIONS: The male:female mortality rate ratio and the proportion of women starting haemodialysis were remarkably stable, which does not support the hypothesis of gender differences in health care/haemodialysis access or could imply that such differences might have persisted over decades. Future research should expand to other countries and other forms of RRT.

Frailty in seropositive rheumatoid arthritis patients of working age: a cross-sectional study.

OBJECTIVES: The prevalence of frailty has been widely researched in the elderly population. However, data about people of working age are scarce. The aim of this paper was to assess the prevalence of prefrailty and frailty in rheumatoid arthritis (RA) patients of working age, and to assess factors associated with prefrailty/frailty. METHODS: In this monocentric cross-sectional study, 100 RA patients aged 18-65 years were included. Frailty was measured with the Frailty Instrument for Primary Care of the Survey of Health, Ageing and Retirement in Europe (SHARE-FI) and disease activity with the Clinical Disease Activity Index (CDAI). In addition, disease duration (years), pain intensity (visual analogue scale) and employment status were also assessed. RESULTS: Fifty-five percent were robust, 30% prefrail and 15% were frail. Eighty-nine of the prefrail/frail individuals suffered from exhaustion. Compared to robust individuals, the prefrail/frail individuals had significantly higher median scores in disease activity [4.0 (Q25-Q75: 0-10) vs. 11 (Q25-Q75: 6-18)] and pain intensity [3.0 (Q25-Q75: 2.0-4.0) vs. 4.0 (Q25-Q75: 2.8-6.3)] and a higher rate of unemployment [31% vs. 53%]. In the multivariable analysis, higher disease activity (ß=0.444; p<0.001), unemployment (ß=0.243; p=0.005), higher pain intensity (ß=0.186; p=0.060) and longer disease duration (ß=0.181; p=0.020) were associated with a higher frailty score. CONCLUSIONS: Frailty is common in RA patients, even those of working age. As the prevalence of frailty increases with age, it is important to take this syndrome into account in younger persons and to take action to counteract frailty.

A cross-sectional study on self-reported physical and mental health-related quality of life in rheumatoid arthritis and the role of illness perception.

BACKGROUND: Psychosocial models including illness perception might explain individual differences in health-related quality of life (HRQoL) and daily functioning in chronically ill patients. The aim of this study was to assess the association of illness perception among rheumatoid arthritis (RA) patients with physical and mental HRQoL, adjusted for demographic variables, clinical variables and social support. METHODS: A cross-sectional study conducted at a Viennese rheumatology outpatient clinic on 120 RA patients. Participants completed questionnaires on demographic and clinical characteristics, HRQoL (SF-36 Questionnaire), illness beliefs (Brief Illness Perception Questionnaire) and social support (Social Support Scale-8). Analyses were performed with multivariate linear regression. RESULTS: The mean physical was lower (38.38) than the mean mental SF-36 summary score (46.94). In univariate analysis, all domains of illness perception except belief in a chronic disease course were associated with physical and mental HRQoL. In multivariate analyses, illness perception accounted for 51% of variance in physical HRQoL. A stronger belief in the consequences of RA (consequences, ß = - 0.33) and a stronger belief in repeated disease recurrence (timeline cyclical, ß = - 0 .31) were significantly associated with physical HRQoL in the fully adjusted model. Illness perception accounted for 45% of variance in mental HRQoL. Emotional representation (ß = - 0 .27) and fatigue (ß = - 0 .36) were significantly associated with mental HRQoL in the fully adjusted model. CONCLUSION: This study highlights the importance of RA patients' beliefs about their illness and symptoms in relation to HRQoL. Identification of patients' perception of RA may be a way to positively influence disease outcomes such as quality of life as illness perception is amenable to intervention.

Sleep Quality in Patients with Rheumatoid Arthritis and Associations with Pain, Disability, Disease Duration, and Activity.

We aimed to assess the subjective sleep quality in patients with rheumatoid arthritis (RA) and its correlation with disease activity, pain, inflammatory parameters, and functional disability. In a cross-sectional study, patients with confirmed RA diagnosis responded to a questionnaire (consisting of socio-demographic data, the Health Assessment Questionnaire Disability Index, and the Medical Outcome Study Sleep Scale). Disease activity was assessed with the Clinical Disease Activity Index, and pain levels using the visual analogue scale. In addition, inflammatory markers (C-reactive protein, interleukin-6, and tumor necrosis factor alpha) were analyzed. Ninety-five patients were analyzed, predominantly female, with an average age of 50.59 (9.61) years. Fifty-seven percent reported non-optimal sleep duration, where functional disability (92.7% vs. 69.8%; p = 0.006) and higher median pain levels (3.75 (2.3⁻6.0) vs. 2.5 (2.0⁻3.5); p = 0.003) were also more prevalent. No differences in sociodemographic variables, disease duration or activity, inflammatory parameters, or use of biological and corticosteroid therapy were observed. The multivariate regression analysis showed that more intense pain was associated with a lower likelihood of optimal sleep (odds ratio (OR) = 0.68, 95% confidence interval (CI) 0.47⁻0.98, p = 0.038). Patients with RA report a high prevalence of non-optimal sleep, which is linked to pain level. Clinicians need to be aware of this issue and the potential effects on health and functional status.

A single residue switch reveals principles of antibody domain integrity.

Despite their importance for antibody architecture and design, the principles governing antibody domain stability are still not understood in sufficient detail. Here, to address this question, we chose a domain from the invariant part of IgG, the CH2 domain. We found that compared with other Ig domains, the isolated CH2 domain is a surprisingly unstable monomer, exhibiting a melting temperature of ∼44 °C. We further show that the presence of an additional C-terminal lysine in a CH2 variant substantially increases the melting temperature by ∼14 °C relative to CH2 WT. To explore the molecular mechanism of this effect, we employed biophysical approaches to probe structural features of CH2. The results revealed that Lys101 is key for the formation of three secondary structure elements: the very C-terminal ß-strand and two adjacent α-helices. We also noted that a dipole interaction between Lys101 and the nearby α-helix, is important for stabilizing the CH2 architecture by protecting the hydrophobic core. Interestingly, this interaction between the α-helix and C-terminal charged residues is highly conserved in antibody domains, suggesting that it represents a general mechanism for maintaining their integrity. We conclude that the observed interactions involving terminal residues have practical applications for defining domain boundaries in the development of antibody therapeutics and diagnostics.

Work Ability and Employment in Rheumatoid Arthritis: A Cross-Sectional Study on the Role of Muscle Strength and Lower Extremity Function.

OBJECTIVE: The aim of the present study was to assess the association between muscle strength, lower extremity function, employment status, and work ability in RA patients. METHODS: One hundred seropositive RA outpatients of working age were included in this cross-sectional study. Employment status was assessed by interview and work ability by the Work Ability Index-Single Item Scale (WAS). Muscle strength was determined using dynamometer measurement of isometric hand grip and knee extensor strength. Lower extremity function was measured using the short physical performance battery (SPPB). Regression models estimate the association between unemployment, work ability and muscle strength, and lower extremity function, controlling for sociodemographic and disease-related factors. RESULTS: Forty-one percent of the RA patients were not gainfully employed, and their median work ability had a good WAS value (7.00 [4.00-7.00]). Patients with better knee extensor strength (OR=1.07, 95% CI [1.02-1.12) and better physical performance (OR=1.71, 95% CI [1.18-2.49]) had a significantly better chance of gainful employment. The odds for hand grip strength remained significant when adjusted for sociodemographic (OR=1.5, 95% CI [1.00-1.09]), but not for disease-specific variables. Better hand grip strength (ß=0.25, p=0.039) and better knee extensor strength (ß=0.45, p=0.001) as well as better lower extremity function (SPPB) (ß=0.51, p<0.001) remained significantly associated with work ability following adjustment for sociodemographic and disease-specific variables. CONCLUSIONS: The association of employment status and work ability with parameters of physical fitness suggests that improvement in muscle strength and lower extremity function may positively influence work ability and employment in individuals with RA.

Sexual health in patients with rheumatoid arthritis and the association between physical fitness and sexual function: a cross-sectional study.

The aim of this study was to examine sexual health in patients with rheumatoid arthritis (RA), and to analyse factors associated with sexual health with a focus on physical fitness. One hundred RA patients aged between 18 and 65 years were included in a cross-sectional study. Handgrip strength and knee extensor strength were measured with a dynamometer, and physical performance with the Short Physical Performance Battery (SPPB). Fifty-four patients, mean age 47.8 (SD 10.6) years, 61% female, answered a questionnaire about sexual health. Fifty-seven percent reported, at least, sometimes having difficulty with sexual intercourse (27.8% due to joint stiffness, 24.1% due to fatigue, 18.5% due to pain). Handgrip strength and knee extensor strength significantly correlated with the desire to engage in sexual intercourse, frequency of sexual contact and satisfaction with overall sex life. The SPPB total score correlated with satisfaction with overall sex life, and the SPPB repeated chair stands test with the desire to have sexual intercourse and satisfaction with overall sex life. After adjusting for age, gender, disease activity, comorbidity, co-medication and pain intensity, the repeated chair stands test remained significantly associated with the frequency of sexual contact (0.53; 0.01-1.05) and with satisfaction with overall sex life (1.39; 0.28-2.51). The results of this study show that problems with sexual health are highly prevalent in patients with RA. The ability to rise from a chair is associated with sexual function, independent of disease activity and pain intensity.

Determinants of the assembly and function of antibody variable domains.

The antibody Fv module which binds antigen consists of the variable domains VL and VH. These exhibit a conserved ß-sheet structure and comprise highly variable loops (CDRs). Little is known about the contributions of the framework residues and CDRs to their association. We exchanged conserved interface residues as well as CDR loops and tested the effects on two Fvs interacting with moderate affinities (KDs of ~2.5 µM and ~6 µM). While for the rather instable domains, almost all mutations had a negative effect, the more stable domains tolerated a number of mutations of conserved interface residues. Of particular importance for Fv association are VLP44 and VHL45. In general, the exchange of conserved residues in the VL/VH interface did not have uniform effects on domain stability. Furthermore, the effects on association and antigen binding do not strictly correlate. In addition to the interface, the CDRs modulate the variable domain framework to a significant extent as shown by swap experiments. Our study reveals a complex interplay of domain stability, association and antigen binding including an unexpected strong mutual influence of the domain framework and the CDRs on stability/association on the one side and antigen binding on the other side.

Intravenous Fluid Challenge Decreases Intracellular Volume: A Bioimpedance Spectroscopy-Based Crossover Study in Healthy Volunteers.

The effects of intravenous fluid therapy on fluid compartments and hemodynamics of the human body remain enigmatic. We therefore tested the efficacy of bioimpedance spectroscopy in a crossover study, where 15 males received 0.5 ml/kg/min ELO-MEL-isoton (osmolarity = 302 mosmol/l) during 60 minutes, or nothing at all. In group "Fluid", fluid load increased from -0.2 ± 1.0 l extracellular volume at baseline to its maximum of 1.0 ± 0.9 l in minute 70, and remained continuously elevated throughout minute 300. In group "Zero", fluid load decreased from 0.5 ± 1.1 l at baseline to its minimum of -1.1 ± 1.1 l in minute 300. In group "Fluid", intracellular volume decreased from 26.8 ± 3.9 l at baseline to its minimum of 26.0 ± 3.9 l in minute 70, and remained continuously decreased throughout minute 300. In group "Zero", intracellular volume increased from 26.5 ± 3.8 l at baseline to its maximum of 27.1 ± 3.9 l in minute 120, and decreased thereafter. In group "Fluid" compared to "Zero", systolic blood pressure was significantly higher, from minute 50-90. In conclusion, intravenous fluid therapy caused a clinically meaningful, sustained increase in fluid load, and a decrease in intracellular volume. These data raise interest in studying fluid administration by the gastrointestinal route, perhaps even when managing critical illness.

Workability and Muscle Strength in Patients With Seropositive Rheumatoid Arthritis: Survey Study Protocol.

BACKGROUND: Rheumatoid arthritis (RA) and other rheumatic conditions not only fundamentally affect patients' quality of life and physiological needs but are also negatively associated with work ability. The costs of poor work ability, which, in sum, are more than treatment costs, pose an economic burden to society and patients. Work ability in RA appears to be multifactorial; symptoms such as pain, swelling, and stiffness play a major role, as these directly affect functional disability. Also, RA patients typically suffer from reduced muscle strength. Lower extremity function and grip strengths especially impair their quality of life. However, the role of muscle strength and disease activity as determinants of work ability have not yet been studied. OBJECTIVE: The primary objective of this study is to compare work ability in working-age participants with seropositive RA and with high and low disease activity; the secondary objective is to evaluate the association of muscle strength, functional ability, and frailty with work ability. METHODS: This monocentric cross-sectional study will be conducted at a rheumatologic outpatient clinic and day hospital with approximately 100 seropositive RA patients aged <65 years. A clinical disease activity index as a measure for rheumatoid disease activity will be assessed during the patients' routine visits at the clinic. Work ability, frailty, and functional disability will be evaluated with (self-reported) questionnaires as well as with physical tests (Work Ability Index/Score; Health Assessment Questionnaire Disability Index; Survey of Health, Ageing, and Retirement in Europe Frailty Instrument; Short Physical Performance Battery). Muscle strength will be determined with dynamometer measurements of isometric hand grip strength and quadriceps femoris muscle contraction strength. Sleep quality (Medical Outcomes Study Sleep Scale) and sexual functioning as physiological needs will additionally be determined with self-reported questionnaires. RESULTS: For this study funding has already been awarded and enrollment has been completed. Data are currently being evaluated. CONCLUSIONS: This study will evaluate the association of work ability with modifiable parameters such as muscle strength and functional ability. It will provide further insights into work ability in RA and its associated risk factors. Any evidence of association will motivate further research, and the findings might encourage interventions focused specifically on improving muscle strength and lower extremity function to positively affect work ability. TRIAL REGISTRATION: ClinicalTrials.gov (NCT02581852); https://clinicaltrials.gov/ct2/show/NCT02581852 (Archived by WebCite at http://www.webcitation.org/6oNcelHtQ).

Epigenetic control of estrogen receptor expression and tumor suppressor genes is modulated by bioactive food compounds.

BACKGROUND: The tumor suppressor genes p15(INK)4(b) and p16(INK)4(a) as well as the estrogen receptor-α (ESR1) gene are abnormally methylated and expressed in colon cancer. The cancer-preventative abilities of several bioactive food components have been linked to their estrogenic and epigenetic activities. METHODS: The effect of folic acid, zebularine, resveratrol, genistein and epigallocatechin-3-gallate (EGCG) on tumor cell growth, promoter methylation of ESR1, p15(INK)4(b) and p16(INK)4(a) and gene expression of ESR1 and ESR2 was analyzed in Caco-2 cells. Gene expression was measured using real-time PCR, and promoter CpG methylation was assessed using bisulfite conversion and methylation-specific PCR. RESULTS: After exposure to a high concentration of folic acid (20 µmol/l), enhanced cancer cell growth and concomitant increased methylation of the ESR1 (3.6-fold), p16(INK)4(a) and p15(INK)4(b) promoters was observed. A lower concentration of folic acid (2 µmol/l) decreased cell growth. The phytoestrogens genistein and resveratrol enhanced expression of ESR1 (genistein 200 µmol/l: 2.1-fold; resveratrol 50 µmol/l: 6.3-fold) and ESR2 (2.6- and 3.6-fold, respectively). Genistein and resveratrol treatment increased promoter methylation of ESR1 (genistein 200 µmol/l: 2.9-fold; resveratrol 50 µmol/l: 1.4-fold). For p16(INK)4(a), increased methylation was found after exposure to 10 µmol/l resveratrol, but for p15(INK)4(b), decreased methylation was found. Both components showed growth-inhibitory activities. For EGCG, growth inhibition at 100 µmol/l and suppressed promoter methylation of tumor suppressor genes (p16(INK)4(a): 0.9-fold; p15(INK)4(b): 0.6-fold) was seen. CONCLUSIONS: Our results show that these food compounds regulate ESR and tumor suppressor gene expression by multiple mechanisms including epigenetic processes. An improved understanding of these epigenetic effects could therefore support specific dietary concepts of epigenetic cancer prevention and intervention.