Proteome Coverage after Simultaneous Proteo-Metabolome Liquid-Liquid Extraction

Proteomics and metabolomics are essential in systems biology, and simultaneous proteo-metabolome liquid-liquid extraction (SPM-LLE) allows isolation of the metabolome and proteome from the same sample. Since the proteome is present as a pellet in SPM-LLE, it must be solubilized for quantitative proteomics. Solubilization and proteome extraction are critical factors in the information obtained at the proteome level. In this study, we investigated the performance of two surfactants (sodium deoxycholate (SDC), sodium dodecyl sulfate (SDS)) and urea in terms of proteome coverage and extraction efficiency of an interphase proteome pellet generated by methanol-chloroform based SPM-LLE. We also investigated how the performance differs when the proteome is extracted from the interphase pellet or by direct cell lysis. We quantified 12 lipids covering triglycerides and various phospholipid classes, and 25 polar metabolites covering central energy metabolism in chloroform and methanol extracts. Our study reveals that the proteome coverages between the two surfactants and urea for the SPM-LLE interphase pellet were similar, but the extraction efficiencies differed significantly. While SDS led to enrichment of basic proteins, which were mainly ribosomal and ribonuclear proteins, urea was the most efficient extraction agent for simultaneous proteo-metabolome analysis. The results of our study also show that the performance of surfactants for quantitative proteomics is better when the proteome is extracted through direct cell lysis rather than an interphase pellet. In contrast, the performance of urea for quantitative proteomics was significantly better when the proteome was extracted from an interphase pellet than by direct cell lysis. We demonstrated that urea is superior to surfactants for proteome extraction from SPM-LLE interphase pellets, with a particularly good performance for the extraction of proteins associated with metabolic pathways. Data are available via ProteomeXchange with identifier PXD027338.</p

Novel methods for in vitro modeling of pancreatic cancer reveal important aspects for successful primary cell culture

Background: Pancreatic cancer remains a fatal disease. Experimental systems are needed for personalized treatment strategies, drug testing and to further understand tumor biology. Cell cultures can serve as an excellent preclinical platform, but their generation remains challenging. Methods: Tumor cells from surgically removed pancreatic ductal adenocarcinoma (PDAC) specimens were cultured under novel protocols. Cellular growth and composition were analyzed and culture conditions were continuously optimized. Characterization of cell cultures and primary tumors was performed via hematoxylin and eosin (HE) and immunofluorescence (IF) staining. Results: Protocols for two- and three-dimensional PDAC primary cell cultures could successfully be established. Primary cell culture depended on dissociation techniques, growth factor supplementation and extracellular matrix components containing Matrigel being crucial for the transformation to three-dimensional PDAC organoids. The generated cultures showed to be highly resemblant to established PDAC primary cell cultures. HE and IF staining for cell culture and corresponding primary tumor characterization could successfully be performed. Conclusions: The work presented herein shows novel and effective methods to successfully establish primary PDAC cell cultures in a distinct time frame. Factors contributing to cell growth and differentiation could be identified with important implications for further primary cell culture protocols. The established protocols might serve as novel tools in personalized tumor therapy

Direct and plant community mediated effects of management intensity on annual nutrient leaching risk in temperate grasslands

Grassland management intensity influences nutrient cycling both directly, by changing nutrient inputs and outputs from the ecosystem, and indirectly, by altering the nutrient content, and the diversity and functional composition of plant and microbial communities. However, the relative importance of these direct and indirect processes for the leaching of multiple nutrients is poorly studied. We measured the annual leaching of nitrate, ammonium, phosphate and sulphate at a depth of 10 cm in 150 temperate managed grasslands using a resin method. Using Structural Equation Modeling, we distinguished between various direct and indirect effects of management intensity (i.e. grazing and fertilization) on nutrient leaching. We found that management intensity was positively associated with nitrate, ammonium and phosphate leaching risk both directly (i.e. via increased nutrient inputs) and indirectly, by changing the stoichiometry of soils, plants and microbes. In contrast, sulphate leaching risk was negatively associated with management intensity, presumably due to increased outputs with mowing and grazing. In addition, management intensification shifted plant communities towards an exploitative functional composition (characterized by high tissue turnover rates) and, thus, further promoted the leaching risk of inorganic nitrogen. Plant species richness was associated with lower inorganic nitrogen leaching risk, but most of its effects were mediated by stoichiometry and plant community functional traits. Maintaining and restoring diverse plant communities may therefore mitigate the increased leaching risk that management intensity imposes upon grasslands

Effects of vitamin D, omega-3 and a simple strength exercise programme in cardiovascular disease prevention: The DO-HEALTH randomized controlled trial

Background: The effects of non-pharmaceutical interventions in the prevention of cardiovascular diseases (CVD) in older adults remains unclear. Therefore, the aim was to investigate the effect of 2000 IU/day of vitamin D$_3$, omega-3 fatty acids (1 g/day), and a simple home strength exercise program (SHEP) (3×/week) on lipid and CVD biomarkers plasma changes over 3 years, incident hypertension and major cardiovascular events (MACE). Methods: The risk of MACE (coronary heart event or intervention, heart failure, stroke) was an exploratory endpoint of DO-HEALTH, incident hypertension and change in biomarkers were secondary endpoints. DO-HEALTH is a completed multicentre, randomised, placebo-controlled, 2 × 2 × 2 factorial design trial enrolling 2157 Europeans aged ≥70 years. Results: Participants' median age was 74 [72, 77] years, 61.7% were women, 82.5% were at least moderately physically active, and 40.7% had 25(OH)D < 20 ng/mL at baseline. Compared to their controls, omega-3 increased HDL-cholesterol (difference in change over 3 years: 0.08 mmol/L, 95% CI 0.05-0.10), decreased triglycerides (-0.08 mmol/L, (95%CI -0.12 to -0.03), but increased total- (0.15 mmol/L, 95%CI 0.09; 0.2), LDL- (0.11 mmol/L, 0.06; 0.16), and non-HDL-cholesterol (0.07 mmol/L, 95%CI 0.02; 0.12). However, neither omega-3 (adjustedHR 1.00, 95%CI 0.64-1.56), nor vitamin D$_3$ (aHR 1.37, 95%CI 0.88-2.14), nor SHEP (aHR 1.18, 95%CI 0.76-1.84) reduced risk of MACE or incident hypertension compared to control. Conclusion: Among generally healthy, active, and largely vitamin D replete, older adults, treatment with omega-3, vitamin D$_3$, and/or SHEP had no benefit on MACE prevention. Only omega-3 supplementation changed lipid biomarkers, but with mixed effects. TRIAL REGISTRATION CLINICALTRIALS

Rationale and Plan for Vitamin D Food Fortification : A Review and Guidance Paper

Vitamin D deficiency can lead to musculoskeletal diseases such as rickets and osteomalacia, but vitamin D supplementation may also prevent extraskeletal diseases such as respiratory tract infections, asthma exacerbations, pregnancy complications and premature deaths. Vitamin D has a unique metabolism as it is mainly obtained through synthesis in the skin under the influence of sunlight (i.e., ultraviolet-B radiation) whereas intake by nutrition traditionally plays a relatively minor role. Dietary guidelines for vitamin D are based on a consensus that serum 25-hydroxyvitamin D (25[OH]D) concentrations are used to assess vitamin D status, with the recommended target concentrations ranging from >= 25 to >= 50 nmol/L (>= 10->= 20 ng/mL), corresponding to a daily vitamin D intake of 10 to 20 mu g (400-800 international units). Most populations fail to meet these recommended dietary vitamin D requirements. In Europe, 25(OH)D concentrations <30 nmol/L (12 ng/mL) and <50 nmol/L (20 ng/mL) are present in 13.0 and 40.4% of the general population, respectively. This substantial gap between officially recommended dietary reference intakes for vitamin D and the high prevalence of vitamin D deficiency in the general population requires action from health authorities. Promotion of a healthier lifestyle with more outdoor activities and optimal nutrition are definitely warranted but will not erase vitamin D deficiency and must, in the case of sunlight exposure, be well balanced with regard to potential adverse effects such as skin cancer. Intake of vitamin D supplements is limited by relatively poor adherence (in particular in individuals with low-socioeconomic status) and potential for overdosing. Systematic vitamin D food fortification is, however, an effective approach to improve vitamin D status in the general population, and this has already been introduced by countries such as the US, Canada, India, and Finland. Recent advances in our knowledge on the safety of vitamin D treatment, the dose-response relationship of vitamin D intake and 25(OH)D levels, as well as data on the effectiveness of vitamin D fortification in countries such as Finland provide a solid basis to introduce and modify vitamin D food fortification in order to improve public health with this likewise cost-effective approach.Peer reviewe

OPLAH ablation leads to accumulation of 5-oxoproline, oxidative stress, fibrosis, and elevated fillings pressures:a murine model for heart failure with a preserved ejection fraction

Aims The prevalence of heart failure with a preserved ejection fraction (HFpEF) is increasing, but therapeutic options are limited. Oxidative stress is suggested to play an important role in the pathophysiology of HFpEF. However, whether oxidative stress is a bystander due to comorbidities or causative in itself remains unknown. Recent results have shown that depletion of 5-oxoprolinase (OPLAH) leads to 5-oxoproline accumulation, which is an important mediator of oxidative stress in the heart. We hypothesize that oxidative stress induced by elevated levels of 5-oxoproline leads to the onset of a murine HFpEF-like phenotype. Methods and results Oplah full body knock-out (KO) mice had higher 5-oxoproline levels coupled to increased oxidative stress. Compared with wild-type (WT) littermates, KO mice had increased cardiac and renal fibrosis with concurrent elevated left ventricular (LV) filling pressures, impaired LV relaxation, yet a normal LV ejection fraction. Following the induction of cardiac ischaemia/reperfusion (IR) injury, 52.4% of the KO mice died compared with only 15.4% of the WT mice (P <0.03). Furthermore, KO mice showed a significantly increased atrial, ventricular, kidney, and liver weights compared with WT mice (P <0.05 for all). Cardiac and renal fibrosis were more pronounced following cardiac IR injury in the KO mice and these mice developed proteinuria post-IR injury. To further address the link between 5-oxoproline and HFpEF, 5-oxoproline was measured in the plasma of HFpEF patients. Compared with healthy controls (3.8 +/- 0.6 mu M), 5-oxoproline levels were significantly elevated in HFpEF patients (6.8 +/- 1.9 mu M, P <0.0001). Furthermore, levels of 5-oxoproline were independently associated with more concentric remodelling on echocardiography. Conclusion Oxidative stress induced by 5-oxoproline results in a murine phenotype reminiscent of the clinical manifestation of HFpEF without the need for surgical or pharmacological interference. Better understanding of the role of oxidative stress in HFpEF may potentially lead to novel therapeutic options

Health care for irregular migrants: pragmatism across Europe. A qualitative study

<p>Abstract</p> <p>Background</p> <p>Health services in Europe face the challenge of delivering care to a heterogeneous group of irregular migrants (IM). There is little empirical evidence on how health professionals cope with this challenge. This study explores the experiences of health professionals providing care to IM in three types of health care service across 16 European countries.</p> <p>Results</p> <p>Semi-structured interviews were conducted with health professionals in 144 primary care services, 48 mental health services, and 48 Accident & Emergency departments (total n = 240). Although legal health care entitlement for IM varies across countries, health professionals reported facing similar issues when caring for IM. These issues include access problems, limited communication, and associated legal complications. Differences in the experiences with IM across the three types of services were also explored. Respondents from Accident & Emergency departments reported less of a difference between the care for IM patients and patients in a regular situation than did respondents from primary care and mental health services. Primary care services and mental health services were more concerned with language barriers than Accident & Emergency departments. Notifying the authorities was an uncommon practice, even in countries where health professionals are required to do this.</p> <p>Conclusions</p> <p>The needs of IM patients and the values of the staff appear to be as important as the national legal framework, with staff in different European countries adopting a similar pragmatic approach to delivering health care to IM. While legislation might help to improve health care for IM, more appropriate organisation and local flexibility are equally important, especially for improving access and care pathways.</p

A meta-analysis of previous falls and subsequent fracture risk in cohort studies

NC Harvey acknowledges funding from the UK Medical Research Council (MC_PC_21003; MC_PC_21001). The WHI program is funded by the National Heart, Lung, and Blood Institute, National Institutes of Health, U.S. Department of Health and Human Services through 75N92021D00001, 75N92021D00002, 75N92021D00003, 75N92021D00004, and 75N92021D00005. Funding for the MrOS USA study comes from the National Institute on Aging (NIA), the National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS), the National Center for Advancing Translational Sciences (NCATS), and NIH Roadmap for Medical Research under the following grant numbers: U01 AG027810, U01 AG042124, U01 AG042139, U01 AG042140, U01 AG042143, U01 AG042145, U01 AG042168, U01 AR066160, and UL1 TR000128. Funding for the SOF study comes from the National Institute on Aging (NIA), and the National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS), supported by grants (AG05407, AR35582, AG05394, AR35584, and AR35583). Funding for the Health ABC study was from the Intramural research program at the National Institute on Aging under the following contract numbers: NO1-AG-6–2101, NO1-AG-6–2103, and NO1-AG-6–2106.Peer reviewedPostprin

Previous fracture and subsequent fracture risk : a meta-analysis to update FRAX

A large international meta-analysis using primary data from 64 cohorts has quantified the increased risk of fracture associated with a previous history of fracture for future use in FRAX. The aim of this study was to quantify the fracture risk associated with a prior fracture on an international basis and to explore the relationship of this risk with age, sex, time since baseline and bone mineral density (BMD). We studied 665,971 men and 1,438,535 women from 64 cohorts in 32 countries followed for a total of 19.5 million person-years. The effect of a prior history of fracture on the risk of any clinical fracture, any osteoporotic fracture, major osteoporotic fracture, and hip fracture alone was examined using an extended Poisson model in each cohort. Covariates examined were age, sex, BMD, and duration of follow-up. The results of the different studies were merged by using the weighted β-coefficients. A previous fracture history, compared with individuals without a prior fracture, was associated with a significantly increased risk of any clinical fracture (hazard ratio, HR = 1.88; 95% CI = 1.72-2.07). The risk ratio was similar for the outcome of osteoporotic fracture (HR = 1.87; 95% CI = 1.69-2.07), major osteoporotic fracture (HR = 1.83; 95% CI = 1.63-2.06), or for hip fracture (HR = 1.82; 95% CI = 1.62-2.06). There was no significant difference in risk ratio between men and women. Subsequent fracture risk was marginally downward adjusted when account was taken of BMD. Low BMD explained a minority of the risk for any clinical fracture (14%), osteoporotic fracture (17%), and for hip fracture (33%). The risk ratio for all fracture outcomes related to prior fracture decreased significantly with adjustment for age and time since baseline examination. A previous history of fracture confers an increased risk of fracture of substantial importance beyond that explained by BMD. The effect is similar in men and women. Its quantitation on an international basis permits the more accurate use of this risk factor in case finding strategies