Non-adherence to cardiometabolic medication as assessed by LC-MS/MS in urine and its association with kidney and cardiovascular outcomes in type 2 diabetes mellitus.

AIMS/HYPOTHESIS: Non-adherence to medication is a frequent barrier in the treatment of patients with type 2 diabetes mellitus, potentially limiting the effectiveness of evidence-based treatments. Previous studies have mostly relied on indirect adherence measures to analyse outcomes based on adherence. The aim of this study was to use LC-MS/MS in urine-a non-invasive, direct and objective measure-to assess non-adherence to cardiometabolic drugs and analyse its association with kidney and cardiovascular outcomes. METHODS: This cohort study includes 1125 participants from the PROVALID study, which follows patients with type 2 diabetes mellitus at the primary care level. Baseline urine samples were tested for 79 cardiometabolic drugs and metabolites thereof via LC-MS/MS. An individual was classified as totally adherent if markers for all drugs were detected, partially non-adherent when at least one marker for one drug was detected, and totally non-adherent if no markers for any drugs were detected. Non-adherence was then analysed in the context of cardiovascular (composite of myocardial infarction, stroke and cardiovascular death) and kidney (composite of sustained 40% decline in eGFR, sustained progression of albuminuria, kidney replacement therapy and death from kidney failure) outcomes. RESULTS: Of the participants, 56.3% were totally adherent, 42.0% were partially non-adherent, and 1.7% were totally non-adherent to screened cardiometabolic drugs. Adherence was highest to antiplatelet and glucose-lowering agents and lowest to lipid-lowering agents. Over a median (IQR) follow-up time of 5.10 (4.12-6.12) years, worse cardiovascular outcomes were observed with non-adherence to antiplatelet drugs (HR 10.13 [95% CI 3.06, 33.56]) and worse kidney outcomes were observed with non-adherence to antihypertensive drugs (HR 1.98 [95% CI 1.37, 2.86]). CONCLUSIONS/INTERPRETATION: This analysis shows that non-adherence to cardiometabolic drug regimens is common in type 2 diabetes mellitus and negatively affects kidney and cardiovascular outcomes.

Prevalence, patient burden and physicians' perception of pruritus in haemodialysis patients.

BACKGROUND: Chronic kidney disease-associated pruritus (CKD-aP) is an underrated symptom in patients with impaired kidney function. The present study assessed the prevalence, impact on quality of life (QoL) and risk factors for CKD-aP in a contemporary national cohort of patients on haemodialysis. In addition, we evaluated attending physicians' awareness and approach to therapy. METHODS: Validated patient's and physician's questionnaires on pruritus severity and QoL were used in combination with information obtained by the Austrian Dialysis and Transplant Registry. RESULTS: The prevalence of mild, moderate and severe pruritus in 962 observed patients was 34.4%, 11.4% and 4.3%. Physicians' estimated prevalence values were 25.0 (95% CI 16.8-33.2), 14.4 (11.3-17.6) and 6.3% (4.9-8.3), respectively. The estimated national prevalence estimate extrapolated from the observed patients was 45.0% (95% CI 39.5-51.2) for any, 13.9% (95% CI 10.6-17.2) for moderate and 4.2% (95% CI 2.1-6.2) for severe CKD-aP. CKD-aP severity was significantly associated with impaired QoL. Risk factors for moderate-severe pruritus were higher C-reactive protein [odds ratio (OR) 1.61 (95% CI 1.07-2.43)] and parathyroid hormone (PTH) values [OR 1.50 (95% CI 1.00-2.27)]. Therapy for CKD-aP included changes in the dialysis regimen, topical treatments, antihistamines, gabapentin and pregabalin and phototherapy in a majority of centres. CONCLUSIONS: While the overall prevalence of CKD-aP in our study is similar to that in previously published literature, the prevalence of moderate-severe pruritus is lower. CKD-aP was associated with reduced QoL and elevated markers of inflammation and PTH. The high awareness of CKD-aP in Austrian nephrologists may explain the lower prevalence of more severe pruritus.

Urinary peptides provide information about the risk of mortality across a spectrum of diseases and scenarios.

BACKGROUND: There is evidence of pre-established vulnerability in individuals that increases the risk of their progression to severe disease or death, although the mechanisms causing this are still not fully understood. Previous research has demonstrated that a urinary peptide classifier (COV50) predicts disease progression and death from SARS-CoV-2 at an early stage, indicating that the outcome prediction may be partly due to vulnerabilities that are already present. The aim of this study is to examine the ability of COV50 to predict future non-COVID-19-related mortality, and evaluate whether the pre-established vulnerability can be generic and explained on a molecular level by urinary peptides. METHODS: Urinary proteomic data from 9193 patients (1719 patients sampled at intensive care unit (ICU) admission and 7474 patients with other diseases (non-ICU)) were extracted from the Human Urinary Proteome Database. The previously developed COV50 classifier, a urinary proteomics biomarker panel consisting of 50 peptides, was applied to all datasets. The association of COV50 scoring with mortality was evaluated. RESULTS: In the ICU group, an increase in the COV50 score of one unit resulted in a 20% higher relative risk of death [adjusted HR 1.2 (95% CI 1.17-1.24)]. The same increase in COV50 in non-ICU patients resulted in a higher relative risk of 61% [adjusted HR 1.61 (95% CI 1.47-1.76)], consistent with adjusted meta-analytic HR estimate of 1.55 [95% CI 1.39-1.73]. The most notable and significant changes associated with future fatal events were reductions of specific collagen fragments, most of collagen alpha I (I). CONCLUSION: The COV50 classifier is predictive of death in the absence of SARS-CoV-2 infection, suggesting that it detects pre-existing vulnerability. This prediction is mainly based on collagen fragments, possibly reflecting disturbances in the integrity of the extracellular matrix. These data may serve as a basis for proteomics-guided intervention aiming towards manipulating/ improving collagen turnover, thereby reducing the risk of death.

International Variability of Renal and Cardiovascular Outcomes and Mortality in Patients with Type 2 Diabetes Mellitus in Europe.

INTRODUCTION: Type 2 diabetes and its complications represent a huge burden to public health. With this prospective, observational cohort study, we aimed to estimate and to compare the incidence rate (IR) of renal and cardiovascular outcomes and all-cause mortality in patients with type 2 diabetes in different European countries. METHODS: The renal endpoint was a composite of a sustained decline in estimated GFR of at least 40%, a sustained increase in albuminuria of at least 30% including a transition in albuminuria class, progression to kidney failure with replacement therapy, or death from renal causes. The cardiovascular endpoint was a composite of death from cardiovascular causes, nonfatal myocardial infarction, or nonfatal stroke. RESULTS: 3,131 participants from four European countries (Austria, Hungary, The Netherlands, and Scotland) with a median follow-up time of 4.4 years were included. IRs were adjusted for several risk factors including sex, age, estimated GFR, albuminuria, HbA1c, blood pressure, and duration of type 2 diabetes. Across countries, the adjusted IR for the renal endpoint was significantly higher in Hungary and Austria, and the adjusted IR for the cardiovascular endpoint was significantly higher in Scotland and Austria. All-cause mortality was significantly higher in Scotland compared to all other countries. CONCLUSION: Our findings show how the longitudinal outcome of patients with type 2 diabetes varies significantly across European countries even after accounting for the distribution of underlying risk factors.

Rationale and design of the Innsbruck Diabetic Kidney Disease Cohort (IDKDC)-a prospective study investigating etiology and progression of early-stage chronic kidney disease in type 2 diabetes.

Background: The development of chronic kidney disease (CKD) in about 20%-40% of patients with type 2 diabetes (T2D) aggravates cardiovascular morbidity and mortality. Pathophysiology is of increasing relevance for individual management and prognosis, though it is largely unknown among T2D patients with CKD as histologic work-up is not routinely performed upon typical clinical presentation. However, as clinical parameters do not appropriately reflect underlying kidney pathology, reluctance regarding timely histologic assessment in T2D patients with CKD should be critically questioned. As the etiology of CKD in T2D is heterogeneous, we aim to assess the prevalence and clinical disease course of typical diabetic vs atypical/non-specific vs non-diabetic vs coexisting kidney pathologies among T2D patients with mild-to-moderate kidney impairment [KDIGO stage G3a/A1-3 or G2/A2-3; i.e. estimated glomerular filtration rate (eGFR) 59-45 mL/min irrespective of albuminuria or eGFR 89-60 mL/min and albuminuria >30 mg/g creatinine]. Methods: The Innsbruck Diabetic Kidney Disease Cohort (IDKDC) study aims to enroll at least 65 T2D patients with mild-to-moderate kidney impairment to undergo a diagnostic kidney biopsy. Six-monthly clinical follow-ups for up to 5 years will provide clinical and laboratory data to assess cardio-renal outcomes. Blood, urine and kidney tissue specimen will be biobanked to identify diagnostic and prognostic biomarkers. Conclusions: While current risk assessment is primarily based on clinical parameters, our study will provide the scientific background for a potential change of the diagnostic standard towards routine kidney biopsy and clarify its role for individual risk prediction regarding cardio-renal outcome in T2D patients with mild-to-moderate kidney impairment.

Impact of Renal Denervation on Urinary Peptide-Based Biomarkers in Hypertension.

BACKGROUND: Catheter-based renal denervation (RDN) reduces blood pressure in hypertension. Urinary peptides are associated with cardiovascular and renal disease and provide prognostic information. We aimed to investigate the effect of RDN on urinary peptide-based classifiers associated with chronic kidney and heart disease and to identify urinary peptides affected by RDN. METHODS: This single-arm, single-center study included patients undergoing catheter-based RDN. Urine samples were collected before and 24 months after RDN and were analyzed using capillary electrophoresis coupled with mass spectrometry. Predefined urinary peptide-based classifiers for chronic kidney disease (CKD273), coronary artery disease (CAD238), and heart failure (HF1) were applied. RESULTS: This study included 48 patients (33% female) with uncontrolled hypertension. At 24 months after RDN, systolic blood pressure (165±17 versus 148±20 mm Hg; P<0.0001), diastolic blood pressure (90±17 versus 81±13 mm Hg; P<0.0001), and mean arterial pressure (115±15 versus 103±13 mm Hg; P<0.0001) decreased significantly. A total of 103 urinary peptides from 37 different proteins, mostly collagens, altered following RDN. CAD238, a 238 coronary artery-specific polypeptide-based classifier, significantly improved following RDN (Cohen's d, -0.632; P=0.0001). The classification scores of HF1 (P=0.8295) and CKD273 (P=0.6293) did not change significantly. CONCLUSIONS: RDN beneficially affected urinary peptides associated with coronary artery disease. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique identifier: NCT01888315.

Abiotic stress-induced accumulation of raffinose in Arabidopsis leaves is mediated by a single raffinose synthase (RS5, At5g40390).

BACKGROUND: The sucrosylgalactoside oligosaccharide raffinose (Raf, Suc-Gal1) accumulates in Arabidopsis leaves in response to a myriad of abiotic stresses. Whilst galactinol synthases (GolS), the first committed enzyme in Raf biosynthesis are well characterised in Arabidopsis, little is known of the second biosynthetic gene/enzyme raffinose synthase (RS). Conflicting reports suggest the existence of either one or six abiotic stress-inducible RSs (RS-1 to -6) occurring in Arabidopsis. Indirect evidence points to At5g40390 being responsible for low temperature-induced Raf accumulation in Arabidopsis leaves. RESULTS: By heterologously expressing At5g40390 in E.coli, we demonstrate that crude extracts synthesise Raf in vitro, contrary to empty vector controls. Using two independent loss-of-function mutants for At5g40390 (rs 5-1 and 5-2), we confirm that this RS is indeed responsible for Raf accumulation during low temperature-acclimation (4°C), as previously reported. Surprisingly, leaves of mutant plants also fail to accumulate any Raf under diverse abiotic stresses including water-deficit, high salinity, heat shock, and methyl viologen-induced oxidative stress. Correlated to the lack of Raf under these abiotic stress conditions, both mutant plants lack the typical stress-induced RafS activity increase observed in the leaves of wild-type plants. CONCLUSIONS: Collectively our findings point to a single abiotic stress-induced RS isoform (RS5, At5g40390) being responsible for Raf biosynthesis in Arabidopsis leaves. However, they do not support a single RS hypothesis since the seeds of both mutant plants still contained Raf, albeit at 0.5-fold lower concentration than seeds from wild-type plants, suggesting the existence of at least one other seed-specific RS. These results also unambiguously discount the existence of six stress-inducible RS isoforms suggested by recent reports.

Combination Therapy of RAS Inhibition and SGLT2 Inhibitors Decreases Levels of Endotrophin in Persons with Type 2 Diabetes.

We investigated for the first time the effect of combination therapy of renin-angiotensin system inhibition (RASi) and sodium-glucose co-transporter-2 inhibitors (SGLT2is) on endotrophin (ETP), a pro-fibrotic signaling molecule reflecting collagen type VI formation, measured in the plasma of persons with type 2 diabetes (T2D). ETP was measured using the PRO-C6 ELISA in 294 individuals from the "Drug combinations for rewriting trajectories of renal pathologies in type 2 diabetes" (DC-ren) project. In the DC-ren study, kidney disease progression was defined as a >10% decline in the estimated glomerular filtration rate (eGFR) to an eGFR < 60 mL/min/1.73 m2. Among the investigated circulating markers, ETP was the most significant predictor of future eGFR. Combination therapy of RASi and SGLT2is led to a significant reduction in ETP levels compared to RASi monotherapy (p for slope difference = 0.002). Higher levels of baseline plasma ETP were associated with a significantly increased risk of kidney disease progression (p = 0.007). In conclusion, plasma ETP identified individuals at higher risk of kidney disease progression. The observed decreased levels of plasma ETP with combination therapy of RASi and SGLT2is in persons with T2D may reflect a reduced risk of kidney disease progression following treatment with SGLT2is.

Prognosis and Personalized In Silico Prediction of Treatment Efficacy in Cardiovascular and Chronic Kidney Disease: A Proof-of-Concept Study.

(1) Background: Kidney and cardiovascular diseases are responsible for a large fraction of population morbidity and mortality. Early, targeted, personalized intervention represents the ideal approach to cope with this challenge. Proteomic/peptidomic changes are largely responsible for the onset and progression of these diseases and should hold information about the optimal means of treatment and prevention. (2) Methods: We investigated the prediction of renal or cardiovascular events using previously defined urinary peptidomic classifiers CKD273, HF2, and CAD160 in a cohort of 5585 subjects, in a retrospective study. (3) Results: We have demonstrated a highly significant prediction of events, with an HR of 2.59, 1.71, and 4.12 for HF, CAD, and CKD, respectively. We applied in silico treatment, implementing on each patient's urinary profile changes to the classifiers corresponding to exactly defined peptide abundance changes, following commonly used interventions (MRA, SGLT2i, DPP4i, ARB, GLP1RA, olive oil, and exercise), as defined in previous studies. Applying the proteomic classifiers after the in silico treatment indicated the individual benefits of specific interventions on a personalized level. (4) Conclusions: The in silico evaluation may provide information on the future impact of specific drugs and interventions on endpoints, opening the door to a precision-based medicine approach. An investigation into the extent of the benefit of this approach in a prospective clinical trial is warranted.

An Arabidopsis T-DNA insertion mutant for galactokinase (AtGALK, At3g06580) hyperaccumulates free galactose and is insensitive to exogenous galactose.

Galactokinase (GALK, EC 2.7.1.6) is a cytosolic enzyme with a wide occurrence across the taxonomic kingdoms. It catalyzes the phosphorylation of α-d-galactose (Gal) to α-d-Gal-1-P. The cytotoxicity of free (unphosphorylated) Gal is well documented in plants and causes marked defects. An Arabidopsis GALK (AtGALK, At3g06580) was previously identified, cloned and functionally characterized in Escherichia coli and was suggested to occur as a single copy gene in Arabidopsis. We identified an AtGALK T-DNA insertion mutant (atgalk) that (i) is AtGALK transcript deficient; (ii) displays no GALK activity in vegetative tissues; and (iii) accumulates Gal up to 6.8 mg g(-1) FW in vegetative tissues, in contrast to wild-type plants. By constitutively overexpressing the AtGALK cDNA, atgalk was functionally rescued. Three independent transformed lines showed restored AtGALK transcripts and GALK activity and had low leaf Gal concentrations comparable with those observed in wild-type plants. Surprisingly, in vitro grown atgalk plants were largely insensitive to the exogenous application of up to 100 mM free Gal, while wild-type plants exhibited sensitivity to low Gal concentrations (10 mM). Furthermore, atgalk seedlings retained the capacity for uptake of exogenously supplied Gal (100 mM), accumulating up to 57 mg g(-1) FW in leaves. Leaves from soil-grown atgalk plants that exhibited no growth or morphological defects were used to demonstrate that the accumulating Gal occurred exclusively in the vacuoles of mesophyll protoplasts. Collectively, these findings suggest a novel Gal detoxification pathway that targets free Gal to the vacuole and is active in the atgalk mutant background.

VvGOLS1 and VvHsfA2 are involved in the heat stress responses in grapevine berries.

Among various environmental factors, temperature is a major regulator affecting plant growth, development and fruit composition. Grapevine is the most cultivated fruit plant throughout the world, and grapes are used for wine production and human consumption. The molecular mechanisms involved in grapevine tolerance to high temperature, especially at the fruit level, are poorly understood. To better characterize the sensitivity of berries to the microenvironment, high temperature conditions were locally applied to Vitis vinifera Cabernet Sauvignon clusters. Two genes, VvGOLS1 and VvHsfA2, up-regulated by this treatment, were identified and further characterized. The expression profile of VvGOLS1 correlated positively with galactinol accumulation in heat-stressed berries. However, no galactinol derivatives, such as raffinose and stachyose, accumulated upon heat stress. Heterologous expression of VvGOLS1 in Escherichia coli showed that it encodes a functional galactinol synthase. Transient expression assays showed that the heat stress factor VvHsfA2 transactivates the promoter of VvGOLS1 in a heat stress-dependent manner. Taken together, our results highlight the intrinsic capacity of grape berries to perceive heat stress and to initiate adaptive responses, suggesting that galactinol may play a signaling role in these responses.

Biological variation and reference change value of the estimated glomerular filtration rate in humans: A systematic review and meta-analysis.

Background: Knowledge of the biological variation of serum or plasma creatinine (Cr) and the estimated glomerular filtration rate (eGFR) is important for understanding disease dynamics in Chronic Kidney Disease (CKD). The aim of our study was to determine the magnitude of random fluctuation of eGFR by determining its reference change value (RCV). Methods: We performed a systematic review and meta-analysis of studies on biological variation of Cr. Relevant studies were identified by systematic literature search on PubMed. Additional studies were retrieved from the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) Biological Variation Database. Random-effects meta-analysis was conducted to derive an overall estimate of intra-individual variation of creatinine (CVICr). Based on our estimate of CVICr and RCV for Cr, the RCV for the eGFR was determined. Results: Among identified studies, 37 met our inclusion criteria. Meta-analysis of all studies yielded a CVICr of 5.2% (95% confidence interval [CI] 4.6-5.8%), however high between-study heterogeneity (I 2 = 82.3%) was found. Exclusion of outliers led to a significant reduction of heterogeneity while still including 85% of all studies and resulted in a slightly lower CVICr of 5.0% (95% CI 4.7-5.4%). Assuming an analytical variation of CVA 1.1%, we found an overall RCV for eGFR of ±16.5%. After exclusion of outlier studies, we found a minimum conservative RCV for eGFR of ±12.5%. Conclusion: The RCV of the eGFR represents a valuable tool for clinicians to discern true changes in kidney function from random fluctuation.

Clonal Hematopoiesis of Indeterminate Potential and Diabetic Kidney Disease: A Nested Case-Control Study.

Introduction: The disease trajectory of diabetic kidney disease (DKD) shows a high interindividual variability not sufficiently explained by conventional risk factors. Clonal hematopoiesis of indeterminate potential (CHIP) is a proposed novel cardiovascular risk factor. Increased kidney fibrosis and glomerulosclerosis were described in mouse models of CHIP. Here, we aim to analyze whether CHIP affects the incidence or progression of DKD. Methods: A total of 1419 eligible participants of the PROVALID Study were the basis for a nested case-control (NCC) design. A total of 64 participants who reached a prespecified composite endpoint within the observation period (initiation of kidney replacement therapy, death from kidney failure, sustained 40% decline in estimated glomerular filtration rate or sustained progression to macroalbuminuria) were identified and matched to 4 controls resulting in an NCC sample of 294 individuals. CHIP was assessed via targeted amplicon sequencing of 46 genes in peripheral blood. Furthermore, inflammatory cytokines were analyzed in plasma via a multiplex assay. Results: The estimated prevalence of CHIP was 28.91% (95% CI 22.91%-34.91%). In contrast to other known risk factors (albuminuria, hemoglobin A1c, heart failure, and smoking) and elevated microinflammation, CHIP was not associated with incident or progressive DKD (hazard ratio [HR] 1.06 [95% CI 0.57-1.96]). Conclusions: In this NCC study, common risk factors as well as elevated microinflammation but not CHIP were associated with kidney function decline in type 2 diabetes mellitus.

Functional identification of Arabidopsis ATSIP2 (At3g57520) as an alkaline α-galactosidase with a substrate specificity for raffinose and an apparent sink-specific expression pattern.

Arabidopsis ATSIP2 has recently been suggested to be a raffinose synthase gene. However, it has high amino acid identity to functionally characterized alkaline α-galactosidases from Cucumis melo and Zea mays. Using the Sf9 insect cell expression system, we demonstrate that recombinant ATSIP2 is a genuine alkaline α-galactosidase with a distinct substrate specificity for raffinose, and not a raffinose synthase. A ß-glucuronidase reporter construct using the ATSIP2 promoter shows that ATSIP2 is strongly expressed in sink tissues of Arabidopsis, i.e. sink leaves and non-xylem parts of the root stele, suggesting a physiological function in raffinose phloem unloading.

Quantitative detection of changes in the leaf-mesophyll tonoplast proteome in dependency of a cadmium exposure of barley (Hordeum vulgare L.) plants.

Although the vacuole is the most important final store for toxic heavy metals like cadmium (Cd(2+)), our knowledge on how they are transported into the vacuole is still insufficient. It has been suggested that Cd(2+) can be transported as phytochelatin-Cd(2+) by an unknown ABC transporter or in exchange with protons by cation/proton exchanger (CAX) transporters. To unravel the contribution of vacuolar transporters to Cd(2+) detoxification, a quantitative proteomics approach was performed. Highly purified vacuoles were isolated from barley plants grown under minus, low (20 microM), and high (200 microM) Cd(2+ )conditions and protein levels of the obtained tonoplast samples were analyzed using isobaric tag for relative and absolute quantitation (iTRAQ). Although 56 vacuolar transporter proteins were identified, only a few were differentially expressed. Under low-Cd(2+) conditions, an inorganic pyrophosphatase and a gamma-tonoplast intrinsic protein (gamma-TIP) were up-regulated, indicating changes in energization and water fluxes. In addition, the protein ratio of a CAX1a and a natural resistance-associated macrophage protein (NRAMP), responsible for vacuolar Fe(2+) export was increased. CAX1a might play a role in vacuolar Cd(2+) transport. An increase in NRAMP activity leads to a higher cytosolic Fe(2+) concentration, which may prevent the exchange of Fe(2+) by toxic Cd(2+). Additionally, an ABC transporter homolog to AtMRP3 showed up-regulation. Under high Cd(2+) conditions, the plant response was more specific. Only a protein homologous to AtMRP3 that showed already a response under low Cd(2+) conditions, was up-regulated. Interestingly, AtMRP3 is able to partially rescue a Cd(2+)-sensitive yeast mutant. The identified transporters are good candidates for further investigation of their roles in Cd(2+) detoxification.

Raffinose in chloroplasts is synthesized in the cytosol and transported across the chloroplast envelope.

In chloroplasts, several water-soluble carbohydrates have been suggested to act as stress protectants. The trisaccharide raffinose (alpha-1,6-galactosyl sucrose) is such a carbohydrate but has received little attention. We here demonstrate by compartmentation analysis of leaf mesophyll protoplasts that raffinose is clearly (to about 20%) present in chloroplasts of cold-treated common bugle (Ajuga reptans L.), spinach (Spinacia oleracea L.) and Arabidopsis [Arabidopsis thaliana (L.) Heynh.] plants. The two dedicated enzymes needed for raffinose synthesis, galactinol synthase and raffinose synthase, were found to be extra-chloroplastic (probably cytosolic) in location, suggesting that the chloroplast envelope contains a raffinose transporter. Uptake experiments with isolated Ajuga and Arabidopsis chloroplasts clearly demonstrated that raffinose is indeed transported across the chloroplast envelope by a raffinose transporter, probably actively. Raffinose uptake into Ajuga chloroplasts was a saturable process with apparent K(m) and v(max) values of 27.8 mM and 3.3 micromol mg(-1) Chl min(-1), respectively.

Frost tolerance in excised leaves of the common bugle (Ajuga reptans L.) correlates positively with the concentrations of raffinose family oligosaccharides (RFOs).

Mass increases in raffinose family oligosaccharides (RFOs, alpha1,6-galactosyl extensions of sucrose) are well documented in the generative tissues of many plants upon cold acclimation, and they (i.e. mainly the two shortest RFO members, raffinose and stachyose) have been suggested as frost stress protectants. Our focus here was on the longer RFO members as they commonly occur in the frost-hardy evergreen labiate Ajuga reptans in its natural habitat, and accumulate to their highest concentrations in winter when the plant is faced with sub-zero temperatures. We examined the effects of RFO concentration and chain length on frost tolerance using excised leaves which accumulate long-chain RFOs under both cold and warm conditions, thereby uncoupling the acclimation temperature from RFO production. We demonstrated that frost tolerance in excised A. reptans leaves correlates positively with long-chain RFO accumulation under both acclimation temperatures. After 24 d post-excision in the warm, the leaves had increased their RFO concentrations (mainly long-chain RFOs) 22-fold to 78 mg g(-1) fresh weight, and decreased their EL(50) values (temperature at which 50% leakage occurred) from -10.5 to -24.5 degrees C, suggesting a protective role for these oligosaccharides in the natural frost tolerance of A. reptans.

Patients with Dementia in Primary Care: Who Is Referred to a Neurologist/Psychiatrist and What Patient-Oriented Factors Are Associated with the Visit?

BACKGROUND: The current guidelines imply that basic medical diagnostics for dementia should be provided by general practitioners in cooperation with other specialists such as neurologists and psychiatrists. OBJECTIVES: The aims of this paper were to 1) compare the dementia patients of general practice residents whose care is co-managed by neurology/psychiatry residents with those whose care is not; 2) identify the patient variables associated with the utilization of neurological and psychiatric specialists; and 3) describe the frequency of imaging used for dementia patients in primary care. METHODS: The analyses utilized data from 485 individuals who screened positive for dementia in primary care (PWD). Clinical variables and the utilization of specialists were assessed via medical records and face-to-face interviews. The factors associated with the utilization of specialists were assessed using multivariate linear regression and included age, sex, relationship status, cognitive impairment, depression, activities of daily living, and formal diagnosis of dementia. RESULTS: Our results show that 89 out of 485 study participants (18.4%) were referred to specialists 12 months prior to assessment. Of these 89 individuals, 14.6% (n = 13) did not receive imaging diagnostics, while 39.3% (n = 35) received brain imaging by CT scan and 46.1% (n = 41) by MRI. PWD referred to specialists differed from those not referred, in age, relationship status, and the presence of a formal diagnosis. Our multivariate analysis revealed that younger age (OR = 0.95; 95% -confidence interval 0.90-0.99; p = 0.04) and higher functional impairment (OR = 1.15; 95% -confidence interval 1.02-1.30; p = 0.02) were associated with a visit to a specialist. DISCUSSION: Only 1 out of every 4 to 5 individuals who have screened positive for dementia have visited a specialist in psychiatry or neurology. While in general, women utilized specialists less often than men, younger and more functionally impaired patients were more likely to be sent to a specialist by their treating general practitioner. Almost 90% of the patients sent to a specialist received cranial neuroimaging, suggesting high adherence to diagnostic guidelines in specialized care.

The C-terminal sequence from common bugle leaf galactan:galactan galactosyltransferase is a non-sequence-specific vacuolar sorting determinant.

The Ajuga reptans L. galactan:galactan galactosyltransferase (ArGGT) is a vacuolar enzyme that synthesizes long-chain raffinose family oligosaccharides (RFOs), the major storage carbohydrates of this plant. ArGGT is structurally and functionally related to acid plant alpha-galactosidases (alpha-Gals) of the glycosylhydrolase family 27, present in the apoplast or the vacuole. Sequence comparison of acid alpha-Gals with ArGGT revealed that they all contain an N-terminal signal sequence and a highly similar core sequence. Additionally, ArGGT and some acid alpha-Gals contain C-terminal extensions with low sequence similarities to each other. Here, we show that the C-terminal pentapeptide, SLQMS, is a non-sequence-specific vacuolar sorting determinant. Analogously, we demonstrate that the C-terminal extensions of selected acid alpha-Gals from Arabidopsis, barley, and rice, are also non-sequence-specific vacuolar sorting determinants, suggesting the presence of at least one vacuolar form of acid alpha-Gal in every plant species.