[Important alteration of the Dutch treatment algorithm in patients with type 2 diabetes mellitus]. / Belangrijke wijziging van behandelrichtlijn 'Diabetes mellitus type 2'.

Recent trials of two classes of glucose-lowering drugs (SGLT2 inhibitors and GLP1RAs) have shown consistent cardiovascular and renal benefits that appear independent of glycaemic control. These results have prompted the Dutch College of General Practitioners (NHG) together with the Dutch Society of Internal Medicine (NIV) to update the treatment algorithm in patients with type 2 diabetes mellitus (T2D) at very high CVD risk. The use of SGLT2 inhibitors or GLP1RAs is now recommended in 3 groups of people with T2D. 1. patients with established CVD; 2. Patients with chronic kidney disease and a moderately to high CVD risk according to KDIGO; 3. patients with heart failure with reduced ejection fraction (HFrEF). Treatment algorithms differ for drug-naïve and drug-treated patients with T2D. In both drug-naïve and drug-treated patients the use of a SGLT2 inhibitor respectively as monotherapy or add-on is recommended as first step. If HbA1c is above the individual target, metformin will be added in drug-naive patients whereas GLP1-RAs could be considered in drug-treated patients. GLP1-RAs should also be considered when SGLT2-inhibitors are contraindicated.

Guideline on the diagnosis and treatment of primary idiopathic clubfoot.

- A delegation of 6 pediatric orthopedic surgeons from the Dutch Orthopedic Association (NOV) and 2 members of the board of the Dutch Parents' Association for children with clubfoot created the guideline "The diagnosis and treatment of primary idiopathic clubfeet" between April 2011 and February 2014. The development of the guideline was supported by a professional methodologist from the Dutch Knowledge Institute of Medical Specialists. This evidence-based guideline process was new and unique, in the sense that the process was initiated by a parents' association. This is the first official guideline in pediatric orthopedics in the Netherlands, and to our knowledge it is also the first evidence-based guideline on clubfoot worldwide. The guideline was developed in accordance with the criteria of the international AGREE instrument (AGREE II: Appraisal of Guidelines for Research and Evaluation II). The scientific literature was searched and systematically analyzed. In the second phase, conclusions and recommendations in the literature were formulated according to the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) method. Recommendations were developed considering the balance of benefits and harms, the type and quality of evidence, the values and preferences of the people involved, and the costs. The guideline is a solid foundation for standardization of clubfoot treatment in the Netherlands, with a clear recommendation of the Ponseti method as the optimal method of primary clubfoot treatment. We believe that the format used in the current guideline sets a unique example for guideline development in pediatric orthopedics that may be used worldwide. Our format ensured optimal collaboration between medical specialists and parents, and resulted in an important change in clubfoot care in the Netherlands, to the benefit of medical professionals as well as parents and patients. In this way, it is possible to improve professional collaboration between medical specialists and parents, resulting in an important change in clubfoot care in the Netherlands that will benefit medical professionals, parents, and patients. The guideline was published online, and is freely available from the Dutch Guideline Database ( www.richtlijnendatabase.nl ).

The effect of immediate breast reconstruction on the timing of adjuvant chemotherapy: a systematic review.

Adjuvant chemotherapy is often needed to achieve adequate breast cancer control. The increasing popularity of immediate breast reconstruction (IBR) raises concerns that this procedure may delay the time to adjuvant chemotherapy (TTC), which may negatively impact oncological outcome. The current systematic review aims to investigate this effect. During October 2014, a systematic search for clinical studies was performed in six databases with keywords related to breast reconstruction and chemotherapy. Eligible studies met the following inclusion criteria: (1) research population consisted of women receiving therapeutic mastectomy, (2) comparison of IBR with mastectomy only groups, (3) TTC was clearly presented and mentioned as outcome measure, and (4) original studies only (e.g., cohort study, randomized controlled trial, case-control). Fourteen studies were included, representing 5270 patients who had received adjuvant chemotherapy, of whom 1942 had undergone IBR and 3328 mastectomy only. One study found a significantly shorter mean TTC of 12.6 days after IBR, four studies found a significant delay after IBR averaging 6.6-16.8 days, seven studies found no significant difference in TTC between IBR and mastectomy only, and two studies did not perform statistical analyses for comparison. In studies that measured TTC from surgery, mean TTC varied from 29 to 61 days for IBR and from 21 to 60 days for mastectomy only. This systematic review of the current literature showed that IBR does not necessarily delay the start of adjuvant chemotherapy to a clinically relevant extent, suggesting that in general IBR is a valid option for non-metastatic breast cancer patients.

Trans-membrane area asymmetry controls the shape of cellular organelles.

Membrane organelles often have complicated shapes and differ in their volume, surface area and membrane curvature. The ratio between the surface area of the cytosolic and luminal leaflets (trans-membrane area asymmetry (TAA)) determines the membrane curvature within different sites of the organelle. Thus, the shape of the organelle could be critically dependent on TAA. Here, using mathematical modeling and stereological measurements of TAA during fast transformation of organelle shapes, we present evidence that suggests that when organelle volume and surface area are constant, TAA can regulate transformation of the shape of the Golgi apparatus, endosomal multivesicular bodies, and microvilli of brush borders of kidney epithelial cells. Extraction of membrane curvature by small spheres, such as COPI-dependent vesicles within the Golgi (extraction of positive curvature), or by intraluminal vesicles within endosomes (extraction of negative curvature) controls the shape of these organelles. For instance, Golgi tubulation is critically dependent on the fusion of COPI vesicles with Golgi cisternae, and vice versa, for the extraction of membrane curvature into 50-60 nm vesicles, to induce transformation of Golgi tubules into cisternae. Also, formation of intraluminal ultra-small vesicles after fusion of endosomes allows equilibration of their TAA, volume and surface area. Finally, when microvilli of the brush border are broken into vesicles and microvilli fragments, TAA of these membranes remains the same as TAA of the microvilli. Thus, TAA has a significant role in transformation of organelle shape when other factors remain constant.

Insertion of apoLp-III into a lipid monolayer is more favorable for saturated, more ordered, acyl-chains.

Neutral lipid transport in mammals is complicated involving many types of apolipoprotein. The exchangeable apolipoproteins mediate the transfer of hydrophobic lipids between tissues and particles, and bind to cell surface receptors. Amphipathic α-helices form a common structural motif that facilitates their lipid binding and exchangeability. ApoLp-III, the only exchangeable apolipoprotein found in insects, is a model amphipathic α-helix bundle protein and its three dimensional structure and function mimics that of the mammalian proteins apoE and apoAI. Even the intracellular exchangeable lipid droplet protein TIP47/perilipin 3 contains an α-helix bundle domain with high structural similarity to that of apoE and apoLp-III. Here, we investigated the interaction of apoLp-III from Locusta migratoria with lipid monolayers. Consistent with earlier work we find that insertion of apoLp-III into fluid lipid monolayers is highest for diacylglycerol. We observe a preference for saturated and more highly ordered lipids, suggesting a new mode of interaction for amphipathic α-helix bundles. X-ray reflectivity shows that apoLp-III unfolds at a hydrophobic interface and flexible loops connecting the amphipathic α-helices stay in solution. X-ray diffraction indicates that apoLp-III insertion into diacylglycerol monolayers induces additional ordering of saturated acyl-chains. These results thus shed important new insight into the protein-lipid interactions of a model exchangeable apolipoprotein with significant implications for its mammalian counterparts.

Dietary fiber, carbohydrate quality and quantity, and mortality risk of individuals with diabetes mellitus.

BACKGROUND: Dietary fiber, carbohydrate quality and quantity are associated with mortality risk in the general population. Whether this is also the case among diabetes patients is unknown. OBJECTIVE: To assess the associations of dietary fiber, glycemic load, glycemic index, carbohydrate, sugar, and starch intake with mortality risk in individuals with diabetes. METHODS: This study was a prospective cohort study among 6,192 individuals with confirmed diabetes mellitus (mean age of 57.4 years, and median diabetes duration of 4.4 years at baseline) from the European Prospective Investigation into Cancer and Nutrition (EPIC). Dietary intake was assessed at baseline (1992-2000) with validated dietary questionnaires. Cox proportional hazards analysis was performed to estimate hazard ratios (HRs) for all-cause and cardiovascular mortality, while adjusting for CVD-related, diabetes-related, and nutritional factors. RESULTS: During a median follow-up of 9.2 y, 791 deaths were recorded, 306 due to CVD. Dietary fiber was inversely associated with all-cause mortality risk (adjusted HR per SD increase, 0.83 [95% CI, 0.75-0.91]) and CVD mortality risk (0.76[0.64-0.89]). No significant associations were observed for glycemic load, glycemic index, carbohydrate, sugar, or starch. Glycemic load (1.42[1.07-1.88]), carbohydrate (1.67[1.18-2.37]) and sugar intake (1.53[1.12-2.09]) were associated with an increased total mortality risk among normal weight individuals (BMI≤25 kg/m(2); 22% of study population) but not among overweight individuals (P interaction≤0.04). These associations became stronger after exclusion of energy misreporters. CONCLUSIONS: High fiber intake was associated with a decreased mortality risk. High glycemic load, carbohydrate and sugar intake were associated with an increased mortality risk in normal weight individuals with diabetes.

Dietary glycemic load and glycemic index and risk of coronary heart disease and stroke in Dutch men and women: the EPIC-MORGEN study.

BACKGROUND: The associations of glycemic load (GL) and glycemic index (GI) with the risk of cardiovascular diseases (CVD) are not well-established, particularly in men, and may be modified by gender. OBJECTIVE: To assess whether high dietary GL and GI increase the risk of CVD in men and women. METHODS: A large prospective cohort study (EPIC-MORGEN) was conducted within the general Dutch population among 8,855 men and 10,753 women, aged 21-64 years at baseline (1993-1997) and free of diabetes and CVD. Dietary intake was assessed with a validated food-frequency questionnaire and GI and GL were calculated using Foster-Powell's international table of GI. Information on morbidity and mortality was obtained through linkage with national registries. Cox proportional hazards analysis was performed to estimate hazard ratios (HRs) for incident coronary heart disease (CHD) and stroke, while adjusting for age, CVD risk factors, and dietary factors. RESULTS: During a mean follow-up of 11.9 years, 581 CHD cases and 120 stroke cases occurred among men, and 300 CHD cases and 109 stroke cases occurred among women. In men, GL was associated with an increased CHD risk (adjusted HR per SD increase, 1.17 [95% CI, 1.02-1.35]), while no significant association was found in women (1.09 [0.89-1.33]). GI was not associated with CHD risk in both genders, while it was associated with increased stroke risk in men (1.27 [1.02-1.58]) but not in women (0.96 [0.75-1.22]). Similarly, total carbohydrate intake and starch intake were associated with a higher CHD risk in men (1.23 [1.04-1.46]; and 1.24 [1.07-1.45]), but not in women. CONCLUSION: Among men, high GL and GI, and high carbohydrate and starch intake, were associated with increased risk of CVD.

TIP47 functions in the biogenesis of lipid droplets.

TIP47 (tail-interacting protein of 47 kD) was characterized as a cargo selection device for mannose 6-phosphate receptors (MPRs), directing their transport from endosomes to the trans-Golgi network. In contrast, our current analysis shows that cytosolic TIP47 is not recruited to organelles of the biosynthetic and endocytic pathways. Knockdown of TIP47 expression had no effect on MPR distribution or trafficking and did not affect lysosomal enzyme sorting. Therefore, our data argue against a function of TIP47 as a sorting device. Instead, TIP47 is recruited to lipid droplets (LDs) by an amino-terminal sequence comprising 11-mer repeats. We show that TIP47 has apolipoprotein-like properties and reorganizes liposomes into small lipid discs. Suppression of TIP47 blocked LD maturation and decreased the incorporation of triacylglycerol into LDs. We conclude that TIP47 functions in the biogenesis of LDs.

Biophysics and function of phosphatidic acid: a molecular perspective.

Phosphatidic acid is the simplest (diacyl)glycerophospholipid present in cells and is now a well established second messenger with direct biological functions. It is specifically recognized by diverse proteins and plays an important role in cellular signaling and membrane dynamics in all eukaryotes. An important determinant of the biological functions of phosphatidic acid is its anionic headgroup. In this review we will focus on the peculiar ionization properties of phosphatidic acid and their crucial role in lipid-protein interactions. We will take a molecular approach focusing entirely on the physical chemistry of the lipid and develop a model explaining the ionization properties of phosphatidic acid, termed the electrostatic-hydrogen bond switch model. Diverse examples from recent literature in support of this model will be presented and the broader implications of our findings will be discussed.

Quantitative label-free imaging of lipid composition and packing of individual cellular lipid droplets using multiplex CARS microscopy.

Lipid droplets (LDs) are highly dynamic organelles that perform multiple functions, including the regulated storage and release of cholesterol and fatty acids. Information on the molecular composition of individual LDs within their cellular context is crucial in understanding the diverse biological functions of LDs, as well as their involvement in the development of metabolic disorders such as obesity, type II diabetes, and atherosclerosis. Although ensembles of LDs isolated from cells and tissues were analyzed in great detail, quantitative information on the heterogeneity in lipid composition of individual droplets, and possible variations within single lipid droplets, is lacking. Therefore, we used a label-free quantitative method to image lipids within LDs in 3T3-L1 cells. The method combines submicron spatial resolution in three dimensions, using label-free coherent anti-Stokes Raman scattering microscopy, with quantitative analysis based on the maximum entropy method. Our method allows quantitative imaging of the chemistry (level of acyl unsaturation) and physical state (acyl chain order) of individual LDs. Our results reveal variations in lipid composition and physical state between LDs contained in the same cell, and even within a single LD.

Diacylglycerol is required for the formation of COPI vesicles in the Golgi-to-ER transport pathway.

Diacylglycerol is necessary for trans-Golgi network (TGN) to cell surface transport, but its functional relevance in the early secretory pathway is unclear. Although depletion of diacylglycerol did not affect ER-to-Golgi transport, it led to a redistribution of the KDEL receptor to the Golgi, indicating that Golgi-to-ER transport was perturbed. Electron microscopy revealed an accumulation of COPI-coated membrane profiles close to the Golgi cisternae. Electron tomography showed that the majority of these membrane profiles originate from coated buds, indicating a block in membrane fission. Under these conditions the Golgi-associated pool of ARFGAP1 was reduced, but there was no effect on the binding of coatomer or the membrane fission protein CtBP3/BARS to the Golgi. The addition of 1,2-dioctanoyl-sn-glycerol or the diacylglycerol analogue phorbol 12,13-dibutyrate reversed the effects of endogenous diacylglycerol depletion. Our findings implicate diacylglycerol in the retrograde transport of proteins from Golgi to the ER and suggest that it plays a critical role at a late stage of COPI vesicle formation.

An electrostatic/hydrogen bond switch as the basis for the specific interaction of phosphatidic acid with proteins.

Phosphatidic acid (PA) is a minor but important phospholipid that, through specific interactions with proteins, plays a central role in several key cellular processes. The simple yet unique structure of PA, carrying just a phosphomonoester head group, suggests an important role for interactions with the positively charged essential residues in these proteins. We analyzed by solid-state magic angle spinning 31P NMR and molecular dynamics simulations the interaction of low concentrations of PA in model membranes with positively charged side chains of membrane-interacting peptides. Surprisingly, lysine and arginine residues increase the charge of PA, predominantly by forming hydrogen bonds with the phosphate of PA, thereby stabilizing the protein-lipid interaction. Our results demonstrate that this electrostatic/hydrogen bond switch turns the phosphate of PA into an effective and preferred docking site for lysine and arginine residues. In combination with the special packing properties of PA, PA may well be nature's preferred membrane lipid for interfacial insertion of positively charged membrane protein domains.

Actin filaments are involved in the maintenance of Golgi cisternae morphology and intra-Golgi pH.

Here we examine the contribution of actin dynamics to the architecture and pH of the Golgi complex. To this end, we have used toxins that depolymerize (cytochalasin D, latrunculin B, mycalolide B, and Clostridium botulinum C2 toxin) or stabilize (jasplakinolide) filamentous actin. When various clonal cell lines were examined by epifluorescence microscopy, all of these actin toxins induced compaction of the Golgi complex. However, ultrastructural analysis by transmission electron microscopy and electron tomography/three-dimensional modelling of the Golgi complex showed that F-actin depolymerization first induces perforation/fragmentation and severe swelling of Golgi cisternae, which leads to a completely disorganized structure. In contrast, F-actin stabilization results only in cisternae perforation/fragmentation. Concomitantly to actin depolymerization-induced cisternae swelling and disorganization, the intra-Golgi pH significantly increased. Similar ultrastructural and Golgi pH alkalinization were observed in cells treated with the vacuolar H+ -ATPases inhibitors bafilomycin A1 and concanamycin A. Overall, these results suggest that actin filaments are implicated in the preservation of the flattened shape of Golgi cisternae. This maintenance seems to be mediated by the regulation of the state of F-actin assembly on the Golgi pH homeostasis.

What makes the bioactive lipids phosphatidic acid and lysophosphatidic acid so special?

Phosphatidic acid and lysophosphatidic acid are minor but important anionic bioactive lipids involved in a number of key cellular processes, yet these molecules have a simple phosphate headgroup. To find out what is so special about these lipids, we determined the ionization behavior of phosphatidic acid (PA) and lysophosphatidic acid (LPA) in extended (flat) mixed lipid bilayers using magic angle spinning 31P NMR. Our data show two surprising results. First, despite identical phosphomonoester headgroups, LPA carries more negative charge than PA when present in a phosphatidylcholine bilayer. Dehydroxy-LPA [1-oleoyl-3-(phosphoryl)propanediol] behaves in a manner identical to that of PA, indicating that the difference in negative charge between LPA and PA is caused by the hydroxyl on the glycerol backbone of LPA and its interaction with the phosphomonoester headgroup. Second, deprotonation of phosphatidic acid and lysophosphatidic acid was found to be strongly stimulated by the inclusion of phosphatidylethanolamine in the bilayer, indicating that lipid headgroup charge depends on local lipid composition and will vary between the different subcellular locations of (L)PA. Our findings can be understood in terms of a hydrogen bond formed within the phosphomonoester headgroup of (L)PA and its destabilization by competing intra- or intermolecular hydrogen bonds. We propose that this hydrogen bonding property of (L)PA is involved in the various cellular functions of these lipids.

Membrane activity of the phospholipase C-delta1 pleckstrin homology (PH) domain.

PH-PLCdelta1 [the PH domain (pleckstrin homology domain) of PLCdelta1 (phospholipase C-delta1)] is among the best-characterized phosphoinositide-binding domains. PH-PLCdelta1 binds with high specificity to the headgroup of PtdIns(4,5)P2, but little is known about its interfacial properties. In the present study, we show that PH-PLCdelta1 is also membrane-active and can insert significantly into PtdIns(4,5)P2-containing monolayers at physiological (bilayer-equivalent) surface pressures. However, this membrane activity appears to involve interactions distinct from those that target PH-PLCdelta1 to the PtdIns(4,5)P2 headgroup. Whereas the majority of PtdIns(4,5)P2-bound PH-PLCdelta1 can be displaced by adding excess of soluble headgroup [Ins(1,4,5)P3], membrane activity of PH-PLCdelta1 cannot. PH-PLCdelta1 differs from other phosphoinositide-binding domains in that its membrane insertion does not require that the phosphoinositide-binding site be occupied. Significant monolayer insertion remains when the phosphoinositide-binding site is mutated, and PH-PLCdelta1 can insert into monolayers that contain no PtdIns(4,5)P2 at all. Our results suggest a model in which reversible membrane binding of PH-PLCdelta1, mediated by PtdIns(4,5)P2 or other acidic phospholipids, occurs without membrane insertion. Accumulation of the PH domain at the membrane surface enhances the efficiency of insertion, but does not significantly affect its extent, whereas the presence of phosphatidylethanolamine and cholesterol in the lipid mixture promotes the extent of insertion. This is the first report of membrane activity in an isolated PH domain and has implications for understanding the membrane targeting by this common type of domain.

Spontaneous curvature of phosphatidic acid and lysophosphatidic acid.

The formation of phosphatidic acid (PA) from lysophosphatidic acid (LPA), diacylglycerol, or phosphatidylcholine plays a key role in the regulation of intracellular membrane fission events, but the underlying molecular mechanism has not been resolved. A likely possibility is that PA affects local membrane curvature facilitating membrane bending and fission. To examine this possibility, we determined the spontaneous radius of curvature (R(0p)) of PA and LPA, carrying oleoyl fatty acids, using well-established X-ray diffraction methods. We found that, under physiological conditions of pH and salt concentration (pH 7.0, 150 mM NaCl), the R(0p) values of PA and LPA were -46 A and +20 A, respectively. Thus PA has considerable negative spontaneous curvature while LPA has the most positive spontaneous curvature of any membrane lipid measured to date. The further addition of Ca(2+) did not significantly affect lipid spontaneous curvature; however, omitting NaCl from the hydration buffer greatly reduced the spontaneous curvature of PA, turning it into a cylindrically shaped lipid molecule (R(0p) of -1.3 x 10(2) A). Our quantitative data on the spontaneous radius of curvature of PA and LPA at a physiological pH and salt concentration will be instrumental in developing future models of biomembrane fission.

Cisplatin nanocapsules.

Cisplatin nanocapsules represent a novel lipid formulation of the anticancer drug cis-diamminedichloroplatinum(II), in which nanoprecipitates of cisplatin are covered by a phospholipid bilayer coat consisting of an equimolar mixture of phosphatidylcholine and phosphatidylserine. Cisplatin nanocapsules are characterized by an unprecedented cisplatin-to-lipid molar ratio and exhibit strongly improved cytotoxicity against tumor cells in vitro compared with the free drug. Here, methods for preparing and characterizing cisplatin nanocapsules are reported.

Secretory traffic triggers the formation of tubular continuities across Golgi sub-compartments.

The organization of secretory traffic remains unclear, mainly because of the complex structure and dynamics of the secretory pathway. We have thus studied a simplified system, a single synchronized traffic wave crossing an individual Golgi stack, using electron tomography. Endoplasmic-reticulum-to-Golgi carriers join the stack by fusing with cis cisternae and induce the formation of intercisternal tubules, through which they redistribute their contents throughout the stack. These tubules seem to be pervious to Golgi enzymes, whereas Golgi vesicles are depleted of both enzymes and cargo. Cargo then traverses the stack without leaving the cisternal lumen. When cargo exits the stack, intercisternal connections disappear. These findings provide a new view of secretory traffic that includes dynamic intercompartment continuities as key players.

Golgi enzymes are enriched in perforated zones of golgi cisternae but are depleted in COPI vesicles.

In the most widely accepted version of the cisternal maturation/progression model of intra-Golgi transport, the polarity of the Golgi complex is maintained by retrograde transport of Golgi enzymes in COPI-coated vesicles. By analyzing enzyme localization in relation to the three-dimensional ultrastructure of the Golgi complex, we now observe that Golgi enzymes are depleted in COPI-coated buds and 50- to 60-nm COPI-dependent vesicles in a variety of different cell types. Instead, we find that Golgi enzymes are concentrated in the perforated zones of cisternal rims both in vivo and in a cell-free system. This lateral segregation of Golgi enzymes is detectable in some stacks during steady-state transport, but it was significantly prominent after blocking endoplasmic reticulum-to-Golgi transport. Delivery of transport carriers to the Golgi after the release of a transport block leads to a diminution in Golgi enzyme concentrations in perforated zones of cisternae. The exclusion of Golgi enzymes from COPI vesicles and their transport-dependent accumulation in perforated zones argues against the current vesicle-mediated version of the cisternal maturation/progression model.