Membrane lipid organization and nicotinic acetylcholine receptor function: A two-way physiological relationship.

Nicotinic acetylcholine receptors (nAChRs) are involved in a great range of physiological and pathological conditions. Since they are transmembrane proteins, they interact strongly with the lipids surrounding them. Thus, the plasma membrane composition and heterogeneity play an essential role for the correct nAChR function, on the one hand, and the nAChR influences its immediate lipid environment, on the other hand. The aim of this work was to investigate in more detail the role of the biophysical properties of the membrane in nAChR function and vice versa, focusing on the relationship between Chol and nAChRs. To this end, we worked with different model systems which were treated either with (i) more Chol, (ii) cholesteryl hemisuccinate, or (iii) the enzyme cholesterol oxidase to generate different membrane sterol conditions and in the absence and presence of γTM4 peptide as a representative model of the nAChR. Fluorescence measurements with crystal violet and patch-clamp recordings were used to study nAChR conformation and function, respectively. Using confocal microscopy of giant unilamellar vesicles we probed the membrane phase state/order and organization (coexistence of lipid domains) and lipid-nAChR interaction. Our results show a feedback relationship between membrane organization and nAChR function, i.e. whereas the presence of a model of nAChRs conditions membrane organization, changing its lipid microenvironment, membrane organization and composition perturb nAChRs function. We postulate that nAChRs have a gain of function in disordered membrane environments but a loss of function in ordered ones, and that Chol molecules at the outer leaflet in annular sites and at the inner leaflet in non-annular sites are related to nAChR gating and desensitization, respectively. Thus, depending on the membrane composition, organization, and/or order, the nAChR adopts different conformations and locates in distinct lipid domains and this has a direct effect on its function.

Effects of solution conductivity on macropore size dynamics in electroporated lipid vesicle membranes.

Using fast imaging microscopy, we investigate in detail the expansion of micron-sized pores occurring in individual electroporated giant unilamellar vesicles composed of the phospholipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). To infer pore dynamics on the electrodeformed and electropermeabilized vesicles, we develop a computational approach and provide for the first time a direct evidence of quantitative agreement between experimental data and the well-established theoretical prediction of Smith, Neu and Krassowska (SNK). The analysis we describe also provides an extension to the current theoretical literature on how the conductivity ratio of the internal and the external vesicle solution plays a determinant role in the definition of the electrical force driving pore expansion kinetics.

Metabolic determinants of NAFLD in adults with type 1 diabetes.

AIM: To assess the main metabolic determinants of non-alcoholic fatty liver disease (NAFLD) in adult patients with type 1 diabetes (T1D). METHODS: 115 patients with T1D were divided into 4 groups according to NAFLD grade. NAFLD was diagnosed via transient elastography when CAP > 233 dB/m. Body composition was evaluated by Inbody720, Biospace. Serum lipids, liver enzymes, uric acid, creatinine, hsCRP and HbA1c were evaluated at fasting. RESULTS: The overall prevalence of NAFLD was 47% (n = 54). In the subgroup with BMI > 25 kg/m2 NAFLD prevalence was 66%; and positive family history of type 2 diabetes brought the risk up to 76%. 37% of the lean individuals also had NAFLD. HbA1c > 7% doubled the risk of NAFLD. Waist circumference > 82.5 cm was independently related to NAFLD, accounting for 24% of its variation in females. Accumulation of two and three metabolic syndrome (MetS) components, besides hyperglycemia, increased the risk of NAFLD by 14% (p < 0.0001) and 6% (p = 0.024), respectively. Lean NAFLD correlated with total insulin dose; NAFLD in overweight T1D patients correlated with triglycerides. CONCLUSIONS: NAFLD is highly prevalent in adults with T1D and obesity or other metabolic derangements and might be independently related to poor long-term glycemic control and waist circumference in females.

A Case of Mody 2 - Associated Hyperglycemia Diagnosed as Gestational Diabetes.

Maturity-onset diabetes of the young (MODY) is the most common monogenic form of diabetes, accounting for 1-2% of all diabetes cases. At least 14 different MODY subtypes have been identified the most common of which is MODY 2 caused by mutations in the glucokinase (GSK) gene. The mild hyperglycemia of MODY 2 is often first detected during pregnancy. Patients with MODY are usually misdiagnosed as either idiopathic type 1 or type 2 diabetes. The recognition of MODY 2 during pregnancy has important clinical implications as the management of hyperglycemia may differ from the established algorithm in gestational diabetes. Fetus development could be seriously affected in case it has inherited the GSK mutation and maternal hyperglycemia is insulin treated to the pregnancy adopted glycemic targets. The case report describes the stepwise diagnostic approach to a 43-year-old woman with a history of gestational diabetes and persistent prediabetes who was found to be a carrier of a heterozygous pathogenic variant in GSK (c.184G>A) and discusses the possible genotype of her two children according to their birth weight.

Relationship between cardiac autonomic neuropathy and cardio-metabolic risk profile in adults with type 1 diabetes.

AIM: The present study aims to determine the prevalence and association of cardiac autonomic neuropathy (CAN) with some traditional cardio-metabolic risk factors in adults with type 1 diabetes (T1D). MATERIAL AND METHODS: 235 adults with T1D, divided into three groups according to diabetes duration, were recruited in this cross-sectional study from May 2017 till December 2018. Anthropometric parameters and blood pressure were measured. Lipids, liver enzymes, uric acid, creatinine, HbA1c and high sensitive C-reactive protein (hsCRP) were measured at fasting. Albumin/creatinine ratio (ACR) was measured in a first spot urine sample. Body composition was evaluated using bio-impedance analysis, Inbody720 (Biospace, USA). Advanced glycation end products (AGEs) were assessed by autofluorescence method, AGE Reader (Diagnoptics, The Netherlands). CAN was assessed by ANX-3.0 monitoring technology (ANSAR Medical Technologies, Inc., Philadelphia, PA), applying standard clinical tests. 2005 IDF and 2009 JIS definitions were used to define Metabolic Syndrome (MetS). RESULTS: The prevalence of CAN was 23% and increased with diabetes duration. Sympathetic activity was independently related to age, albumin/creatinine ratio (ACR) and total body fat mass, and parasympathetic activity - to age and ACR. Elevated hsCRP, AGEs and body fat, diabetic retinopathy and nephropathy, as well as hypertension, dyslipidemia and metabolic syndrome were found to increase the risk of CAN in T1D. CONCLUSION: CAN appears to be a common complication of T1D, especially with longer duration, and is found to be related to diabetic microvascular disease and metabolic syndrome components.

Light controlled cell-to-cell adhesion and chemical communication in minimal synthetic cells.

Decorating GUVs, used as minimal synthetic cell models, with photoswitchable proteins allows controlling the adhesion between them and their assembly into multicellular structures with light. Thereby, the chemical communication between a sender and a receiver GUV, which strongly depends on their spatial proximity, can also be photoregulated.

Dynamic blue light-switchable protein patterns on giant unilamellar vesicles.

The blue light-dependent interaction between the proteins iLID and Nano allows recruiting and patterning proteins on GUV membranes, which thereby capture key features of patterns observed in nature. This photoswitchable protein interaction provides non-invasive, reversible and dynamic control over protein patterns of different sizes with high specificity and spatiotemporal resolution.

Epigenetic alterations in patients with type 2 diabetes mellitus.

Epigenetic changes, in particular DNA methylation processes, play a role in the pathogenesis and progression of type 2 diabetes mellitus (T2DM) linking genetic and environmental factors. To clarify this role, we have analyzed in patients with different duration of T2DM: (i) expression levels of methyl-CpG-binding domain protein 2 (MBD2) as marker of DNA methylation, and ii) methylation changes in 22 genes connected to cellular stress and toxicity. We have analyzed MBD2 mRNA expression levels in16 patients and 12 controls and the methylation status of stress and toxicity genes in four DNA pools: (i) controls; (ii) newly-diagnosed T2DM patients; (iii) patients with T2DM duration of <5 years and (iv) of >5 years. The MBD2 expression levels were 10.4-times increased on average in T2DM patients compared to controls. Consistent increase in DNA methylation fraction with the increase in T2DM duration was observed in Prdx2 and SCARA3 genes, connected to oxidative stress protection and in BRCA1 and Tp53 tumor-suppressor genes. In conclusion, increased MBD2 expression in patients indicated general dysregulation of DNA methylation in T2DM. The elevated methylation of Prdx2 and SCARA3 genes suggests disturbance in oxidative stress protection in T2DM. The increased methylation of BRCA1 and Tp53 genes unraveled an epigenetic cause for T2DM related increase in cancer risk.

Phase diagram and tie-line determination for the ternary mixture DOPC/eSM/cholesterol.

We propose a novel, to our knowledge, method for the determination of tie lines in a phase diagram of ternary lipid mixtures. The method was applied to a system consisting of dioleoylphosphatidylcholine (DOPC), egg sphingomyelin (eSM), and cholesterol (Chol). The approach is based on electrofusion of single- or two-component homogeneous giant vesicles in the fluid phase and analyses of the domain areas of the fused vesicle. The electrofusion approach enables us to create three-component vesicles with precisely controlled composition, in contrast to conventional methods for giant vesicle formation. The tie lines determined in the two-liquid-phase coexistence region are found to be not parallel, suggesting that the dominant mechanism of lipid phase separation in this region changes with the membrane composition. We provide a phase diagram of the DOPC/eSM/Chol mixture and predict the location of the critical point. Finally, we evaluate the Gibbs free energy of transfer of individual lipid components from one phase to the other.

Minimally invasive approach to thyroid gland surgery--indications, methods, first results.

UNLABELLED: After the introduction of minimally invasive operative techniques in the surgery of organs located in body cavities, extensive work has been done in the last five years with respect to their application in thyroid gland surgery as well. MATERIALS AND METHODS: In 2011, 406 patients underwent thyroid surgery at the Department of Surgery, Kaspela General Hospital for Active Treatment EOOD - Plovdiv. The study involved 48 of these patients, chosen at random and divided into two groups (A-minimally invasive thyroidectomy (MIT) - 26 patients, and B - minimally invasive video-assisted thyroidectomy (MIVAT) - 22 patients). All patients included in the study were selected on the basis of presence of one or more indeterminate nodules (fine needle aspiration biopsy - FNAB) sized up to 3.5 cm, with normal size of the thyroid gland up to 20-25 cm2, detected by preoperative ultrasonography. The study excluded patients with recurrent goitre, malignant disease of the thyroid gland and evidence of preoperative radiation therapy in the area of the head, neck and/or upper mediastinum. The preoperative investigation included history, physical examination, blood indices, echography, gamma camera and FNAB. Sensation of pain was evaluated by the patients according to a visual analogue scale, where lack of pain was evaluated as 0, and the most severe pain was evaluated as 10. RESULTS: The average duration of the hospitalization of patients undergoing MIT was 16 +/- 3.14 hours, whereas the patients undergoing MIVAT had to stay at the hospital for 18 +/- 3.56 hours. No complications were registered regarding the recurrent laryngeal nerves (RLN), as well as the operative wound. It should be noted that in all patients the course of the respective RLN was identified during the operative intervention, visually in most cases, or by electrostimulation. Postoperative hypoparathyroidism, registered by measuring the level of serum calcium, was observed in one visual analogue scale, as well as in the administration of pain-relieving agents during hospitalization. Other complications were not registered for the follow-up period, which covered an average of 6.56 +/- 3.14 months. CONCLUSIONS: The clinical analysis of the patients observed gave us grounds to draw the following conclusions: 1. The minimally invasive approach in patients with thyroid gland pathology is a possible and favourable option in selected patients with size of the gland within certain limits and not suffering from severe concomitant diseases. 2. The results in both groups of patients were comparable and did not show significant difference.

Integrated internist - addiction medicine - hepatology model for hepatitis C management for individuals on methadone maintenance.

Despite a high prevalence of hepatitis C virus (HCV) among drug users, HCV evaluation and treatment acceptance are extremely low among these patients when referred from drug treatment facilities for HCV management. We sought to increase HCV treatment effectiveness among patients from a methadone maintenance treatment program (MMTP) by maintaining continuity of care. We developed, instituted and retrospectively assessed the effectiveness of an integrated, co-localized care model in which an internist-addiction medicine specialist from MMTP was embedded in the hepatitis clinic. Methadone maintenance treatment program patients were referred, evaluated by the internist and hepatologist in hepatitis clinic and provided HCV treatment with integration between both sites. Of 401 evaluated patients, anti-HCV antibody was detected in 257, 86% of whom were older than 40 years. Hepatitis C virus RNA levels were measured in 222 patients, 65 of whom were aviremic. Of 157 patients with detectable HCV RNA, 125 were eligible for referral to the hepatitis clinic, 76 (61%) of whom accepted and adhered with the referral. Men engaged in MMTP <36 months were significantly less likely to be seen in hepatitis clinic than men in MMTP more than 36 months (odds ratio = 7.7; 95% confidence interval 2.6-22.9) or women. We evaluated liver histology in 63 patients, and 83% had moderate to advanced liver disease. Twenty-four patients initiated treatment with 19 completing and 13 (54%) achieving sustained response. In conclusion, integrated care between the MMTP and the hepatitis clinic improves adherence with HCV evaluation and treatment compared to standard referral practices.

Effect of the HIV-1 fusion peptide on the mechanical properties and leaflet coupling of lipid bilayers.

The fusion peptide (FP) of the human immunodeficiency virus (HIV) is part of the N-terminus of the viral envelope glycoprotein gp41 and is believed to play an important role in the viral entry process. To understand the immediate effect of this peptide on the cell membrane, we have studied the influence of the synthetic FP sequence FP23 on the mechanical properties of model lipid bilayers. For this purpose, giant unilamellar vesicles were prepared from the unsaturated lipid dioleoylphosphatidylcholine mixed in various molar ratios with FP23. The bending stiffness of the vesicles was measured with two different methods: fluctuation analysis and aspiration with micropipettes. The data obtained from both of these approaches show that the bending stiffness of the membrane decreases gradually with increasing concentration of the FP23 in the bilayer. Low concentrations of only a few mol% FP23 are sufficient to decrease the bending stiffness of the lipid bilayer by about a factor of 2. Finally, data obtained for the stretching elasticity modulus of the membrane suggest that the peptide insertion decreases the coupling between the two leaflets of the bilayer.

Composition dependence of vesicle morphology and mixing properties in a bacterial model membrane system.

We have determined the mixing properties and lamellar organization of bacterial membrane mimetics composed of 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE) and -phosphatidylglycerol (POPG) at various molar ratios applying differential scanning calorimetry, small and wide-angle X-ray scattering, as well as optical phase contrast microscopy. Combining the experimental thermodynamic data with a simulation of the liquidus and solidus lines, we were able to construct a phase diagram. Using this approach, we find that the lipids mix in all phases non-ideally in the thermodynamic sense. As expected, pure POPE assembles into multilamellar and pure POPG into unilamellar vesicles, respectively, which are stable within the studied temperature range. In contrast, mixtures of the two components form oligolamellar vesicles consisting of about three to five bilayers. The layers within these oligolamellar liposomes are positionally correlated within the gel phase, but become uncorrelated within the fluid phase exhibiting freely fluctuating bilayers, while the vesicles as a whole remain intact and do not break up into unilamellar forms. X-ray, as well as DSC data, respectively, reveal a miscibility gap due to a lateral phase segregation at POPG concentrations above about 70 mol%, similar to previously reported data on mixtures composed of disaturated PEs and PGs. Hence, the existence of a region of immiscibility is a general feature of PE/PG mixtures and the mixing properties are dominated by PE/PG headgroup interactions, but are largely independent of the composition of the hydrocarbon chains. This is in accordance with a recent theoretical prediction.

Pretransitional effects in dimyristoylphosphatidylcholine vesicle membranes: optical dynamometry study.

We used micron-sized latex spheres to probe the phase state and the viscoelastic properties of dimyristoylphosphatidylcholine (DMPC) bilayers as a function of temperature. One or two particles were manipulated and stuck to a DMPC giant vesicle by means of an optical trap. Above the fluid-gel main transition temperature, T(m) congruent with 23.4 degrees C, the particles could move on the surface of the vesicle, spontaneously (Brownian motion) or driven by an external force, either gravity or the laser beam's radiation pressure. From the analysis of the particle motions, we deduced the values of the membrane hydrodynamic shear viscosity, eta(s), and found that it would increase considerably near T(m). Below T(m), the long-distance motion of the particles was blocked. We performed experiments with two particles stuck on the membrane. By optical dynamometry, we measured the elastic resistance of the membrane to a variation in the interparticle distance and found that it would decrease considerably (down to zero) when the temperature was increased to T(m). We propose an interpretation relating the elastic response to the membrane curvature modulus, k(C). In this scheme, the two-bead dynamometry experiments provide a direct measurement of k(C) in the P'(beta) phase of lipid bilayers.

Ultrastructural features of the choline acetyltransferase-containing neurons and relationships with nigral dopaminergic and cortical afferent pathways in the rat striatum.

The aim of this study was first to specify the morphology and neuronal environment of the large cholinergic neurons, and second to determine the distribution and mode of termination of the corticostriatal and dopaminergic inputs on these neurons in the rat striatum. Immunocytochemical procedures with a monoclonal antibody against choline acetyltransferase, Golgi staining and standard electron microscopic techniques were used to specify the ultrastructural features of the putatively cholinergic classical large neurons. The large/choline acetyltransferase-positive neurons are characterized by a voluminous, eccentric, and deeply indented nucleus leaving a large cytoplasmic area, and by the presence of an abundant granular endoplasmic reticulum and of many polysomes and free ribosomes. Serial ultrathin sectioning further indicated the presence of nematosomes or nucleolus-like bodies within the nucleus and the cytoplasm of the large neurons. In addition, these neurons were found to be in direct apposition with up to four surrounding neurons showing features typical of medium-sized spiny neurons. These data support the view that the putatively cholinergic neurons may have an intense metabolic activity and may be involved in striatal clusters. When choline acetyltransferase immunostaining was coupled with the identification of degenerating corticostriatal afferents after lesion of the cerebral cortex, degenerating terminals were seen to form synapses of an asymmetrical type on distal labelled dendrites, but these contacts were very rare. On the other hand, nigrostriatal dopaminergic axons, identified by means of either the degeneration method or tyrosine hydroxylase immunostaining, were often found to run directly for long distances around the choline acetyltransferase-positive cell bodies. Occasionally, dopaminergic terminals formed possible symmetrical synapses on choline acetyltransferase-positive cell bodies or proximal dendrites. These data provide evidence that the putatively cholinergic neurons are directly contacted by corticostriatal and dopaminergic nigrostriatal afferents. The respective positions and nature of the two types of contacts further provide morphological support for the hypothesis that postsynaptic interactions may occur between the corticostriatal and dopaminergic nigrostriatal afferents at the level of the cholinergic neurons.

Ultrastructural relationships between choline acetyltransferase- and neuropeptide y-containing neurons in the rat striatum.

The relationships between cholinergic and neuropeptide Y-containing neuronal systems in the rat striatum were examined using a dual immunoperoxidase labelling method. These neurons were identified by their immunoreactivity to choline acetyltransferase and neuropeptide Y, respectively, and were visualized on the same sections using 3,3'-diaminobenzidine and benzidine dihydrochloride as distinct chromogens under two conditions: (i) neuropeptide Y detection by the 3,3'-diaminobenzidine diffuse brown reaction product and choline acetyltransferase detection by the benzidine dihydrochloride blue, granular reaction product; (ii) choline acetyltransferase detection by 3,3'-diaminobenzidine and neuropeptide Y detection by benzidine dihydrochloride. Although both neuropeptide Y- and choline acetyltransferase-immunoreactive cell bodies were simultaneously detected and were easily distinguishable whatever the conditions used, neuropeptide Y- and choline acetyltransferase-immunoreactive dendrites and axons could not be visualized on the same sections, since only the diaminobenzidine-labelled processes were detectable. Light microscopic observations on sections dual labelled with either method confirmed that choline acetyltransferase and neuropeptide Y immunoreactivities were localized in morphologically different populations of striatal neurons scattered throughout the striatum, choline acetyltransferase immunoreactivity being associated with large neurons and neuropeptide Y immunoreactivity with medium-sized neurons. In addition, the choline acetyltransferase-immunoreactive neurons were found to be more numerous than the neuropeptide Y-immunoreactive neurons and to be prevalent in the dorsolateral areas of the striatum, whereas neuropeptide Y-immunoreactive neurons were preferentially found in the ventromedial areas of this structure. Electron microscopic observations on sections processed under either condition revealed that choline acetyltransferase-positive terminals form synaptic contacts of the symmetrical type with neuropeptide Y-positive somata and proximal dendrites and that choline acetyltransferase-positive neurons are contacted by neuropeptide Y-positive terminals. These data show that the striatal neuropeptide Y- and choline acetyltransferase-containing neuronal systems have reciprocal synaptic interactions and provide morphological support for the hypothesis that striatal cholinergic and neuropeptide Y interneuron activities may be functionally linked.

Cortical projection of giant neostriatal neurons in the cat. Light and electron microscopic horseradish peroxidase study.

Following voluminous injections of horseradish peroxidase (HRP) in various neocortical fields, a small number of labeled large neurons are observed ipsilaterally in the putamen, striatal ponticuli, caudate nucleus, and nucleus accumbens septi. The bulk of the corticopetal cells are found in the putamen and in the striatal ponticuli. A more significant number of labeled neurons is encountered following injections in auditory and sensorimotor cortex, followed by the prefrontal and premotor cortex; very few cells project to the visual cortex. Ultrastructurally, the large HRP-labeled neurons display an eccentrically located, indented nucleus, abundant granular endoplasmic reticulum forming Nissl bodies, well developed Golgi zones, and numerous dense bodies. The simultaneous demonstration of retrogradely transported HRP and acetylcholinesterase (AChE) suggest that the large neurons are presumably cholinergic. These results provide evidence that at least some of the giant striatal neurons are efferent cells. The coincidence of cytological, histochemical, and hodological criteria invite the speculation that the giant corticopetal neostriatal neurons might be related to the magnocellular cholinergic groups of the basal forebrain (especially the Ch4 group).

[15 patients with myelodysplastic syndrome]. / Izsledvaniia vurkhu 15 bolni s mielodisplastichen sindrom.

15 patients (10 men and 5 women) with myelodysplastic syndrome, treated in a Hematologic Clinic during the last three years, are studied. The patients are classified in the following groups: with a refractory anemia (4 patients), with an acquired idiopathic sideroblast anemia (3 patients), with a refractory anemia with an excess of blast cells (8 patients). The diagnosis is proved by morphologic examinations, in some of the patients by isotopic examinations and in 5 patients a cytogenetic analysis is made. 7 patients died and in 5 of them the post mortem examination confirms the clinical diagnosis of myelodysplastic syndrome passed in acute leukosis. 4 patients are still alive and are under active observation. For the remaining 4 patients there are no data. The shortest survival is 6 months and the longest one is 9 years. The prognostic importance of the number of blast cells in the bone marrow is pointed out. The patients with refractory anemia with an excess of blast cells are treated with mercaptopurine or small doses of cytosine arabinoside in addition to the conventional blood-stimulating therapy applied in the other forms of myelodysplastic syndrome.

[Lennert's lymphoma]. / Vurkhu limfoma na Lennert.

The epithelioid cell Lennert's lymphoma is a rare form of malignant lymphomas. The authors present the cases of 4 patients, aged 50-71 years, 3 men and 2 women, with histologically proved Lennert's lymphoma. In two of the patients the diagnosis was proved at post mortem, too. The cases were characterized by a great variety of the clinico-hematologic data, the course of the disease and the therapeutic response. In all patients the disease was expressed in the III stage and in three of the patients it progressed to the IV stage. The therapeutic conduct is discussed because an early occurrence of cytopenia was found.

[Long-term myelodysplastic syndrome transformed into blastic leukemia]. / Dulgogodishen mielodisplastichen sindrom, transformiran v blastna levkoza.

A very rare case of myelodysplastic syndrome of long duration transformed into acute leukemia is reported. The syndrome presented with refractory anemia with ring sideroblasts and after eight years of favourable course abruptly turned into acute myelomonoblastic leukemia with a rapid fatal outcome caused by gram-negative sepsis. The disappearance of the ring-sideroblasts from the bone marrow is pointed out as a bad prognostic sign. Treatment of the myelodysplastic syndrome with small doses of cytosine arabinoside is discussed as well as the nonobligatory passing through the stage of a gradual increase of the blast cells before the transformation into acute leukemia.