Chitosan-Surfactant Composite Nanocoatings on Glass and Zinc Surfaces Prepared from Aqueous Solutions.

Hydrophobic coatings from chitosan-surfactant composites (ca. 400 nm thick by UV-Vis spectroscopy) for possible corrosion protection were developed on glass and zinc substrates. The surfactants (sodium dodecyl sulfate, SDS or sodium dodecylbenzenesulfonate, and SDBS) were added to the chitosan by two methods: mixing the surfactants with the aqueous chitosan solutions before film deposition or impregnating the deposited chitosan films with surfactants from their aqueous solutions. For the mixed coatings, it was found that the lower surface tension of solutions (40-45 mN/m) corresponded to more hydrophobic (80-90°) coatings in every case. The hydrophobicity of the impregnated coatings was especially significant (88° for SDS and 100° for SDBS). Atomic force microscopy studies revealed a slight increase in roughness (max 1.005) for the most hydrophobic coatings. The accumulation of surfactants in the layer was only significant (0.8-1.0 sulfur atomic %) in the impregnated samples according to X-ray photoelectron spectroscopy. Polarization and electron impedance spectroscopy tests confirmed better barrier properties for these samples (40-50% pseudo-porosity instead of 94%). The degree of swelling in a water vapor atmosphere was significantly lower in the case of the impregnated coatings (ca. 25%) than that of the native ones (ca. 75%), measured by spectroscopic ellipsometry. Accordingly, good barrier layer properties require advantageous bulk properties in addition to surface hydrophobicity.

Correlations between the anti-corrosion properties and the photocatalytic behavior of epoxy coatings incorporating modified graphene oxide deposited on a zinc substrate.

This research aimed to create a substrate-coating system based on zinc and an epoxy resin incorporating modified graphene oxide, which possesses two key characteristics: effective resistance against corrosion and the ability to harness photocatalytic properties. Furthermore, correlations between the anti-corrosion properties and the photocatalytic behaviour of the coatings were made. Thin epoxy (EP) layers embedding 0.1 wt% graphene oxide (GO), reduced graphene oxide (rGO), and modified graphene oxide with (3-aminopropyl)-triethoxysilane (APTES) or poly(amidoamine) (PAMAM) dendrimer were applied on a zinc (Zn) substrate using the dip-coating method. Anti-corrosion properties of coated Zn samples were investigated through electrochemical impedance spectroscopy (EIS) measurements. They showed that the corrosion protection effect is more prominent for EP containing functionalized GO, the highest in the case of GO-PAMAM. The results of the EIS measurements indicated also that the corrosion protection provided by EP-rGO is smaller than that of EP. The photocatalytic properties of the coatings were studied by exposure of the samples to Methylene Blue (MB) solution followed by monitoring the model dye degradation through UV-Vis measurements. To determine the changes in the anti-corrosion properties due to photocatalysis, the coated Zn samples were put through additional EIS measurements. The same coatings applied to a glass substrate lacked photocatalytic properties, indicating that the Zn substrate is accountable for the degradation of MB. Furthermore, the incorporation of GO or functionalized GO into the coating amplifies this effect. From EIS spectra, it was determined that the protective properties loss observed after 3 days is due to coating delamination during exposure to MB solution, the EP-GO-APTES retaining the best adhesion of the coating, 98% remaining on Zn after a cross-hatch test. The corrosion measurements were complemented by examining the morphology and structure of the coatings and the modified GO particles. All things considered, the Zn/EP-GO-APTES system shows the best ability to break down organic pollutants, keeping a good anti-corrosive property and adhesion.

Wetting and swelling behaviour of N-acetylated thin chitosan coatings in aqueous media.

Chitosan nanocoatings (thickness range of 120-540 nm) were produced on glass, zinc and silicon substrates with dip-coating and spin coating techniques to study their pH-dependent wetting and swelling behaviour. The coatings were N-acetylated with the methanolic solution of acetic anhydride to increase the degree of acetylation from 36 % to 100 % (according to ATR-FTIR studies). The measured contact angles of Britton-Robinson (BR) buffer solutions (pH 6.0, 7.4 and 9.0) were lower on the acetylated surfaces (ca. 50°), than that of their native counterparts (ca. 70°) and does not depend on the pH. Contrary, contact angles on the native coating deteriorated 10°-15° with increasing the pH. In addition, for native coatings, the decrease of the contact angles over time also showed a pH dependence: at pH 9.0 the contact angle decreased by 7° in 10 min, while at pH 6.0 it decreased by 13° and at a much faster rate. The constraint swelling of the coatings in BR puffer solutions was studied in situ by scanning angle reflectometry. The swelling degree of the native coatings increased significantly with decreasing pH (from 250 % to 500 %) due to the increased number of protonated amino groups, while the swelling degree of acetylated coatings was ca. 160 % regardless of the pH. The barrier properties of the coatings were studied by electrochemical tests on zinc substrates. The analysis of polarization curves showed the more permeable character of the acetylated coatings despite the non-polar character of the bulk coating matrix. It can be concluded that in the case of native coatings, 49 % of the absorbed water is in bound form, which does not assist ion transport, while in the case of acetylated coatings, this value is only 33 %.

Effect of the Preparation Method on the Properties of Eugenol-Doped Titanium Dioxide (TiO2) Sol-Gel Coating on Titanium (Ti) Substrates.

The focus of this study was the preparation of sol-gel titanium dioxide (TiO2) coatings, by the dip-coating technique, on Ti6Al4V (TiGr5) and specific Ti implant substrates. In order to confer antibacterial properties to the layers, Eugenol was introduced in the coatings in two separate ways: firstly by introducing the Eugenol in the sol (Eug-TiO2), and secondly by impregnating into the already deposed TiO2 coating (TiO2/Eug). Optimization of Eugenol concentration as well as long term were performed in orderboth short- and long-term Eugenol concentration was performed to investigate the prepared samples thoroughly. The samples were investigated by electrochemical impedance spectroscopy (EIS) and potentiodynamic polarization curves (PDP). To investigate their resistance against Gram-negative Escherichia coli bacteria, microbiological analysis was performed on coatings prepared on glass substrates. Structural studies (FT-IR analysis, Raman spectroscopy) were performed to confirm Eugenol-TiO2 interactions. Coating thicknesses and adhesion were also determined for all samples. The results show that Eug-TiO2 presented with improved anticorrosive effects and significant antibacterial properties, compared to the other investigated samples.

Bisphenol A Diglycidyl Ether-Primary Amine Cooligomer-poly(ε-caprolactone) Networks: Synthesis and Characterization.

In this work, the preparation and systematic investigation of cross-linked polyurethane-epoxy (PU-EP) polymer systems are reported. The PU-EP polymers were prepared using a reaction of isocyanate (NCO)-terminated PU-prepolymer with diglycidyl ether of bisphenol A (DGEBA)-amine cooligomer. The oligomerization of DGEBA was carried out by adding furfurylamine (FA) or ethanolamine (EA), resulting in DGEBA-amine cooligomers. For the synthesis of NCO-terminated PU-prepolymer, poly(ε-caprolactone)diol (PCD) (Mn = 2 kg/mol) and 1,6-hexamethylene diisocyanate (HDI) were used. The cross-linking was achieved by adding DGEBA-amine cooligomer to PU-prepolymer, in which the obtained urethane bonds, due to the presence of free hydroxil groups in the activated DGEBA, served as netpoints. During cross-linking, ethanolamine provides an additional free hydroxyl group for the formation of a new urethane bond, while furfurylamine can serve as a thermoreversible coupling element (e.g., Diels-Alder adduct). The PU-EP networks were characterized using attenuated total reflectance Fourier-transform infrared spectroscopy (ATR-FTIR), differential scanning calorimetry (DSC), dynamical mechanical analysis (DMA) and scanning electron microscopy (SEM). The DMA curves of some PU-EPs (depending on the compositions and the synthetic method) revealed a plateau-like region above the melting temperature (Tm) of PCD, confirming the presence of a cross-linked structure. This property resulted in a shape memory (SM) behavior for these samples, which can be fine-tuned in the presence of furfurylamine through the formation of additional thermoreversible bonds (e.g., Diels-Alder adduct).

Temporary Anti-Corrosive Double Layer on Zinc Substrate Based on Chitosan Hydrogel and Epoxy Resin.

In practice, metal structures are frequently transported or stored before being used. Even in such circumstances, the corrosion process caused by environmental factors (moisture, salty air, etc.) can occur quite easily. To avoid this, metal surfaces can be protected with temporary coatings. The objective of this research was to develop coatings that exhibit effective protective characteristics while also allowing for easy removal, if required. Novel, chitosan/epoxy double layers were prepared on zinc by dip-coating to obtain temporary tailor-made and peelable-on-demand, anti-corrosive coatings. Chitosan hydrogel fulfills the role of a primer that acts as an intermediary between the zinc substrate and the epoxy film to obtain better adhesion and specialization. The resulting coatings were characterized using electrochemical impedance spectroscopy, contact angle measurements, Raman spectroscopy, and scanning electron microscopy. The impedance of the bare zinc was increased by three orders of magnitude when the protective coatings were applied, proving efficient anti-corrosive protection. The chitosan sublayer improved the adhesion of the protective epoxy coating. The structural integrity and absolute impedance of the protective layers were conserved in both basic and neutral environments. However, after fulfilling its lifespan, the chitosan/epoxy double-layered coating could be removed after treatment with a mild acid without damaging the substrate. This was because of the hydrophilic properties of the epoxy layer, as well as the tendency of chitosan to swell in acidic conditions.

Barrier behaviour of partially N-acetylated chitosan layers in aqueous media.

Chitosan coatings of 353 ± 12 nm thickness were prepared on glass and zinc substrates by dip-coating method to study their barrier-behaviour. The coatings were chemically modified to increase their degree of acetylation (DA) from ca. 44 % up to ca. 98 % resulting a quasi-chitin coating. The effect of the acetylation reaction was studied by infrared spectroscopy, and the structural changes of the native and acetylated coatings were investigated by UV-Vis spectrophotometry and X-ray diffraction. The surface properties of the coated samples were characterized by wettability measurements - advancing water contact angle decreased from ca. 80° (native) to ca. 43° (fully acetylated) - and microscopic (SEM, AFM) studies. The barrier behaviour of the chitosan layer depending on the DA was evaluated by electrochemical impedance spectroscopy studies and with a special mesoporous silica - chitosan bilayer system by measuring the amount of dye (Rhodamine 6G) accumulated in the silica through the chitosan coating during an impregnation step. These methods showed significant decrease in the barrier-effect of the coatings with increasing DA (accumulation of approximately six times more dye and a reduction of charge transfer resistance by an order of magnitude), due to the structural and ionization changes in the coatings.

Biochemical and Clinical Effects of Vitamin E Supplementation in Hungarian Smith-Lemli-Opitz Syndrome Patients.

Smith-Lemli-Opitz syndrome (SLOS) is a severe monogenic disorder resulting in low cholesterol and high 7-dehydrocholesterol (7-DHC) levels. 7-DHC-derived oxysterols likely contribute to disease pathophysiology, and thus antioxidant treatment might be beneficial because of high oxidative stress. In a three-year prospective study, we investigated the effects of vitamin E supplementation in six SLOS patients already receiving dietary cholesterol treatment. Plasma vitamin A and E concentrations were determined by the high-performance liquid chromatography (HPLC) method. At baseline, plasma 7-DHC, 8-dehydrocholesterol (8-DHC) and cholesterol levels were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. The clinical effect of the supplementation was assessed by performing structured parental interviews. At baseline, patients were characterized by low or low-normal plasma vitamin E concentrations (7.19-15.68 µmol/L), while vitamin A concentrations were found to be normal or high (1.26-2.68 µmol/L). Vitamin E supplementation resulted in correction or significant elevation of plasma vitamin E concentration in all patients. We observed reduced aggression, self-injury, irritability, hyperactivity, attention deficit, repetitive behavior, sleep disturbance, skin photosensitivity and/or eczema in 3/6 patients, with notable individual variability. Clinical response to therapy was associated with a low baseline 7-DHC + 8-DHC/cholesterol ratio (0.2-0.4). We suggest that determination of vitamin E status is important in SLOS patients. Supplementation of vitamin E should be considered and might be beneficial.

Elevated inflammatory gene expression in intervertebral disc tissues in mice with ADAM8 inactivated.

We found ADAM8 enzymatic activity elevated in degenerative human intervertebral disc (IVD). Here, we examined the discs in ADAM8-inactivation mice that carry a mutation preventing self-activation of the enzyme. Surprisingly, elevated gene expression for inflammatory markers (Cxcl1, IL6) was observed in injured discs of ADAM8 mutant mice, along with elevated expression of type 2 collagen gene (Col2a1), compared with wild type controls. Injured annulus fibrosus of mutant and wild type mice contained a higher proportion of large collagen fibers compared with intact discs, as documented by microscopic examination under circular polarized light. In the intact IVDs, Adam8EQ mouse AF contained lower proportion of yellow (intermediate) fiber than WT mice. This suggests that ADAM8 may regulate inflammation and collagen fiber assembly. The seemingly contradictory findings of elevated inflammatory markers in mutant mice and excessive ADAM8 activity in human degenerative discs suggest that ADAM8 may interact with other enzymatic and pro-inflammatory processes needed for tissue maintenance and repair. As a future therapeutic intervention to retard intervertebral disc degeneration, partial inhibition of ADAM8 proteolysis may be more desirable than complete inactivation of this enzyme.

Tonsillectomia versus tonsillotomia / Tonsillectomia versus tonsillotomia.

Összefoglaló. Bevezetés és célkituzés: A szerzok a posztoperatív fájdalom és a sebgyógyulás tekintetében prospektív vizsgálattal hasonlították össze gyermekeken (67 fo, 1-12 év) a hagyományos hidegeszközzel történo extracapsularis tonsillectomiát (23 fo) a microdebriderrel (23 fo) és a coblatorral (21 fo) végzett intracapsularis tonsillotomiával. Módszer: A vizsgálatok a betegek által kitöltött kérdoívek, valamint prospektív klinikai adatgyujtés alapján történtek. Eredmények: Az intracapsularis tonsillotomia gyógyulási idejét 50%-kal rövidebbnek találtuk, és az elso 13 napban szignifikánsan kevesebb fájdalommal és fájdalomcsillapító igénnyel járt, mint az extracapsularis tonsillectomia eseteiben. A tonsillotomiás csoporton belül egyedül a posztoperatív elso napi fájdalom tekintetében észleltünk szignifikáns különbséget a két különbözo módszer között a coblator javára (p<0,05). A vizsgálatokat retrospektív áttekintéssel is kiegészítettük, 4 évi gyermek- (1-15 éves) tonsillamutéten átesett beteganyagunk (1487 fo) eredményeinek feldolgozásával. Tonsillectomia (1253 fo) után 7,7%-os utóvérzési arányt észleltünk, mutéti vérzéscsillapításra 1,3%-ban volt szükség. Tonsillotomia esetén (234 fo) 0,43%-os utóvérzési arányt regisztráltunk. Ebben a csoportban vérzés miatt nem, de 2 esetben ismételt obstrukciót okozó hypertrophia, 1 esetben góctünetek miatt reoperációt végeztünk (1,28%). Következtetés: Eredményeiket a szerzok a nemzetközi ajánlások tükrében elemezték. Az intracapsularis tonsillotomia kisebb fájdalommal, kisebb vérzéssel és kisebb megterheléssel jár. A közösségbe való aktív visszatérés akár egy hét után lehetséges a tonsillectomiára jellemzo 3 héttel szemben, mindez jelentos szocioökonómiai elonyökkel járhat. Orv Hetil. 2020; 161(45): 1920-1926. INTRODUCTION AND OBJECTIVE: Examining operated children in this prostective study inditerscompared (67 pts, 1-12 yrs) the extracapsular tonsillectomy with conventional cold-knife (23 pts) to extracapsular tonsillotomy with microdebrider (23 pts) and coblator (21 pts) for postoperative pain and wound-healing disorders. METHOD: The study was based on patient-completed questionnaires as well as prospective clinical data collection. RESULTS: The recovery time of intracapsular tonsillotomy was found less than 50%, with less pain than in the cases of extracapsular tonsillectomy. Postoperative pain was significantly less in the tonsillototomy group than the tonsillectomy group. Within the tonsillotomy group, a significant difference was observed between the two different methods in favor of the coblator for only the postoperative first-day pain. The studies were supplemented with a retrospective review by processing the 4 yrs results of their pediatric (1-15-yrs) patients who underwent tonsillectomy (1487 pts). After tonsillectomy (1253 pts), a postoperative bleeding rate of 7.7% was observed, and surgical hemostasis was required in 1.3%. In the case of tonsillotomy (234 pts), a postoperative bleeding rate of 0.43% was recorded. In this group, reoperation was not performed due to bleeding, whereas it was neccesary in 2 cases due to hypertrophy causing repeated obstruction, in 1 case due by virtue of focal symptomes (1.28%). CONCLUSION: Our results were analyzed on the basis of international recommendations. Intracapsular tonsillotomy is associated with less pain, less bleeding, and less strain. Active return to the community is possible after up to a week compared to the 3 weeks typical of tonsillectomy, all of which can have significant socioeconomic benefits. Orv Hetil. 2020; 161(45): 1920-1926.

Accumulation of 2-Acetylamino-5-mercapto-1,3,4-thiadiazole in chitosan coatings for improved anticorrosive effect on zinc.

Chitosan (Chit) coatings were applied on zinc substrates by the dip-coating method. Subsequently, the coatings were impregnated with a corrosion inhibitor, 2-Acetylamino-5-mercapto-1,3,4-thiadiazole (AcAMT) to obtain an increased anticorrosive effect. The coating thickness and the AcAMT accumulation were determined using UV-Vis spectroscopy on glass and quartz substrates, respectively. The surface morphology and coverage were investigated with atomic force microscopy. Electrochemical impedance spectroscopy and potentiodynamic polarization techniques were used to investigate the protective properties of the impregnated coatings. The chitosan coatings facilitated the accumulation of the corrosion inhibitor inside the polymeric matrix (a multiplication of 380 times compared to the impregnating solution concentration was calculated), channeling high amounts of AcAMT to the Zn surface, which resulted in an inhibition efficiency of >90%. This effect demonstrates the applicability of chitosan coatings as carriers for corrosion inhibitors, significantly reducing the amount of inhibitor needed to achieve good anticorrosive effects.

FBN1 gene mutations in 26 Hungarian patients with suspected Marfan syndrome or related fibrillinopathies.

Marfan syndrome (MFS) is an autosomal dominant connective tissue disorder mainly affecting the cardiovascular, ocular and musculo-skeletal systems. FBN1 gene mutations lead to MFS and related connective tissue disorders. In this work we described clinical and molecular data of 26 unrelated individuals with suspected MFS who were referred for FBN1 mutation analysis. FBN1 gene sequencing was performed by next generation sequencing and Sanger sequencing methods. We identified 23 causal or potentially causal (including variants of uncertain significance) FBN1 variants, seven of them was novel (˜30%). About 30% of the cases were sporadic. FBN1 mutations were associated with MFS in the majority of the patients, in two cases with severe and early onset manifestation of the syndrome. Missense mutations were detected in 69.6% (16/23), the majority of them were located in one of the cbEGF motifs and ˜70% of them substituted conserved cystein residues. Small deletions/duplications were identified in 13% of the cases (3/23), while splice site variants were detected in 17.4% (4/23). In three unrelated patients a low frequency recurrent silent variant (c.3294C > T (p.Asp1098=) was identified. FBN1 mRNA analysis showed that the mutation does not lead to aberrant splicing, based on available data the mutation was classified as benign.

Eco-friendly indigo carmine-loaded chitosan coatings for improved anti-corrosion protection of zinc substrates.

Chitosan (Chit) coatings were prepared on zinc and glass substrates by dip-coating method. The coatings were impregnated with a non-toxic compound, indigo carmine (IC). The novel, eco-friendly, IC-loaded chitosan coatings were characterized morpho-structurally, and their corrosion protection behavior was investigated using potentiodynamic polarization and electrochemical impedance spectroscopy techniques. The surface properties of the coated samples were evaluated by wettability measurements. The thickness of the native chitosan layers and the stability of the impregnated layers in terms of dye release on glass substrates were studied by UV-vis spectrophotometry. The good corrosion inhibiting efficiency of the coatings (>90%) was attributed to the ionic crosslinking of the positively charged Chit with negatively charged IC. The Chit-IC coatings can be successfully used as model systems for chitosan-based coatings incorporating ionic inhibitors and in less demanding applications, such as temporary protective coatings for metals, removable on demand by scrubbing with mild acidic solutions.

A novel point mutation affecting Asn76 of dystrophin protein leads to dystrophinopathy.

Mutations in the DMD gene lead to Duchenne and Becker muscular dystrophy (DMD/BMD). Missense mutations are rare cause of DMD/BMD. A six-month-old male patient presented with mild generalized muscle weakness, hypotonia, and delayed motor development. Dystrophinopathy was suspected because of highly elevated serum creatine kinase level (1497 U/L) and tiered DMD gene analysis was performed. Multiplex ligation-dependent probe amplification (MLPA) assay showed deletion of exon 4, which could not be confirmed by another method. Sequencing of exon 4 revealed a novel de novo point mutation (c.227A>T, p.Asn76Ile) in the N-terminal actin-binding domain (N-ABD) of dystrophin protein. The false positive MLPA result was explained by the fact that the affected nucleotide lies directly at the 3' ligation site of the MLPA probe. Sequencing of the whole coding region of DMD gene proved c.227A>T to be the sole variant being potentially pathogenic. According to in silico analyses the mutation was predicted to be highly destabilizing on N-ABD structure possibly leading to protein malfunction. Muscle biopsy was performed and dystrophin immunohistochemistry results were suggestive of BMD. Our results highlight the importance of confirmatory testing of single-exon deletions detected by MLPA and we describe a novel, destabilizing missense mutation in the DMD gene.

Laboratory characterization of leukemic cell procoagulants.

BACKGROUND: In acute myeloid leukemias, there is an increased chance to develop thrombotic disorders. We hypothesized that in addition to leukemic promyelocytes, monocytic leukemia cells may also have a higher procoagulant activity. METHODS: Fibrin formation was assessed by a one-stage clotting assay using a magnetic coagulometer. The thrombin generation test (TGT) of magnetically isolated normal human monocytes, intact leukemic cells and their isolated microparticles was performed by a fluorimetric assay. Phosphatidylserine (PS) expression of leukemic cells and microparticle number determinations were carried out by flow cytometry. RESULTS: All cell lines displayed a significant procoagulant potential compared to isolated normal human monocytes. In the TGT test, the mean of lagtime and the time to peak parameters were significantly shorter in leukemic cells (3.9-4.7 and 9.9-10.3 min) compared to monocytes (14.9 and 26.5 min). The mean of peak thrombin in various monocytic leukemia cell lines was 112.1-132.9 nM vs. 75.1 nM in monocytes; however, no significant difference was observed in the ETP parameter. Factor VII-deficient plasma abolished all procoagulant activity, whereas factor XII-deficient plasma did not affect the speed of fibrin formation and thrombin generation but modulated the amount of thrombin. Factor XI-deficient plasma affected the time to peak values in one leukemic cell line and also attenuated peak thrombin. Leukemia cell-derived microparticles from all three cell lines exerted a procoagulant effect by significantly shortening the lagtime in TGT; there was a nonsignificant difference in case of ETP parameter. CONCLUSIONS: All investigated monocytic leukemia cell lines exhibited significant thrombin generation. This phenomenon was achieved by the procoagulants on the surface of leukemic cells as well as by their microparticles.

[Local infiltration analgesia in total joint replacement]. / Helyi infiltrációs érzéstelenítés alkalmazása nagyízületi endoprotézisek beültetése során.

INTRODUCTION: Total hip and knee replacment surgeries are characterized by severe postoperative pain. Local infiltration analgesia is proved to be very effective. However this method has not been widely used in Hungary. AIM: To evaluate the efficacy of the local infiltration analgesia with modified components in patients underwent total hip or knee replacement surgery. METHOD: Data of 99 consecutive patients underwent primary total hip or knee replacement surgery were evaluated prospectively. In all the 99 surgeries modified local infiltration analgesia was applied. Postoperative pain reported on a visual analog scale was recorded as well as the need for further analgetics during the first 18 hours after surgery. The cost of the analgetic drugs was calculated. The control group comprised 97 consecutive patients underwent total hip or knee replacement, where local infiltration analgesia was not applied. Statistical analysis was done. RESULTS: Patients received local infiltration analgesia reported significantly less pain (p<0.001). The need for postoperatively given analgetics was almost 50% less, and the cost of all postoperative analgetics was 47% less than in the control group. CONCLUSION: In total hip and knee replacement surgeries the modified local infiltration analgesia decreases postoperative pain effectively and contribute to the early mobilization of the patients. Orv. Hetil., 2017, 158(9), 352-357.

Hyperglycaemia suppresses microRNA expression in platelets to increase P2RY12 and SELP levels in type 2 diabetes mellitus.

Megakaryocyte (MK)-derived miRNAs have been detected in platelets. Here, we analysed the expression of platelet and circulating miR-223, miR-26b, miR-126 and miR-140 that might be altered with their target mRNAs in type 2 diabetes mellitus (DM2). MiRNAs were isolated from leukocyte-depleted platelets and plasma samples obtained from 28 obese DM2, 19 non-DM obese and 23 healthy individuals. The effect of hyperglycaemia on miRNAs was also evaluated in MKs using MEG-01 and K562 cells under hyperglycaemic conditions after 8 hours up to four weeks. Quantitation of mature miRNA, pre-miRNAs and target mRNA levels (P2RY12 and SELP) were measured by RT-qPCR. To prove the association of miR-26b and miR-140 with SELP (P-selectin) mRNA level, overexpression or inhibition of these miRNAs in MEG-01 MKs was performed using mimics or anti-miRNAs, respectively. The contribution of calpain substrate Dicer to modulation of miRNAs was studied by calpain inhibition. Platelet activation was evaluated via surface P-selectin by flow cytometry. Mature and pre-forms of investigated miRNAs were significantly reduced in DM2, and platelet P2RY12 and SELP mRNA levels were elevated by two-fold at increased platelet activation compared to controls. Significantly blunted miRNA expressions were observed by hyperglycaemia in MEG-01 and K562-MK cells versus baseline values, while the manipulation of miR-26b and miR-140 expression affected SELP mRNA level. Calpeptin pretreatment restored miRNA levels in hyperglycaemic MKs. Overall, miR-223, miR-26b, miR-126 and miR-140 are expressed at a lower level in platelets and MKs in DM2 causing upregulation of P2RY12 and SELP mRNAs that may contribute to adverse platelet function.

Vitamin D receptor is a novel transcriptional regulator for Axin1.

BACKGROUND: Axin1 is a scaffold protein in the ß-catenin destruction complex, which, if disrupted, contributes to pathogenesis of various human diseases, including colorectal carcinogenesis and inflammatory bowel diseases (IBD). We have previously demonstrated that Salmonella infection promotes the degradation and plasma sequestration of Axin1, leading to bacterial invasiveness and inflammatory responses. Vitamin D and the vitamin D receptor (VDR) appear to be important regulators of IBD and colon cancer. Although VDR and Axin1 are all involved in intestinal inflammation, it remains unclear whether these processes are related or function independently. In the current study, we hypothesize that VDR is an important regulator for the maintenance of physiological level of Axin1. METHODS: Using the intestinal epithelial conditional VDR knockout mouse model (VDRΔIEC) and cultured cell lines, influences of VDR status on the expression of Axin1 was evaluated by Western blots and real-time PCR. Loss- and gain-of-function assays were used to investigate the regulation of VDR on Axin1 at the transcriptional and translational levels. Cells were treated with cycloheximide or actinomycin for molecular mechanistic studies. Candidate genomic VDR binding sites for Axin1 were tested by chromatin immunoprecipitation (ChIP) assay. Physical interactions among VDR, Axin1, and ß-catenin were tested by immunoprecipitation. Cellular localization of Axin1 with different VDR status was determined by fractionation and immunohistochemistry. RESULTS: We found that VDR deletion led to lower protein and mRNA levels of Axin1, whereas knockdown of Axin1 did not change the expression level of VDR protein. Immunoprecipitation data did not support physical interaction between VDR and Axin1. The VDR regulation of Axin1 was through a VDR genomic binding site for Axin1 gene on the regulatory region. Fractionation data showed that cytosolic Axin1 was significantly reduced due to VDR deletion, leaving the nuclear fraction unchanged. In ileum, Axin1 was distributed in the cytosol of apical epithelium and crypts. CONCLUSION: VDR is important for the maintenance of physiological level of Axin1. The discovery of Axin1 as a VDR target gene provides novel and fundamental insights into the interactions between the VDR and ß-catenin signaling pathways.

7DHC-induced changes of Kv1.3 operation contributes to modified T cell function in Smith-Lemli-Opitz syndrome.

In vitro manipulation of membrane sterol level affects the regulation of ion channels and consequently certain cellular functions; however, a comprehensive study that confirms the pathophysiological significance of these results is missing. The malfunction of 7-dehydrocholesterol (7DHC) reductase in Smith-Lemli-Opitz syndrome (SLOS) leads to the elevation of the 7-dehydrocholesterol level in the plasma membrane. T lymphocytes were isolated from SLOS patients to assess the effect of the in vivo altered membrane sterol composition on the operation of the voltage-gated Kv1.3 channel and the ion channel-dependent mitogenic responses. We found that the kinetic and equilibrium parameters of Kv1.3 activation changed in SLOS cells. Identical changes in Kv1.3 operation were observed when control/healthy T cells were loaded with 7DHC. Removal of the putative sterol binding sites on Kv1.3 resulted in a phenotype that was not influenced by the elevation in membrane sterol level. Functional assays exhibited impaired activation and proliferation rate of T cells probably partially due to the modified Kv1.3 operation. We concluded that the altered membrane sterol composition hindered the operation of Kv1.3 as well as the ion channel-controlled T cell functions.

Biglycan Inhibits Capsaicin-Induced Substance P Release by Cultured Dorsal Root Ganglion Neurons.

OBJECTIVE: The purpose of this study was to examine the inhibitory effects of biglycan on substance P release from cultured sensory neurons in response to capsaicin. STUDY DESIGN: In vitro study of cultured primary sensory neurons from the rabbit dorsal root ganglion (DRG). We interrogated the culture system function with capsaicin. Biglycan is an important structural component of the intervertebral disc that may regulate growth factors and inflammatory mediators. We tested the hypothesis that biglycan inhibits substance P release in response to capsaicin. RESULTS: The DRG cultures were shown to contain both neurons and astrocytes by immunostaining using antibodies recognizing neuron and glial cell markers. Cultured DRG cells respond to capsaicin in a dose- and time-dependent manner (capsaicin dose ranges from 5 to 500 µmol/L; stimulation time ranges from 0 to 60 minutes). The neurons preincubated with biglycan released 27% less substance P compared with neurons without biglycan (n = 4, P = 0.036). CONCLUSION: We have established a DRG cell culture system, which contains both sensory neurons and the supporting astrocytes. Biglycan, an inhibitor of substance P release by DRG cultures, may serve as an ingredient in intradiscal injectables to reduce back pain.