Biotin as a structural component in the detection of small model antigens in BLA-S-ELISA.

An interesting biotin-linked-antigen-Sandwich-ELISA was developed (BLA-S-ELISA), which based on the captured Trinitrophenol-Biotin (TNP-Biotin) molecule between the immobilised monovalent antibody and enzyme-conjugated streptavidin. Monoclonal anti-Trinitrotoluene single chain fragment antibody (anti-TNT-scFv) was cloned and expressed in E. coli cells, and then used as an immobilised component in an assay. Thereafter, the previously synthesised TNP-Biotin was added as antigen followed by the addition of streptavidin-horseradish peroxidase (streptavidin-HRP) conjugate which led finally to the formation of a three-component system (antibody/TNP-Biotin/streptavidin-HRP). The assay was performed with a range of different dilutions of TNP-Biotin to establish its minimal detectable concentration. The detection limit of TNP-Biotin was 4 ngmL-1 (i.e. 200 pg or 0.42 pmol antigen calculated on the basis of 50 µL sample or 8.4 nM expressed in concentration units). According to our best knowledge, this is the very first time for any model antigen to be detected with such a form of biotin-streptavidin sandwich-assay.

Pentadecapeptide BPC 157 as Therapy for Inferior Caval Vein Embolization: Recovery of Sodium Laurate-Post-Embolization Syndrome in Rats.

After inferior caval vein embolization therapy, post-embolization syndrome (sodium laurate 10 mg/kg, 0.1 mL into rat inferior caval vein, assessment at 15, 30, 60 min, prime lung lesions, thromboemboli occluding lung vessels), as a severe occlusion/occlusion-like syndrome, might be resolved as a whole by stable gastric pentadecapeptide BPC 157 therapy. At 5 min after laurate injection, stable gastric pentadecapeptide BPC 157 was implemented as therapy (10 µg/kg, 10 ng/kg intraperitoneally or intragastrically). As before, confronted with the occlusion of major vessel(s) or similar noxious procedures, such as rapidly acting Virchow triad circumstances, the particular effect of the therapy (i.e., collateral pathways activation, "bypassing vascular key", i.e., direct blood flow delivery via activation of azygos vein) assisted in the recovery of the vessel/s and counteracted multiorgan failure due to occlusion/occlusion-like syndrome as a whole in the laurate-injected rats. Along with prime lung lesions and thromboemboli occluding lung vessels, post-embolization syndrome rapidly occurred peripherally and centrally as a shared multiorgan and vessel failure, brain, heart, lung, liver, kidney, and gastrointestinal tract lesions, venous hypertension (intracranial (superior sagittal sinus), portal, and caval), aortal hypotension, progressing thrombosis in veins and arteries and stasis, congested and/or failed major veins, and severe ECG disturbances. Whatever the cause, these were all counteracted, eliminated, or attenuated by the application of BPC 157 therapy. As recovery with BPC 157 therapy commonly and rapidly occurred, reversing the collapsed azygos vein to the rescuing collateral pathway might initiate rapid direct blood delivery and start blood flow reorganization. In conclusion, we suggest BPC 157 therapy to resolve further vascular and embolization injuries.

Thermal Characterisation and Isoconversional Kinetic Analysis of Osmotically Dried Pork Meat Proteins Longissimus dorsi.

The kinetic properties and thermal characteristics of fresh pork meat proteins (Longissimus dorsi), as well as osmotically dehydrated meat proteins, were investigated using differential scanning calorimetry. Two isoconversional kinetical methods, namely the differential Friedman and integral Ortega methods, were employed to analyze the data. The obtained kinetic triplet, activation energy, pre-exponential factor, and extent of conversion, has been discussed. The resulting activation energy for proteins of fresh meat ranges between 751 kJ·mol-1 for myosin, 152 kJ·mol-1 for collagen and sarcoplasmic proteins, and 331 kJ·mol-1 for actin at a conversion degree of 0.1 to 0.9. For osmotically dried pork meat proteins, the values range from 307 kJ·mol-1 for myosin 272 kJ·mol-1 for collagen and sarcoplasmic proteins, and 334.83 kJ·mol-1 for actin at a conversion degree from 0.1 to 0.9. The proteins of the dry meat obtained by osmotic dehydration in molasses could be described as partly unfolded as they retain the characteristic protein denaturation transition. Concerning the decrease in enthalpies of proteins denaturation, thermodynamic destabilization of dried meat proteins occurred. On the contrary, dried meat proteins were thermally stabilized with respect to increase in the temperatures of denaturation. Knowledge of the nature of meat protein denaturation of each kind of meat product is one of the necessary tools for developing the technology of meat product processing and to achieve desired quality and nutritional value. The kinetic analysis of meat protein denaturation is appropriate because protein denaturation gives rise to changes in meat texture during processing and directly affects the quality of product.

Severe intracranial haemorrhage in neonatal alloimmune thrombocytopenia due to antibodies against human platelet antigen 1b: Case report and literature review.

Fetal/neonatal alloimmune thrombocytopenia (FNAIT) is a rare life-threatening disorder, leading to severe thrombocytopenia and potentially bleeding, with intracranial haemorrhage (ICH) being the most serious complication. We report on a FNAIT case with fourth-degree ICH that arose due to antibodies against human platelet antigen (HPA)-1b. The male infant, born to an otherwise healthy mother, presented with severe signs of ICH soon after delivery. Since only moderate thrombocytopenia was noted and there were no active signs of bleeding, the infant did not receive intravenous immunoglobulins (IVIg) or platelet transfusion. Spontaneous recovery of platelets was noted on the eighth day of life, but permanent neurological impairment remained as a consequence of ICH. We report the results of HPA and human leukocyte antigen (HLA) antibodies in the mother's and the infant's sera, the family's HPA genotype and the mother's HLA genotype, and summarise previously described cases of FNAIT due to anti-HPA-1b antibodies in the literature. FNAIT with severe ICH due to anti-HPA-1b antibodies is rarely diagnosed. An association between HLA genes and sensitization to HPA-1b antibodies was not demonstrated. The severity of FNAIT and the occurrence of ICH is often difficult to predict. In this case, the infant presented with moderate thrombocytopenia and ICH, with subsequent permanent consequences.

Diagnostic performance of digital breast tomosynthesis in female patients with nipple discharge.

BACKGROUND: Nipple discharge is one of the most common symptoms related to the breast, but it is a presenting feature of breast cancer in 5%-12% of women. AIMS: The purpose of this study was to determine the diagnostic performance of digital breast tomosynthesis (DBT) in the evaluation of patients with nipple discharge and to compare it with mammography (MMG), ultrasound (US), and magnetic resonance imaging (MRI). METHODS AND RESULTS: This retrospective study included 53 patients with nipple discharge. All patients underwent DBT, and results were compared to MMG, breast US, and MRI. Radiological findings for each method were categorized according to BI-RADS classification: categories 1-2 were considered negative and categories 3-5 positive. If a tissue specimen was obtained, the final diagnosis was established based on the results of histopathological analysis; otherwise, a clinical follow-up was required for at least 2 years to confirm benign radiological findings. Measures of diagnostic accuracy of DBT, MMG, US, and MRI were calculated and compared. RESULTS: Final histopathological analysis revealed six malignant breast lesions, all of which were detected in patients with pathologic nipple discharge. DBT and MRI exhibited high sensitivity (100%) and high negative predictive value (100%) for the detection of breast cancer in patients with nipple discharge. DBT showed higher specificity compared to MRI (82.9% vs. 61.9%). Sensitivity and specificity of MMG were 83.3% and 76.6%, respectively. Breast US was determined to have a sensitivity of 66.7% and specificity of 57.5%. CONCLUSION: DBT exhibited higher specificity than MRI at the same level of sensitivity and negative predictive value. Therefore, the use of DBT should be considered as an alternative to MRI in the assessment of patients with nipple discharge.

A novel and effective natural product-based immunodetection tool for TNT-like compounds.

This study aimed to develop a fast and reliable protocol for Trinitrophenol-Tris(hydroxymethyl)aminomethane (TNP-Tris) detection applying a ß-lactamase-fusion protein of choice, the natural product-based immunoreagent tool of competitive sensitivity developed herein for the first time. Since the fusion protein 11B3-scFv-ß-lactamase is constructed from a scFv-antibody (11B3) linked to an enzyme (ß-lactamase), the step related to the use of secondary antibody in the enzyme-linked immunosorbent assay (ELISA) is completely omitted. Indeed, this fusion protein itself serves both as binding mean of the antigen model and detecting agent, due to the presence of the naturally occurring enzyme. In such a way, it actually affords the one-step TNP-Tris detection reaching a promising LOD value of 45 ± 2 fmol or 157 ± 6 pg/mL. Taken all together, the current protocol does represent much cheaper and significantly less-time consuming alternative compared both to the recombinant antibodies and recombinant phages, previously designed means in our labs for the same purpose.

Low sugar jellies of berry fruits: the impact of low vs. high temperature regime on their chemical composition and antioxidativity.

This study focuses on the impact of low and high temperature regimes on the chemical composition and antioxidativity of low sugar berry fruits jellies. High quality fruits (strawberry, raspberry and blackberry) were collected from Western Serbia region, quite well recognised both nationally and internationally due to an extremely well developed practice in growing berry fruits. The obtained results have clearly indicated the importance of low temperature regime for enriched contents of both total phenolics and anthocyanins followed by an enhanced antioxidativity. Rubus fruticosus L. Cacak Thornless cultivar, the only autochthonous berry fruit variety screened herein, may be firmly recommended as a raw material for industrial production of low sugar blackberry jellies with exceptional characteristics. This innovative procedure of preparing berry fruit jellies have encompassed the application of low temperature regime (55 °C), lower content of sugar (40%), seeds separation from jellies followed by no use of pectin throughout the whole process.

Functionality and Storability of Cookies Fortified at the Industrial Scale with up to 75% of Apple Pomace Flour Produced by Dehydration.

Apple pomace flour (APF) with high content of dietary fibers (DF), total polyphenolics (TPCs) and flavonoids (TFCs) was produced at the industrial scale. Bulk and tapped density, swelling, water and oil holding capacity, solubility and hydration density of fine and coarse APF with average particle size 0.16 and 0.50 mm were compared. The effect of wheat flour substitution with 25%, 50% and 75% of fine and coarse APF was studied upon cookies production at the industrial scale and after one year of storage. Coarse APF performed better in respect to sensorial properties, content and retention of dietary compounds and antioxidant (AO) activity. The cookies with optimal share of coarse APF (50%) contained 21 g/100 g of DF and several times higher TPC, TFC as well as AO activity than control cookies, retained well health promoting compounds and maintained an intensely fruity aroma and crispy texture. They were acceptable for consumers according to the hedonic test.

Content of Total Phenolics, Flavan-3-Ols and Proanthocyanidins, Oxidative Stability and Antioxidant Capacity of Chocolate During Storage.

Antioxidant (AO) capacity of chocolates with 27, 44 and 75% cocoa was assessed after production and during twelve months of storage by direct current (DC) polarographic assay, based on the decrease of anodic current caused by the formation of hydroxo-perhydroxyl mercury(II) complex (HPMC) in alkaline solutions of hydrogen peroxide at potentials of mercury oxidation, and two spectrophotometric assays. Relative antioxidant capacity index (RACI) was calculated by taking the average value of the AO assay (the sample mass in all assays was identical). Oxidative stability of chocolate fat was determined by differential scanning calorimetry. Measured parameters and RACI were correlated mutually and with the content of total phenols (Folin-Ciocalteu assay), flavan-3-ols (vanillin and p-dimethylaminocinnamaldehyde assay) and proanthocyanidins (modified Bate-Smith assay). During storage, the studied functional and health-related characteristics remained unchanged. Amongst applied AO assays, the DC polarographic one, whose validity was confirmed by two-way ANOVA and F-test, correlated most significantly with oxidative stability (oxidation onset temperature and induction time). In addition, principal component analysis was applied to characterise chocolate types.

Factors contributing to communication skills development in cochlear implanted children.

BACKGROUND/AIM: Over the last 10 years more than 300 persons received cochlear implant in Serbia and more than 90% of the recipients were children under 10 years of age. The program of cochlear implantation includes postoperative rehabilitation in which cognitive, integrative and developmental methods are used. The study was conducted to reveal factors affecting communication performance (CP) of cochlear implanted (Cl) children. Special attention was focused on the influence of the duration and intensity of rehabilitation and hearing age on further development of communication skills. METHODS: A group of 30 CI children (13 boys and 17 girls) aged 2 to 5 years was enrolled in the study. All of the children had average intelligence and no other developmental disorder. They lived in families and attended rehabilitative seances 3 to 5 times a week. Their parents/caregivers answered structured questionnaire about functioning after pediatric cochlear implantation (FAPCI) and the results were the subject of detailed statistical analysis. RESULTS: Analysis of variance did not show any difference between the boys and the girls regarding FAPCI achievements (F(1, 28) = 2.909; p = 0.099) and age aberration in CP score (F(1,28) = 0.114, p = 0.738). Correlation analysis showed a statistically significant difference in FAPCI scores related to hearing age and duration of rehabilitation. Regression analysis (enter method) showed that model consisting of indipendent variables significantly contributed to prediction of overall FAPCI scores and Adjusted R2 value could explain 32% difference in communication skills of participants in this study. CONCLUSION: Communication skills of CI children evaluated by FAPCI are falling behind normatives for normal hearing children 18.6 months on the average. Hearing age, duration and intensity of rehabilitation have positive predictive value for communication skills development. Later identification of hearing loss and later cochlear implantation lead to delayed development of communication skills.

Fourier transform infrared spectroscopy provides an evidence of papain denaturation and aggregation during cold storage.

Papain is a cysteine protease with wide substrate specificity and many applications. Despite its widespread applications, cold stability of papain has never been studied. Here, we used differential spectroscopy to monitor thermal denaturation process. Papain was the most stabile from 45 °C to 60 °C with ΔG°321 of 13.9±0.3 kJ/mol and Tm value of 84±1 °C. After cold storage, papain lost parts of its native secondary structures elements which gave an increase of 40% of intermolecular ß-sheet content (band maximum detected at frequency of 1621 cm(-1) in Fourier transform infrared (FT-IR) spectrum) indicating the presence of secondary structures necessary for aggregation. The presence of protein aggregates after cold storage was also proven by analytical size exclusion chromatography. After six freeze-thaw cycles around 75% of starting enzyme activity of papain was lost due to cold denaturation and aggregation of unfolded protein. Autoproteolysis of papain did not cause significant loss of the protein activity. Upon the cold storage, papain underwent structural rearrangements and aggregation that correspond to other cold denatured proteins, rather than autoproteolysis which could have the commercial importance for the growing polypeptide based industry.

Poly(urethane-dimethylsiloxane) copolymers displaying a range of soft segment contents, noncytotoxic chemistry, and nonadherent properties toward endothelial cells.

Polyurethane copolymers based on α,ω-dihydroxypropyl poly(dimethylsiloxane) (PDMS) with a range of soft segment contents were prepared by two-stage polymerization, and their microstructures, thermal, thermomechanical, and surface properties, as well as in vitro hemo- and cytocompatibility were evaluated. All utilized characterization methods confirmed the existence of moderately microphase separated structures with the appearance of some microphase mixing between segments as the PDMS (i.e., soft segment) content increased. Copolymers showed higher crystallinity, storage moduli, surface roughness, and surface free energy, but less hydrophobicity with decreasing PDMS content. Biocompatibility of copolymers was evaluated using an endothelial EA.hy926 cell line by direct contact, an extraction method and after pretreatment of copolymers with multicomponent protein mixture, as well as by a competitive protein adsorption assay. Copolymers showed no toxic effect to endothelial cells and all copolymers, except that with the lowest PDMS content, exhibited resistance to endothelial cell adhesion, suggesting their unsuitability for long-term biomedical devices which particularly require re-endothelialization. All copolymers exhibited excellent resistance to fibrinogen adsorption and adsorbed more albumin than fibrinogen in the competitive adsorption assay, suggesting their good hemocompatibility. The noncytotoxic chemistry of these synthesized materials, combined with their nonadherent properties which are inhospitable to cell attachment and growth, underlie the need for further investigations to clarify their potential for use in short-term biomedical devices.

Can mild bilateral sensorineural hearing loss affect developmental abilities in younger school-age children?

The research study was conducted for the purpose of examining the influence of mild bilateral sensorineural hearing loss (MBSNHL) on developmental abilities of younger school-age children. The sample encompassed 144 children with MBSNHL, aged 7.5-11 (M = 8.85). MBSNHL (20-40 dB HL) was identified by pure tone audiometry. The control group encompassed 160 children with normal hearing. The Acadia test of developmental abilities was used for assessment of developmental abilities. Although statistically significant differences between participants with MBSNHL and those with normal hearing were established in the majority of estimated developmental abilities domains, those differences do not indicate any significant delay in development of assessed abilities, except in the domain of auditory discrimination. The obtained results call for a systematic approach to children with MBSNHL in elementary schools.

Active actinidin retains function upon gastro-intestinal digestion and is more thermostable than the E-64-inhibited counterpart.

BACKGROUND: Actinidin is a cysteine protease and major allergen from kiwi fruit. When purified under specific native conditions, actinidin preparations from fresh kiwi fruit contain both an active and inactive form of this enzyme. In this study, biochemical and immunological properties upon simulated gastro-intestinal digestion, as well as thermal stability, were investigated for both active and E-64-inhibited actinidin. RESULTS: Active actinidin retained its primary structure and proteolytic activity after 2 h of simulated gastric digestion, followed by 2 h of intestinal digestion, as assessed by SDS-PAGE, zymography and mass spectroscopy. Immunological reactivity of active actinidin was also preserved, as tested by immunoelectrophoresis. The E-64 inhibited actinidin was fully degraded after 1 h of pepsin treatment. Differential scanning calorimetry showed that active actinidin has one transition maximum temperature (Tm ) at 73.9°C, whereas in the E-64-actinidin complex the two actinidin domains unfolded independently, with the first domain having a Tm value of only 61°C. CONCLUSION: Active actinidin is capable of reaching the intestinal mucosa in a proteolytically active and immunogenic state. Inhibitor binding induces changes in the actinidin molecule that go beyond inhibition of proteolytic activity, also influencing the digestion stability and Tm values of actinidin, features important in the characterisation of food allergens.

Structure and properties of thermoplastic polyurethanes based on poly(dimethylsiloxane): assessment of biocompatibility.

Properties and biocompatibility of a series of thermoplastic poly(urethane-siloxane)s (TPUSs) based on α,ω-dihydroxy ethoxy propyl poly(dimethylsiloxane) (PDMS) for potential biomedical application were studied. Thin films of TPUSs with a different PDMS soft segment content were characterized by (1) H NMR, quantitative (13) C NMR, Fourier transform infrared spectroscopy (FTIR), atomic force microscopy (AFM), differential scanning calorimetry (DSC), dynamic mechanical analysis (DMA), contact angle, and water absorption measurements. Different techniques (FTIR, AFM, and DMA) showed that decrease of PDMS content promotes microphase separation in TPUSs. Samples with a higher PDMS content have more hydrophobic surface and better waterproof performances, but lower degree of crystallinity. Biocompatibility of TPUSs was examined after attachment of endothelial cells to the untreated copolymer surface or surface pretreated with multicomponent protein mixture, and by using competitive protein adsorption assay. TPUSs did not exhibit any cytotoxicity toward endothelial cells, as measured by lactate dehydrogenase and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide assays. The untreated and proteins preadsorbed TPUS samples favored endothelial cells adhesion and growth, indicating good biocompatibility. All TPUSs adsorbed more albumin than fibrinogen in competitive protein adsorption experiment, which is feature regarded as beneficial for biocompatibility. The results indicate that TPUSs have good surface, thermo-mechanical, and biocompatible properties, which can be tailored for biomedical application requirements by adequate selection of the soft/hard segments ratio of the copolymers.

Continuous glucose monitoring in the treatment of obesity in patients with glycogen storage disease type Ia.

UNLABELLED: Glycogen storage disease (GSD) type I is characterized by impaired production of glucose from glycogenolysis and gluconeogenesis resulting in severe hypoglycaemia and increased production of lactic acid, triglyceride and uric acid. The most common type, glycogenosis type Ia, demands a balanced, sufficient carbohydrate intake to preserve normal 24-h glycaemia. Insufficient intake of carbohydrates can cause hypoglycaemia, as the missing glucose-6-phosphatase enzyme cannot free the glucose stored as liver glycogen and nor is gluconeogenesis possible. The principle means of handling this disorder is to avoid starving by taking regular meals during the day and night. Such a dietary regimen could lead to obesity. Herein, we present the case of an adult patient with glycogenosis type Ia suffering from hyperuricaemia, dyslipidaemia and arterial hypertension. The accumulation of these cardiovascular risk factors could lead to the early onset of atherosclerosis, which should be postponed by contemporary methods of surveillance and treatment. LEARNING POINTS: Continuous subcutaneous glucose monitoring may be of value in every adult patient with GSD type I to evaluate the actual prevalence of eventual hypoglycaemic and hyperglycaemic episodes.Good dietary management minimizes the metabolic abnormalities of the disease and decreases the risk of long-term complications.Treatment of obesity in patients with GSD reduces the risk of earlier atherosclerosis and cardiovascular disease.

Cochlear implant--speech and language development in deaf and hard of hearing children following implantation.

BACKGROUND/AIM: Almost 200 cochlear implantations were done in the four centers (two in Belgrade, per one in Novi Sad and Nis) in Serbia from 2002 to 2009. Less than 10% of implantees were postlingually deaf adults. The vast majority, i.e. 90% were pre- and perilingually profoundly deaf children. The aim of this study was to assess the influence of improved auditory perception due to cochlear implantation on comprehension of abstract words in children as compared with hearing impaired children with conventional hearing aids and normal hearing children. METHODS: Thirty children were enrolled in this study: 20 hearing impaired and 10 normal hearing. The vocabulary test was used. RESULTS: The overall results for the whole test (100 words) showed a significant difference in favor of the normal hearing as compared with hearing impaired children. The normal hearing children successfully described or defined 77.93% of a total of 100 words. Success rate for the cochlear implanted children was 26.87% and for the hearing impaired children with conventional hearing aids 20.32%. CONCLUSION: Testing for abstract words showed a statistically significant difference between the cochlear implanted and the hearing impaired children with hearing aids (Mann-Whitney U-test, p = 0.019) implying considerable advantage of cochlear implants over hearing aids regarding successful speech development in prelingually deaf children.

Impact of a guide dog on glycemia regulation in blind/visually impaired persons due to diabetes mellitus.

The aim was to assess glycemia regulation in a blind diabetic patient after getting a guide dog. Glycosylated hemoglobin (HbA1c) results of a blind patient before and after getting the guide dog were retrospectively collected. The paired t-test results yielded a two-tailed P value of 0.0925, a difference considered not statistically significant; the 95% confidence interval of this difference varied from -0.2494 to 1.889. An improvement of glycemia regulation was observed with the guide dog compared to previous glycemia regulation, however, the difference was not statistically significant. The moderate improvement could probably be attributed to the mobility of the blind person having a guide dog. Standard quality of life tests should be included in the evaluation of diabetic blind persons, especially the impact of a guide dog on glycemic control or other chronic complications of diabetes.

The impact of hormone replacement therapy on metabolic syndrome components in perimenopausal women.

Hormone replacement therapy (HRT) is in use for more than a half of century, but the question of indications and ideal candidates for HRT remains unclear. Postmenopausal women are a population with the increasing risks for cardiovascular diseases which are the main cause of death in this group. Decline in oestrogen concentrations is linked to a number of changes in peri and postmenopause: increased total cholesterol, triglycerides, and low density lipoprotein, increased insulin resistance and impaired fibrinolysis. These changes are the main components of metabolic syndrome, the epidemic of the modern age. HRT is currently recommended as the gold standard for the management of vasomotor symptoms, but the benefit of HRT on components of metabolic syndrome and risk for cardiovascular events is still uncertain. In the initial reports from the Women's Health Initiative trial (WHI), overall health risks exceeded benefits in the cohort taking combined estrogen-progestin trial. Observational study in postmenopausal women, some meta-analyses and subsequent analyses of WHI had suggested that the timing of exposure to postmenopausal estrogen therapy may be an important factor in determining subsequent cardiovascular risk. It seems that the early therapy onset is a key factor in accomplishing positive results, but there are almost no surveys regarding the effects on the metabolic syndrome components in perimenopausal women. We hypothesized that the early, low dosage HRT in healthy perimenopausal women would have beneficial effects on the compounds of metabolic syndrome and could decrease the risk of cardiovascular events. Beneficial effect of HRT on cardiovascular diseases is due to the maintained integrity and functional status of the endothelium in younger women. We are aware of the fact that further studies are necessary to test the effects of different doses and routes of administration of HRT on cardiovascular outcomes.