Separation of 1–23-kb complementary DNA strands by urea–agarose gel electrophoresis

Double-stranded (ds), as well as denatured, single-stranded (ss) DNA samples can be analyzed on urea–agarose gels. Here we report that after denaturation by heat in the presence of 8 M urea, the two strands of the same ds DNA fragment of ∼1–20-kb size migrate differently in 1 M urea containing agarose gels. The two strands are readily distinguished on Southern blots by ss-specific probes. The different migration of the two strands could be attributed to their different, base composition-dependent conformation impinging on the electrophoretic mobility of the ss molecules. This phenomenon can be exploited for the efficient preparation of strand-specific probes and for the separation of the complementary DNA strands for subsequent analysis, offering a new tool for various cell biological research areas

Separation of aflatoxins by centrifugal partition chromatography

Aflatoxins are mycotoxins, produced by several species of filamentous fungi. For this group of toxins, there are very low, ppb-level limits in the EU in food and feed products, thus as reference standards relatively high amounts of pure aflatoxins are required. One of the promising methods for their purifications, the centrifugal partition chromatography could be used successfully. Herein this study, the development of a liquid-liquid chromatographic method for the separation of both the aflatoxins and the impurities was involved

Cellulase and hemicellulase enzymes as single molecular nanobiocomposites

We have worked out two different stabilization procedures for glycolytic enzymes with significant biotechnology relevance: for the commercial Novozyme cellulase complex (Celluclast) and for recombinant Thermobifida fusca hemicellulases (β-D-xylosidase and β-D-mannosidase). In the applied methods, individual cellulase and hemicellulase enzyme molecules were conjugated with a polymer nano-layer providing nanobiocomposites (single enzyme nanoparticles, SENs) that exihibit an excellent stability under extreme conditions. The first method that we have been successfully used earlier on chymotrypsin [3], creates a polymer layer around the enzyme molecules with trimethoxysilyl (TMS) functionalities in three steps [4]. The second stabilization method is a novel one pot reaction resulting in an acrylamide-bisacrylamide (AA) copolymer layer around the enzyme molecules in two steps. The heat stability of the developed single enzyme nanoparticles with TMS and AA nanolayers were tested at two different temperatures (+4 °C; 80 °C) following the residual activities of the modified enzymes in regular time interval. Upon incubation of the cellulase complex nanoparticles with TMS and the non-modified cellulase at 80 °C, the nanoparticle has kept its 40% of its starting cellulase activity after 6 hours, whereas the non-modified enzyme lost its activity completely in half an hour. The relative activity of SENs with AA is about 50% of the initial activity. After 12 hours under 80 °C the activity of NCK is reduced to 14%, while the activity of NCKA is 24% of the activity of the native enzyme at the start of the incubation. The nanoparticles of the Thermobifida fusca hemicellulases (β-D-xylosidase and β-D-mannosidase) obtained from both TMS and AA nanolayers have exhibited concomitant stability increase. TMS nanoparticles of β-xylosidase has lost 60% of its activity after 120 days during an incubation procedure at +4 °C, whereas the non-modified β-xylosidase lost all its activity in 40 days during the same conditions. The 40% of the starting enzyme activity of β-D mannosidase with AA has remained after 6 hours, during an incubation process at 80 °C, the activity of the non-modified enzyme has lost in one hour

Single Haemoglobin Nanocapsules as Test Materials for Artificial Blood

Single protein nanocapsules (SPNs) means that each individual protein molecules are coated with a very thin polymer layer. The polymer chains which are porous enough to allow enzymatic functions are bound covalently to the protein molecule. According to our previous results the polymer layer can essentially stabilize different types of enzymes, e.g. its stability became to 50-70 times longer than that of the native ones. The heat stability (at 80 °C SPNs has activity after 24 hours) and the pH-stability (from pH = 1.5 to pH = 12.0) of the covered enzyme can essentially be improved comparing to the native enzymes. Our results show, that SPNs have a good features as drug carriers: acrylamide-bisacrylamide copolymer layer can carries bovine serum albumin molecules across the blood brain barrier in rat brain. We synthesized single haemoglobin nanocapsules with acrylamide-bisacrylamide copolymer on the surface of the molecules (PAAHgB) and their size, homogeneity, aggregation status, zeta potential were investigated compared with other nanomaterials

Use of thyroid hormones in hypothyroid and euthyroid patients: a THESIS* questionnaire survey of Polish physicians. *THESIS: Treatment of hypothyroidism in Europe by specialists: an international survey

Introduction: Over the past several years new evidence on the management of hypothyroidism has emerged, which has influenced recommendations from professional bodies. The presentation of hypothyroid patients has also changed, and new cases are increasingly diagnosed by indiscriminate screening, often identifying cases with minor biochemical disturbances. Little is known about the physician responses and attitudes to this changing landscape. THESIS (Treatment of Hypothyroidism in Europe by Specialists: an International Survey) is a large-scale survey of European physicians who treat patients with hypothyroidism. Here we document current practices of Polish physicians relating to the use of thyroid hormones in hypothyroid and euthyroid patients.  Material and methods: Members of the Polish Society of Endocrinology were invited to participate in the web-based THESIS survey. Results: In total 423 (54.6% of the 774 invited) physicians completed the survey. The majority of respondents (74.2%) would prescribe thyroid hormones for euthyroid patients for certain indications, such as female infertility with elevated thyroid antibodies (63.4%), simple goitre (40.9%), unexplained fatigue (12.1%), obesity (9.7%), hypercholesterolaemia (9.0%), and depression (9.2%). Nearly all physicians (96.0%) declared that the treatment of choice for hypothyroidism is levothyroxine (LT4). However, around one-third (30.3%) were also using LT4 and liothyronine (LT3) combination treatment; LT3 alone was rarely prescribed (1.7%), and none prescribed desiccated thyroid extract. The majority of respondents preferred LT4 tablets. Among alternative formulations, liquid LT4 was most commonly recommended for patients unable to take LT4 in the fasting state (26.0%) and patients with malabsorption (19.9%). Respondents considered prescribing dietary supplements (such as selenium and iodine) in hypothyroid patients with coexisting autoimmune thyroiditis (29.6%) or at the patients’ request (32.2%). LT4 + LT3 combination therapy was used by 32.2% when symptoms persisted notwithstanding normal serum TSH concentration. Psychosocial factors, comorbidities, and the burden of chronic disease were considered as the most likely causes of persistent symptoms. Conclusions: Apart from clinical practice recommendations, other factors influence the thyroid hormone therapy patterns. Moreover, certain areas of clinical practice were identified (the use of thyroid hormones in euthyroid subjects and the use of dietary supplements), which are not in accordance with the current evidence.

Use of thyroid hormones in hypothyroid and euthyroid patients: a THESIS* survey of Belgian specialists *THESIS: treatment of hypothyroidism in Europe by specialists: an international survey.

[en] BACKGROUND: Hypothyroidism is a topic that continues to provoke debate and controversy with regards to specific indications, type of thyroid hormone substitution and efficacy. We investigated the use of thyroid hormones in clinical practice in Belgium, a country where currently only levothyroxine (LT4) tablet formulations are available. METHOD: Members of the Belgian Endocrine Society were invited to respond to an online questionnaire. Results were compared with those from other THESIS surveys. RESULTS: Eighty (50%) of the invited 160 individuals, completed the questionnaire. LT4 was the first treatment of choice for all respondents. As secondary choice, some also prescribed liothyronine (LT3) and LT4 + LT3 combinations (2 and 7 respondents, respectively). Besides hypothyroidism, 34 and 50% of respondents used thyroid hormones for infertile euthyroid TPOAb positive women and the treatment of a growing non-toxic goiter, respectively. Had alternative formulations of LT4 to tablets been available (soft gel or liquid L-T4), 2 out of 80 (2.5%) participants would consider them for patients achieving biochemical euthyroidism but remaining symptomatic. This proportion was higher in case of unexplained poor biochemical control of hypothyroidism (13.5%) and in patients with celiac disease or malabsorption or interfering drugs (10%). In symptomatic euthyroid patients, 20% of respondents would try combined LT4 + LT3 treatment. Psychosocial factors were highlighted as the main contributors to persistent symptoms. CONCLUSIONS: LT4 tablets is the preferred treatment for hypothyroidism in Belgium. A minority of the respondents would try combined LT4 + LT3 in symptomatic but biochemically euthyroid patients. Thyroid hormones are prescribed for euthyroid infertile women with thyroid autoimmunity and patients with non-toxic goiter, a tendency noted in other European countries, despite current evidence of lack of benefit

Crustal structure of the carpathian-pannonian region from ambient noise tomography

We use ambient noise tomography to investigate the crust and uppermost mantle structure beneath the Carpathian-Pannonian region of Central Europe. Over 7500 Rayleigh wave empirical Green's functions are derived from interstation cross-correlations of vertical component ambient seismic noise recordings (2005-2011) using a temporary network of 54 stations deployed during the South Carpathian Project (2009-2011), 56 temporary stations deployed in the Carpathian Basins Project (2005-2007) and 100 permanent and regional broad-band stations. Rayleigh wave group velocity dispersion curves (4-40 s) are determined using the multiple-filter analysis technique. Group velocity maps are computed on a grid of 0.2° × 0.2° from a non-linear 2-D tomographic inversion using the subspace method. We then inverted the group velocity maps for the 3-D shear wave velocity structure of the crust and uppermost mantle beneath the region. Our shear wave velocity model provides a uniquely complete and relatively high-resolution view of the crustal structure in the Carpathian-Pannonian region, which in general is validated by comparison with previous studies using other methods to probe the crustal structure. At shallow depths (30 km are relatively fast, presumably related to shallowing of the Moho consequent on the extensional history of the Pannonian region

Characteristics of specialists treating hypothyroid patients:the “THESIS” collaborative

Introduction: Thyroid specialists influence how hypothyroid patients are treated, including patients managed in primary care. Given that physician characteristics influence patient care, this study aimed to explore thyroid specialist profiles and associations with geo-economic factors. Methods: Thyroid specialists from 28 countries were invited to respond to a questionnaire, Treatment of Hypothyroidism in Europe by Specialists: an International Survey (THESIS). Geographic regions were defined according to the United Nations Statistics Division. The national economic status was estimated using World Bank data on the gross national income per capita (GNI per capita). Results: 5,695 valid responses were received (response rate 33·0%). The mean age was 49 years, and 65·0% were female. The proportion of female respondents was lowest in Northern (45·6%) and highest in Eastern Europe (77·2%) (p &lt;0·001). Respondent work volume, university affiliation and private practice differed significantly between countries (p&lt;0·001). Age and GNI per capita were correlated inversely with the proportion of female respondents (p&lt;0·01). GNI per capita was inversely related to the proportion of respondents working exclusively in private practice (p&lt;0·011) and the proportion of respondents who treated &gt;100 patients annually (p&lt;0·01). Discussion: THESIS has demonstrated differences in characteristics of thyroid specialists at national and regional levels, strongly associated with GNI per capita. Hypothyroid patients in middle-income countries are more likely to encounter female thyroid specialists working in private practice, with a high workload, compared to high-income countries. Whether these differences influence the quality of care and patient satisfaction is unknown, but merits further study.</p