Low-temperature immobilization of water in Antarctic Turgidosculum complicatulum and in Prasiola crispa. Part I. Turgidosculum complicatulum.

The studies of low-temperature immobilization of bound water in Antarctic lichenized fungus Turgidosculum complicatulum were performed using 1H NMR and DSC over a wide range of thallus hydration. 1H NMR free induction decays were decomposed into a solid component well described by the Gaussian function and two exponentially decaying components coming from a tightly bound water and from a loosely bound water fraction. 1H NMR spectra revealed one averaged mobile proton signal component. 1H NMR measurements recorded in time and in frequency domain suggest the non-cooperative bound water immobilization in T. complicatulum thallus. The threshold of the hydration level estimated by 1H NMR analysis at which the cooperative bound water freezing was detected was Δm/m0 ≈ 0.39, whereas for DSC analysis was equal to Δm/m0 = 0.375. Main ice melting estimated from DSC measurements for zero hydration level of the sample starts at tm = -(19.29 ± 1.19)°C. However, DSC melting peak shows a composed form being a superposition of the main narrow peak (presumably melting of mycobiont areas) and a broad low-temperature shoulder (presumably melting of isolated photobiont cells). DSC traces recorded after two-hour incubation of T. complicatulum thallus at -20 °C suggest much lower threshold level of hydration at which the ice formation occurs (Δm/m0 = 0.0842). Presumably it is a result of diffusion induced migration of separated water molecules to ice microcrystallites already present in thallus, but still beyond the calorimeter resolution.

Extreme dehydration observed in Antarctic Turgidosculum complicatulum and in Prasiola crispa.

Gaseous phase hydration effect of extremely dehydrated thallus of the Antarctic lichenized fungus Turgidosculum complicatulum and of green alga Prasiola crispa was observed using hydration kinetics, sorption isotherm, 1H-NMR spectroscopy and relaxometry. Three bound water fractions were distinguished: (1) very tightly bound water, (2) tightly bound water and (3) a loosely bound water fraction detected at higher levels of hydration. Sorption isotherm was sigmoidal in form and well fitted using Dent model. The relative mass of water saturating primary water binding sites was ΔM/m 0 = 0.055 for T. complicatulum and ΔM/m 0 = 0.131 for P. crispa. 1H-NMR free induction decays (FIDs) for T. complicatulum and for P. crispa were superpositions of a solid signal component, and one averaged liquid signal component for P. crispa thallus ([Formula: see text] ≈ 80 µs) or two liquid signal components coming from a tightly bound ([Formula: see text]≈ 71 µs) and from a loosely bound water fraction ([Formula: see text]≈ 278 µs) for T. complicatulum. 1H-NMR spectra recorded for T. complicatulum and for P. crispa thalli revealed one averaged mobile proton signal component L. The total liquid signal component expressed in units of solid (L 1 + L 2)/S suggests the presence of water soluble fraction in T. complicatulum thallus.

Assessment of oxidative/nitrative modifications of plasma proteins, selected ROTEM parameters and kinetics of fibrinogen polymerization in patients with multiple myeloma at diagnosis.

Patients with multiple myeloma (MM) are at increased risk of thrombosis. Growing evidence indicates that oxidative and nitrative modifications of proteins, including fibrinogen, may lead to changes in hemostasis. The study compares samples from patients with MM at diagnosis and healthy volunteers with regard to the oxidative/nitrative modifications of proteins, ROTEM and thrombin-catalyzed fibrin polymerization. The content of carbonyl groups in plasma proteins of patients with MM was significantly higher than in controls (2.981 vs. 1.807 nmol/mg of protein, p = 0.005), while no differences were seen in the concentrations of nitrated proteins. Maximum clot firmness readings were significantly higher in the samples of patients than in controls according to FIBTEM test (23.5 vs. 15 mm, p = 0.006). The lag time of the fibrin polymerization process and the velocity of clot lysis (V Lys) were found to be significantly higher in the group of MM patients than controls. In contrast, no marked differences were identified between studied groups in reference to maximal velocity of fibrin polymerization process (V max), maximal absorbance (A max) and plasmin amidolytic activity values. In conclusion, our study demonstrates that at the time of diagnosis, patients with MM demonstrated greater oxidative stress than healthy volunteers, which is reflected in a higher amount of carbonylated proteins. Some prothrombotic features found in ROTEM tests in MM patients were not confirmed by turbidimetry.

Regioselective hydroxylation of cholecalciferol, cholesterol and other sterol derivatives by steroid C25 dehydrogenase.

Steroid C25 dehydrogenase (S25DH) from Sterolibacterium denitrificans Chol-1S is a molybdenum oxidoreductase belonging to the so-called ethylbenzene dehydrogenase (EBDH)-like subclass of DMSO reductases capable of the regioselective hydroxylation of cholesterol or cholecalciferol to 25-hydroxy products. Both products are important biologically active molecules: 25-hydroxycholesterol is responsible for a complex regulatory function in the immunological system, while 25-hydroxycholecalciferol (calcifediol) is the activated form of vitamin D3 used in the treatment of rickets and other calcium disorders. Studies revealed that the optimal enzymatic synthesis proceeds in fed-batch reactors under anaerobic conditions, with 6-9 % (w/v) 2-hydroxypropyl-ß-cyclodextrin as a solubilizer and 1.25-5 % (v/v) 2-methoxyethanol as an organic co-solvent, both adjusted to the substrate type, and 8-15 mM K3[Fe(CN)6] as an electron acceptor. Such thorough optimization of the reaction conditions resulted in high product concentrations: 0.8 g/L for 25-hydroxycholesterol, 1.4 g/L for calcifediol and 2.2 g/L for 25-hydroxy-3-ketosterols. Although the purification protocol yields approximately 2.3 mg of pure S25DH from 30 g of wet cell mass (specific activity of 14 nmol min-1 mg-1), the non-purified crude extract or enzyme preparation can be readily used for the regioselective hydroxylation of both cholesterol and cholecalciferol. On the other hand, pure S25DH can be efficiently immobilized either on powder or a monolithic silica support functionalized with an organic linker providing NH2 groups for enzyme covalent binding. Although such immobilization reduced the enzyme initial activity more than twofold it extended S25DH catalytic lifetime under working conditions at least 3.5 times.

A method of water-soluble solid fraction saturation concentration evaluation in dry thalli of Antarctic lichenized fungi, in vivo.

BACKGROUND: At initial steps of rehydration from cryptobiosis of anhydrobiotic organisms or at rehydration of dry tissues the liquid 1H NMR signal increased anomaly. The surplus in liquid signal may appear if some solid constituents dissolved, or if they were decomposed by enzymatic action. METHODS: Hydration kinetics, sorption isotherm, 1H NMR spectra and high power relaxometry were applied to monitor gaseous phase rehydration of Antarctic lichen Cetraria aculeata. Tightly and loosely bound water signal were distinguished, and the upper hydration limit for dissolution of water soluble solid fraction was not observed. A simple theoretical model was proposed. RESULTS: The hydration courses showed a very tightly bound water fraction, a tightly bound water, and a loosely bound water fraction. Sigmoidal in form sorption isotherm was fitted well by multilayer sorption model. 1H NMR showed one Gaussian signal component from solid matrix of thallus and one or two Lorentzian line components from tightly bound, and from loosely bound water. The hydration dependency of liquid signal was fitted by rational function. CONCLUSIONS: Although in dehydrated C.aculeata the level of carbohydrates and polyols was low, the lichenase action during rehydration process increased it; the averaged saturation concentration cs =(57.3±12.0)%, which resembled that for sucrose. GENERAL SIGNIFICANCE: The proposed method of water soluble solid fraction saturation concentration, cs , calculation from 1H NMR data may be applied for other organisms experiencing extreme dehydration or for dry tissues. We recalculated the published elsewhere data for horse chestnut (Aesculus hippocastanum) bast [water-soluble solid fraction recognized as sucrose, cs =(74.5±5.1)%]; and for Usnea antarctica, where cs =0.81±0.04.

Antioxidant action of six Trifolium species in blood platelet experimental system in vitro.

This study includes a comparative evaluation of antioxidant effects of plant extracts (1.5-50.0 µg/ml), derived from six clover (Trifolium) species: T. alexandrinum L., T. fragiferum L., T. hybridum L., T. incarnatum L., T. resupinatum var. majus Boiss., and T. resupinatum var. resupinatum L. Chemical profiles of the extracts contained three or four groups of (poly)phenolic compounds such as phenolic acids, clovamides, isoflavones, and other flavonoids. Antioxidant properties of Trifolium extracts were assessed as the efficacy to reduce oxidative and nitrative damage to blood platelets, exposed to 100 µM peroxynitrite-induced oxidative stress in vitro. Antioxidant actions of the examined extracts were determined by the following biomarkers of oxidative stress: thiol groups, 3-nitrotyrosine, lipid hydroperoxides, and thiobarbituric acid-reactive substances (TBARS). Despite the significant differences in the chemical composition (the total phenolic concentrations varied between 11.30 and 52.55 mg/g of dry mass) of Trifolium extracts, we observed noticeable protective effects of almost all tested plant preparations. The T. alexandrinum extract, containing the highest concentration of phenols, was the most effective antioxidant among the tested extracts. On the other hand, the T. incarnatum extract, which contained a comparable total phenolic content (49.77 mg/g), was less efficient in prevention of tyrosine nitration and generation of TBARS. These findings indicate on the important role of individual phenolic components of the examined clover extracts for the final antioxidative effects. Antioxidative properties of the remaining extracts were noticeably weaker.

Increased serum levels of lipopolysaccharide and antiflagellin antibodies in patients with diarrhea-predominant irritable bowel syndrome.

BACKGROUND: Innate immune responses to conserved microbial products such as lipopolysaccharide (LPS) and flagellin are likely important in microbial-host interactions and intestinal homeostasis. We hypothesized that bacterial translocation and activation of mucosal immunity against common microbial antigens might be involved in the development of irritable bowel syndrome (IBS). We therefore compared serum levels of LPS, soluble CD14 (sCD14), and flagellin antibodies between patients with different subtypes of IBS and healthy controls. METHODS: We analyzed serum obtained from 88 patients (74 females) aged 19(43)-73 years and 106 healthy volunteers (77 females) aged 19(38)-62 years. Diarrhea-predominant IBS (D-IBS) was present in 32 patients (36%), 23 patients (26%) had constipation-predominant IBS (C-IBS), and 33 patients (38%) had A-IBS. We used ELISA for sCD14 and antiflagellin immunoglobulin G and limulus amebocyte assay for LPS. Abdominal symptoms and psychiatric comorbidities were assessed using validated questionnaires. KEY RESULTS: We found a significantly higher serum level of LPS in patients with D-IBS compared to controls (p = 0.0155). The level of antibodies to flagellin was higher in patients with IBS than in controls (mainly driven by higher levels in D-IBS, p = 0.0018). The levels of sCD14 were lower in D-IBS patients compared to controls (p = 0.0498). We found a weak, but significant correlation between the levels of antiflagellin antibodies and anxiety among IBS patients (ρ = 0.38; p = 0.0045). CONCLUSIONS & INFERENCES: Our results support the concept that immune reactivity to luminal antigens may have a role in the development of D-IBS. The serum level of antiflagellin antibodies was found to correlate with patients' self-reported anxiety score.

Towards multifunctional MOFs--transforming a side reaction into a post-synthetic protection/deprotection method.

The contaminant commonly found in the important amino-substituted metal-organic framework UiO-66-NH2 has been shown to arise from partial formylation during the synthesis in DMF. Mild conditions have now been developed for both post-synthetic deformylation and near-complete formylation, offering a new post-synthetic protection-deprotection method for the synthesis of multifunctional MOFs.

Epidemiology of Clostridium difficile infection: results of a hospital-based study in Krakow, Poland.

Over the past two decades Clostridium difficile infection (CDI) has appeared as a major public health threat. We performed a retrospective study based on the records of patients hospitalized for CDI at the University Hospital in Krakow, Poland, between 2008 and 2014. In the study period, CDI occurred in 1009 individuals. There were 790 (78%) individuals who developed infection only once, whereas 219 (22%) developed infection more than once. The percentage of deaths within 14 days of CDI confirmation was 2·4%, with a mean age of 74·2 ± 15·9 years. Crude mortality was 12·9% in medical wards, 5·6% for surgical wards and 27·7% in the ICU setting. The time span between diagnosis and death was 5·1 days on average. Between 2008 and 2012 a 6·5-fold increase of CDI frequency with a posterior stabilization and even reduction in 2013 and 2014 was observed. According to the data analysed, 2/3 patients in our population developed CDI during their hospitalization even though they were admitted for different reasons. Medical wards pose a significantly higher risk of CDI than the surgical ones. Age is a risk factor for CDI recurrence. In the case of patients who died, death occurred shortly after diagnosis. The first CDI episode poses much higher risk of mortality than the consecutive ones.

Suitability of the hydrocarbon-hydroxylating molybdenum-enzyme ethylbenzene dehydrogenase for industrial chiral alcohol production.

The molybdenum/iron-sulfur/heme protein ethylbenzene dehydrogenase (EbDH) was successfully applied to catalyze enantiospecific hydroxylation of alkylaromatic and alkylheterocyclic compounds. The optimization of the synthetic procedure involves use of the enzyme in a crude purification state that saves significant preparation effort and is more stable than purified EbDH without exhibiting unwanted side reactions. Moreover, immobilization of the enzyme on a crystalline cellulose support and changes in reaction conditions were introduced in order to increase the amounts of product formed (anaerobic atmosphere, electrochemical electron acceptor recycling or utilization of ferricyanide as alternative electron acceptor in high concentrations). We report here on an extension of effective enzyme activity from 4h to more than 10 days and final product yields of up to 0.4-0.5g/l, which represent a decent starting point for further optimization. Therefore, we expect that the hydrocarbon-hydroxylation capabilities of EbDH may be developed into a new process of industrial production of chiral alcohols.

The Oblazowa 1 early modern human pollical phalanx and Late Pleistocene distal thumb proportions.

The human distal thumb phalanx from the earlier Upper Paleolithic of Oblazowa Cave, southern Poland, exhibits features of its palmar surface that align it morphologically principally with early modern humans. These aspects include the configurations of the proximal palmar fossa, the flexor pollicis longus tendon insertion, the proximal margin of the palmar apical tuft, and especially its low ulnar deviation angle. If it is assumed that it possessed the pollical phalangeal length proportions of an early modern human, it would exhibit modest base and tuft breadths. However, given Late Pleistocene archaic-modern contrasts in relative pollical phalanx lengths, the isolated nature of the phalanx prevents secure assessment of its radioulnar interphalangeal articular and apicaltuft hypertrophy. Similar constraints apply to the assessment of other Pleistocene Homo pollical phalanges.

Thrombin inhibitory activity of some polyphenolic compounds.

Thrombin, also known as an active plasma coagulation factor II, belongs to the family of serine proteases and plays a crucial role in blood coagulation process. The process of thrombin generation is the central event of the hemostatic process and regulates blood coagulant activity. For this reason, thrombin inhibition is key to successful novel antithrombotic pharmacotherapy. The aim of our present study was to examine the effects of the well-known polyphenolic compounds on the activity of thrombin, by characterization of its interaction with selected polyphenols using different biochemical methods and biosensor BIAcore analyses. Only six compounds, cyanidin, quercetin, silybin, cyanin, (+)-catechin and (-)-epicatechin, of all examined in this study polyphenols caused the inhibition of thrombin amidolytic activity. But only three of the six compounds (cyanidin, quercetin and silybin) changed thrombin proteolytic activity. BIAcore analyses demonstrated that cyanidin and quercetin caused a strong response in the interaction with immobilized thrombin, while cyanin and (-)-epicatechin induced a low response. Lineweaver-Burk curves show that used polyphenol aglycones act as competitive thrombin inhibitors. Our results suggest that polyphenolic compounds might be potential structural bases and source to find and project nature-based, safe, orally bioavailable direct thrombin inhibitors.

Markers of microbial translocation predict hypertension in HIV-infected individuals.

OBJECTIVES: The aim of the study was to test the hypothesis that microbial translocation, quantified by levels of lipopolysaccharide (LPS) and subsequent monocyte activation [soluble (sCD14)], is associated with hypertension in HIV-infected individuals. METHODS: In this exploratory substudy, 42 patients were recruited from a larger, longitudinal HIV-infected cohort study on blood pressure. LPS and sCD14 levels were measured retrospectively at the time of nadir CD4 cell count, selecting untreated HIV-infected patients with both advanced immunodeficiency and preserved immunocompetence at the time of nadir. Patients with later sustained hypertension (n = 16) or normotension (n = 26) throughout the study were identified. LPS was analysed using the Limulus Amebocyte Lysate colorimetric assay (Lonza, Walkersville, MD) and sCD14 using an enzyme-linked immunosorbent assay (ELISA). Nonparametric statistical tests were applied. RESULTS: In the HIV-infected patients [median (interquartile range) age 42 (32-46) years; 79% male and 81% Caucasian], LPS and sCD14 levels were both negatively correlated with nadir CD4 cell count. Plasma levels of LPS (P < 0.001) and sCD14 (P = 0.024) were elevated in patients with later hypertension compared with patients with normotension. There was a stepwise increase in the number of patients with hypertension across tertiles of LPS (P = 0.001) and sCD14 (P = 0.007). Both LPS and sCD14 were independent predictors of elevated blood pressure after adjustment for age and gender. For each 10-unit increase in LPS (range 66-272 pg/ml), the increment in mean blood pressure in the first period of blood pressure recording was 0.86 (95% confidence interval 0.31-1.41) mmHg (P = 0.003). CONCLUSIONS: As LPS and sCD14 were both independently associated with elevated blood pressure, microbial translocation may be linked to the development of hypertension.

Virolysis of feline calicivirus and human GII.4 norovirus following chlorine exposure under standardized light soil disinfection conditions.

The relationship between the infectivity of the feline calicivirus (FCV) vaccine strain F-9 and capsid destruction (virolysis) in response to available chlorine was investigated under standardized light soil disinfection conditions. Virolysis was measured using RNase pretreatment (in order to destroy exposed RNA following chlorine treatment) and quantitative reverse transcription PCR. A comparison between the results of plaque assays and virolysis following exposure of FCV F-9 grown in tissue culture to different concentrations of available chlorine showed a similar log-linear relationship, with >4-log reductions occurring at 48 and 66 ppm, respectively. Three non-epidemiologically linked human GII.4 noroviruses (NoVs) present in dilute clinical samples showed behavior similar to each other and were 10 times more resistant to virolysis than cultured FCV F-9. FCV F-9 when present in dilute human GII.4 samples acquired increased resistance to virolysis approaching that of human NoVs. This study represents a direct comparison between the virolysis of a surrogate virus (FCV F-9) and that of human GII.4 NoVs within the same matrix in response to available chlorine. The results support the view that matrix effects have a significant effect on virus survival.

Semi-automatic delineation using weighted CT-MRI registered images for radiotherapy of nasopharyngeal cancer.

PURPOSE: To develop a delineation tool that refines physician-drawn contours of the gross tumor volume (GTV) in nasopharynx cancer, using combined pixel value information from x-ray computed tomography (CT) and magnetic resonance imaging (MRI) during delineation. METHODS: Operator-guided delineation assisted by a so-called "snake" algorithm was applied on weighted CT-MRI registered images. The physician delineates a rough tumor contour that is continuously adjusted by the snake algorithm using the underlying image characteristics. The algorithm was evaluated on five nasopharyngeal cancer patients. Different linear weightings CT and MRI were tested as input for the snake algorithm and compared according to contrast and tumor to noise ratio (TNR). The semi-automatic delineation was compared with manual contouring by seven experienced radiation oncologists. RESULTS: A good compromise for TNR and contrast was obtained by weighing CT twice as strong as MRI. The new algorithm did not notably reduce interobserver variability, it did however, reduce the average delineation time by 6 min per case. CONCLUSIONS: The authors developed a user-driven tool for delineation and correction based a snake algorithm and registered weighted CT image and MRI. The algorithm adds morphological information from CT during the delineation on MRI and accelerates the delineation task.

Three-dimensional power Doppler placental vascularisation indices in early pregnancy: a pilot study.

The objective of our study was to assess 1st trimester placental vascularisation using three-dimensional (3D) power Doppler vascular indices. A cross-sectional study was used involving 41 normal pregnancies from 7 to 10 + 6 weeks. Placental volume was obtained using the 30° virtual organ computer-aided analysis (VOCAL) method. The mean, median, standard deviation (SD), minimum and maximum values were calculated for three vascular indices: the VI, vascularisation index; the FI, flow index and the VFI, vascularisation and flow index. Pearson's correlation coefficient (r) was used to assess the correlation between vascular indices and crown-rump length (CRL). Our results showed mean (± SD) values ranged from 8.66 ± 12.04 to 15.34 ± 13.89 for the VI, from 63.83 ± 43.61 to 109.22 ± 33.87 for the FI and from 9.52 ± 13.86 to 20.59 ± 22.97 for the VFI. There was no correlation between CRL and VI (r = 0.073, p = 0.630) nor VFI (r = 0.147 and p = 0.037); there was a weak correlation between CRL and FI (r = 0.332, p = 0.037). It was concluded that the FI was the only 3D power Doppler vascular index that was correlated with CRL between 7 and 10 + 6 weeks' gestation.

Cyanines as efficient photosensitizers in photodynamic reaction: photophysical properties and in vitro photodynamic activity.

The purpose of the present study was to explore the potential application of cyanines in photodynamic treatment. The photophysical features of four cyanines (KF570, HM118, FBF-749, and ER-139) were investigated by elemental and spectral analyses. Two malignant cell lines (MCF-7/WT and MCF-7/DOX) were used to test the potential for use in the photodynamic therapy. The cytotoxic effects of these dyes were determined by the MTT assay after 4 and 24 h of incubation with the cyanine. KF570 and HM118 were irradiated with red light (630-nm filter) and FBF-749 and ER-139 with green light (435-nm filter). The results showed that the cyanine HM118 demonstrated a major phototoxic effect. It was also noted that the efficiency of photodynamic therapy was higher in the doxorubicin-resistant cell line (MCF-7/DOX).

Measurement of the virolysis of human GII.4 norovirus in response to disinfectants and sanitisers.

The aim of this study was to develop a method for investigating the stability of the human NoV capsid in response to disinfectants and sanitisers (virucides) as an indirect method for determining virus infectivity. Capsid destruction or "virolysis" was measured using the reverse transcribed quantitative PCR (RT-QPCR) reaction in conjunction with RNase treatment (in order to destroy any exposed RNA). Two commercially available alcohol based handwashes, alcohols (75% (v/v) ethanol or isopropanol), quaternary ammonium compounds (0.14% BAC or 0.07% DIDAC), and chlorine dioxide (200 ppm) were all ineffective at promoting virolysis of human norovirus present in dilute clinical samples at the concentrations tested. GII.4 NoVs were sensitive to a combination of heat and alkali. These data show that NoVs present in dilute stool samples are resistant to virolysis using virucides that are used commonly.

Intracranial radiosurgery in the Netherlands. A planning comparison of available systems with regard to physical aspects and workload.

Different planning and treatment systems for intracranial stereotactic radiosurgery available in the Netherlands are compared. The systems for intracranial radiosurgery include: Gamma Knife, Cyberknife, Novalis, and Tomotherapy. Electronic data of 5 patients was transferred to all participating centres and treatment plans were generated according to 2 different prescription protocols. For this study, plans were also generated for a conventional linac. Even systems with a high resolution (Gammaknife and Novalis) have conformity indices in violation with RTOG guidelines (CI > 2.5) when target volumes of <0.5 cc are treated. For medium sized targets (0.5-1 cc) all systems performed reasonably well, but for the different systems a large range of conformity indices was seen (1.1 to 3.7). The differences are partly system dependent but depend also on specific planning choices made. For larger target volumes (> 1 cc), all systems perform well. The workload of the different techniques was comparable although the treatment times were usually longer for Gamma Knife radiosurgery. We conclude that small targets should be treated by dedicated systems, larger volumes (> 0.5-1 cc) can also be treated using conventional treatment systems equipped with a MLC.