Decolorization of remazol brilliant blue R with laccase from Lentinus crinitus grown in agro-industrial by-products.

Lentinus crinitus is a white-rot fungus that produces laccase, an enzyme used for dye decolorization. Enzyme production depends on cultivation conditions, mainly agro-industrial by-products. We aimed to produce laccase from Lentinus crinitus with agro-industrial by-products for dye decolorization. Culture medium had coffee husk (CH) or citric pulp pellet (CP) and different nitrogen sources (urea, yeast extract, ammonium sulfate and sodium nitrate) at concentrations of 0, 0.7, 1.4, 2.8, 5.6 and 11.2 g/L. Enzymatic extract was used in the decolorization of remazol brilliant blue R. CH medium promoted greater laccase production than CP in all evaluated conditions. Urea provided the greatest laccase production for CH (37280 U/L) as well as for CP (34107 U/L). In CH medium, laccase activity was suppressed when carbon-to-nitrogen ratio changed from 4.5 to 1.56, but the other nitrogen concentrations did not affect laccase activity. For CP medium, reduction in carbon-to-nitrogen ratio from 6 to 1.76 increased laccase activity in 17%. The peak of laccase activity in CH medium occurred on the 11th day (41246 U/L) and in CP medium on the 12th day (32660 U/L). The maximum decolorization within 24 h was observed with CP enzymatic extract (74%) and with CH extract (76%).

Antimicrobial and Antioxidant Activities of the Extract and Fractions of Tetradenia riparia (Hochst.) Codd (Lamiaceae) Leaves from Brazil.

Tetradenia riparia (Lamiaceae) is native to Central Africa popularly known as myrrh, used in folk medicine to treat various diseases like malaria, gastroenteritis, and tropical skin disease. This research was to evaluate the antioxidant and antibacterial activities of the crude extract (CE) and fractions (FR) of the T. riparia by classical chromatography. The CE of T. riparia leaves was submitted to column chromatographic fractionation to obtain four fractions of the interest, which were identified by nuclear magnetic resonance and gas chromatograph coupled to mass spectrum: FR-I (abieta-7,9(11)-dien-13-ß-ol), FR-II (Ibozol), FR-III (8 (14), 15-sandaracopimaradiene-2α, 18-diol and 8 (14), 15-sandaracopimaradiene-7α, 18-diol), and FR-IV (Astragalin, Boronolide and Luteolin). Total phenol content of CE and FR were measured, and antioxidant action by methods of DPPH (2,2-diphenyl-1-picrylhydrazyl), ß-carotene/linoleic acid system, and ferric reducing/antioxidant power (FRAP) and the antibacterial activity was evaluated by the broth microdilution method with the determination of the minimum inhibitory concentration (MIC). The FR-IV presented antioxidant potential with 181.67 µg gallic acid/mg, IC50 of 0.61 µg/mL by DPPH method, 55.61% oxidation protection by ß-carotene/linoleic acid system and 4.59 µM ferrous sulfate/mg of sample by FRAP, and the FR-I showed higher antibacterial potential on the strain Staphylococcus aureus with MIC 0.98 µg/mL, Enterococcus faecalis and Bacillus cereus with MIC 31.2 µg/mL. Thus, the fractionation of CE was extremely important to detect fractions with potential activities, and investigations are necessary regarding the mechanism of action and action in vivo.

Pseudomonas putida Stimulates Primordia on Agaricus bitorquis.

Casing layer is one step of Agaricus bisporus cultivation where there is a competitive environment with a high number of microorganisms and diversity interacting with mycelia. It is suggested that a minimal community of these microorganisms would be necessary to stimulate fructification. However, A. bisporus is not able to produce primordia in sterile casing layers or Petri dishes. Thus, the objective of this study was to characterize bacterial microbiota of casing layers from A. bisporus cultivation, isolate, identify and characterize the bacteria responsible for the stimulation of primordium and their action mechanism using Agaricus bitorquis as a primordium stimulation model. Bacterial and Pseudomonas spp. communities of different casing layers of A. bisporus cultivation were collected and quantified. It was concluded that Pseudomonas spp. corresponds to 75-85% of bacterial population of the casing layers in A. bisporus cultivation and among those 12% are Pseudomonas putida. Four biochemical assays were used to identify P. putida. In vitro primordium stimulation of living P. putida and non-living bacterial suspensions, after chemical or physical treatments, was tested using A. bitorquis as a primordium stimulation model. Primordium stimulation assay was registered by photographs, and micrographs of vertical cut of primordium were registered by scanning electron microscope. Interaction of living P. putida with A. bitorquis mycelia is capable of stimulating primordial instead of non-living bacterial suspensions. Stimulation of A. bitorquis primordia does not imply or is related to mycelial growth inhibition, but a hierarchical relation of primordium succession and development is suggested.

Agaricus subrufescens: substratum nitrogen concentration and mycelial extraction method on antitumor activity.

Antitumor activity of Agaricus subrufescens has been shown on vegetative mycelium and basidiocarp. However, few studies have assessed the effect of A. subrufescens cultivation conditions and extraction methods on antitumor activity. This study evaluated the effect of nitrogen concentration on the cultivation medium of A. subrufescens and the extraction method of mycelial antineoplastic actives against sarcoma 180 cells implanted in mice. Two nitrogen sources (isolated soybean protein and NaNO3) and 10 nitrogen concentrations (0.25 to 8.0 g/L) were used. Dried mycelium extract was obtained by hot water infusion (1:10 mass:volume; 90 °C) or by aqueous mixture (1:10 mass:volume, ambient temperature) in ultrapure water. The doses were administered daily by gavage to mice implanted with sarcoma 180 cells. Isolated soy protein is more efficient to mycelial biomass production than NaNO3. The mycelial biomass production increases when the cultivation medium is added with high nitrogen concentrations as well as the splenic index and the antitumor activity of the moistened mycelial powder. Hot water extract is more effective than the moistened mycelial powder to reduce tumor. The antitumor activity of hot water mycelial extract is similar to the one of basidiocarps, presenting lower metabolic demand on the spleen, keeping blood parameters normal and promoting animal wellness.

Identification of 2127 new HLA class I alleles in potential stem cell donors from Germany, the United States and Poland.

We describe 2127 new human leukocyte antigen (HLA) class I alleles found in registered stem cell donors. These alleles represent 28.9% of the currently known class I alleles. Comparing new allele sequences to homologous sequences, we found 68.1% nonsynonymous nucleotide substitutions, 28.9% silent mutations and 3.0% nonsense mutations. Many substitutions occurred at positions that have not been known to be polymorphic before. A large number of HLA alleles and nucleotide variations underline the extreme diversity of the HLA system. Strikingly, 156 new alleles were found not only multiple times, but also in carriers of various parentage, suggesting that some new alleles are not necessarily rare. Moreover, new alleles were found especially often in minority donors. This emphasizes the benefits of specifically recruiting such groups of individuals.

High density FTA plates serve as efficient long-term sample storage for HLA genotyping.

Storage of dried blood spots (DBS) on high-density FTA(®) plates could constitute an appealing alternative to frozen storage. However, it remains controversial whether DBS are suitable for high-resolution sequencing of human leukocyte antigen (HLA) alleles. Therefore, we extracted DNA from DBS that had been stored for up to 4 years, using six different methods. We identified those extraction methods that recovered sufficient high-quality DNA for reliable high-resolution HLA sequencing. Further, we confirmed that frozen whole blood samples that had been stored for several years can be transferred to filter paper without compromising HLA genotyping upon extraction. Concluding, DNA derived from high-density FTA(®) plates is suitable for high-resolution HLA sequencing, provided that appropriate extraction protocols are employed.

Three hundred and seventy-two novel HLA class II alleles identified in potential hematopoietic stem cell donors from Germany, the United States, and Poland.

We have characterized 372 novel human leukocyte antigen (HLA) class II alleles identified in newly registered stem cell donors, this includes 281 HLA-DRB1 alleles, 89 HLA-DQB1 alleles and 2 HLA-DPB1 alleles. Most novel alleles were single nucleotide variants when compared to their respective most homologous alleles. In 66.4% of all novel alleles non-synonymous nucleotide variations were identified, in 30.4% synonymous substitutions and in 3.2% nonsense mutations. Ninty-three (25.0%) novel alleles were found in several individuals; most often these were novel HLA-DRB1 alleles. Lastly, we underline the importance of recruiting ethnic minority donors in countries such as Germany and the United States, as novel alleles were frequently found among these groups.

Clinical and molecular genetic study of 12 Italian families with autosomal recessive Stargardt disease.

Stargardt disease was diagnosed in 12 patients from 12 families using complete ophthalmologic examination, fundus photography, fundus autofluorescence, and spectral-domain optical coherence tomography. DNA was extracted for polymerase chain reaction (PCR) and direct DNA sequencing (ABCA4 gene). Genetic counseling and eye examination were offered to 16 additional family members. Various patterns of presentation were observed in patients with clinical diagnoses of Stargardt disease. The genetic study identified 2 mutations in 75% of families (9/12); a second mutation could not be found in the remaining 25% of families (3/12). The most frequent mutation was G1961E, found in 17% of families (2/12). This finding is similar to that of a previous analysis report of an Italian patient series. Four new mutations were also identified: Tyr1858Asp, Leu1195fsX1196, p.Tyr850Cys, and p.Thr959Ala. Our results suggest that PCR and direct DNA sequencing are the most appropriate techniques for the analysis of the ABCA4 gene. However, this method requires supplementation with specific PCR analysis to diagnose large deletions. The study of the families identified healthy carriers and affected subjects in presymptomatic stages and was also useful for evaluating the risk of transmission to progeny. Combined ophthalmologic and genetic evaluation enabled better clinical management of these families.

Electroconvulsive therapy resolves cortical inhibition and manneristic omissions in a chronic catatonic patient.

We investigated a patient with severe catatonic schizophrenia (manneristic catatonia according to Karl Leonhard) treated with electroconvulsive therapy (ECT) after pharmacological approaches did not result in any clinical improvement. Before and after nine ECT sessions a double-pulse transcranial magnetic stimulation (TMS) paradigm was used to measure intracortical inhibition (ICI) which has been shown to be reduced in a significant proportion of patients with schizophrenia. Although the patient showed no remission regarding some psychomotor aspects after ECT, we found an increase in ICI and a remarkable clinical improvement of catatonic omissions which might be due to changes in the GABAergic system.

Improved antioxidant activity of a starch and gelatin-based biodegradable coating containing Tetradenia riparia extract.

The incorporation of plant-based extracts into polymer-based coatings is an efficient alternative to increase the shelf-life of stored fruit and to decrease or even prevent bacterial growth. Considering strawberries, it is also important to preserve their high antioxidant activity. Hence, this work evaluated the efficiency of a coating based on native cassava starch (NCS), gelatin, and sorbitol, containing different concentrations of Tetradenia riparia extract, in delaying the ripening process of strawberries stored under refrigerated conditions, and in preventing bacterial growth and antioxidant activity losses. Both concentrations of extract (500 or 1000 µg mL-1) increased the thickness, opacity, and water vapor transmission rate (WVTR) of the films when compared to the film without extract, but decreased the solubility. Even though the film without extract was expected to create a more efficient barrier to the coated fruits, the films containing the extract led to similar results of soluble solids (SS), titratable acidity (TA), and vitamin C. Nevertheless, the extract incorporation improved the control over bacterial growth, and preserved the high antioxidant activity of the strawberries within ten days of storage.

Combined OCT distance and FBG force sensing cannulation needle for retinal vein cannulation: in vivo animal validation.

PURPOSE: Retinal vein cannulation is an experimental procedure during which a clot-dissolving drug is injected into an obstructed retinal vein. However, due to the fragility and minute size of retinal veins, such procedure is considered too risky to perform manually. With the aid of surgical robots, key limiting factors such as: unwanted eye rotations, hand tremor and instrument immobilization can be tackled. However, local instrument anatomy distance and force estimation remain unresolved issues. A reliable, real-time local interaction estimation between instrument tip and the retina could be a solution. This paper reports on the development of a combined force and distance sensing cannulation needle, and its experimental validation during in vivo animal trials. METHODS: Two prototypes are reported, relying on force and distance measurements based on FBG and OCT A-scan fibres, respectively. Both instruments provide an 80 [Formula: see text] needle tip and have outer shaft diameters of 0.6 and 2.3 mm, respectively. RESULTS: Both prototypes were characterized and experimentally validated ex vivo. Then, paired with a previously developed surgical robot, in vivo experimental validation was performed. The first prototype successfully demonstrated the feasibility of using a combined force and distance sensing instrument in an in vivo setting. CONCLUSION: The results demonstrate the feasibility of deploying a combined sensing instrument in an in vivo setting. The performed study provides a foundation for further work on real-time local modelling of the surgical scene. This paper provides initial insights; however, additional processing remains necessary.

Variability in a permanent cyanobacterial bloom: species-specific responses to environmental drivers.

Cyanobacterial blooms are characterized by intense growth of one or few species that will dominate the phytoplankton community for periods of few months to an entire year or more. However, even during persistent blooms, important seasonal changes among dominant species can be observed. Pampulha reservoir is a tropical eutrophic reservoir presenting permanent blooms. To identify the main species in this environment, a closer analysis performed by microscopy and 16S-rRNA DGGE revealed Cylindrospermopsis raciborskii as highly dominant throughout the year. The second most abundant group comprised species belonging to the Microcystis genus. They followed a well-defined seasonal pattern described by interesting species-specific ecological trends. During thermal stratification in the rainy/warmer season, C. raciborskii dominated in the water column, while Microcystis spp. were abundant at the end of the dry season, a period characterized by higher total phosphorus concentrations. Phylogenetic analyses confirmed the two dominant taxa and their seasonal trends. The results showed that cyanobacteria major controlling factors were strongly species dependent, shifting from physical/climate related (stratification) to more chemical driven (nutrients/eutrophication). Identifying these drivers is therefore essential for the understanding of the bloom dynamics and the real risks associated with each species, and to eventually adopt the most appropriate and effective management strategies.

Crude extract of cyanobacteria (Radiocystis fernandoi, strain R28) induces liver impairments in fish.

Radiocystis fernandoi R28 strain is a cyanobacterium which produces mostly the RR and YR microcystin variants (MC-RR and MC-YR, respectively). The effects of crude extract of the R. fernandoi strain R28 were evaluated on the protein phosphatases and on the structure and ultrastructure of the liver of the Neotropical fish, Hoplias malabaricus, after acute and subchronic exposure. Concomitantly, the accumulation of the majority of MCs was determined in the liver and muscle. The fish were exposed to 120.60 MC-RR+MC-LR kg-fish-1 (=100µg MC-LReq kg-fish-1) for 12 and 96h (one single dose, acute exposure) and 30days (one similar dose every 72h, subchronic exposure). MCs did not accumulate in the muscle but, in the liver, MC-YR accumulated after acute exposure and MC-RR and MC-YR accumulation occurred after subchronic exposure. Protein phosphatase 2A (PP2A) activity was inhibited only after subchronic exposure. Acute exposure induced liver hyperemia, hemorrhage, changes in hepatocytes and cord-like disorganization. At the ultrastructural level, the decreasing of glycogen and lipid levels, the swelling of mitochondria and whirling of endoplasmic reticulum suggested hepatocyte necrosis. Subchronic exposure resulted in a complete disarrangement of cord-like hepatocytes, some recovery of mitochondria and whirling endoplasmic reticulum and extensive connective tissues containing fibrous materials in the liver parenchyma. Despite microcystin toxicity and liver alterations, no tumor was induced by MCs. In conclusion, the increased algal mass of R. fernandoi in tropical freshwater, producing mainly MC-RR and MC-YR variants, results in fish liver impairments.

Influence of HLA on human partnership and sexual satisfaction.

The major histocompatibility complex (MHC, called HLA in humans) is an important genetic component of the immune system. Fish, birds and mammals prefer mates with different genetic MHC code compared to their own, which they determine using olfactory cues. This preference increases the chances of high MHC variety in the offspring, leading to enhanced resilience against a variety of pathogens. Humans are also able to discriminate HLA related olfactory stimuli, however, it is debated whether this mechanism is of behavioural relevance. We show on a large sample (N = 508), with high-resolution typing of HLA class I/II, that HLA dissimilarity correlates with partnership, sexuality and enhances the desire to procreate. We conclude that HLA mediates mate behaviour in humans.

Detection of 549 new HLA alleles in potential stem cell donors from the United States, Poland and Germany.

We characterized 549 new human leukocyte antigen (HLA) class I and class II alleles found in newly registered stem cell donors as a result of high-throughput HLA typing. New alleles include 101 HLA-A, 132 HLA-B, 105 HLA-C, 2 HLA-DRB1, 89 HLA-DQB1 and 120 HLA-DPB1 alleles. Mainly, new alleles comprised single nucleotide variations when compared with homologous sequences. We identified nonsynonymous nucleotide mutations in 70.7% of all new alleles, synonymous variations in 26.4% and nonsense substitutions in 2.9% (null alleles). Some new alleles (55, 10.0%) were found multiple times, HLA-DPB1 alleles being the most frequent among these. Furthermore, as several new alleles were identified in individuals from ethnic minority groups, the relevance of recruiting donors belonging to such groups and the importance of ethnicity data collection in donor centers and registries is highlighted.

Human pharmacokinetic characterization and in vitro study of the interaction between doxorubicin and paclitaxel in patients with breast cancer.

PURPOSE: We performed a pharmacologic investigation of paclitaxel (PTX) infused over 3 hours and bolus doxorubicin (DOX) to assess the role of sequence, interval between drugs, and duration of doxorubicin infusion on paclitaxel and anthracycline plasma disposition. We also explored possible mechanisms of pharmacokinetic interference involving the physiologic role of the multidrug resistance phenotype in anthracycline and taxane biliary excretion. PATIENTS AND METHODS: Pharmacokinetics was performed in 80 cycles and 36 women with previously untreated metastatic breast cancer. PTX, DOX, and their metabolites 6 alpha-hydroxyl-PTX (6 alpha OH-PTX) and doxorubicinol (DOL) were measured by high-pressure liquid chromatography (HPLC). Human breast cancer MCF-7 wild-type (WT) and resistant (TH) cell lines were cultured in whole human plasma to study anthracycline retention after treatment with different combinations of PTX, Cremophor EL (CEL) (PEG35 castor oil; BASF, Parsippany, NJ), and DOX. RESULTS: Pharmacokinetic interference between PTX and DOX was responsible for nonlinearity of DOX plasma disposition and increased concentrations of DOX and DOL. These effects were PTX dose-dependent, DOX concentration-dependent, and likely a result of interference at the level of liver elimination. In view of the physiologic role of P-glycoproteins (P-gp) in xenobiotic biliary excretion, retention of DOX was assessed in MCF-7 WT and MCF-7 TH cells. Intracellular was significantly higher in MCF-7 WT than MCF-7 TH (P < .05). However, concomitant exposure to DOX, PTX, and CEL caused similar DOX retention in both MCF-7 WT and TH cells. CONCLUSION: PTX, as clinically formulated in CEL, is responsible for a nonlinear disposition of DOX and DOL. Nonlinearity is PTX- and DOX-dependent, and possibly caused by competition for biliary excretion of taxanes and anthracyclines mediated by P-gp. Nonlinearity indicates that even minor modifications of dose and infusion duration of DOX and PTX may lead to unpredictable pharmacodynamic consequences. The postulated role of P-gp suggests that CEL is clinically active, and advises caution in designing combinations of PTX with other drugs that are substrate for P-gp.

Nonlinear pharmacokinetics and metabolism of paclitaxel and its pharmacokinetic/pharmacodynamic relationships in humans.

PURPOSE: To characterize and model the disposition of paclitaxel in humans and define a pharmacodynamic relationships between paclitaxel disposition and its toxicity and efficacy. PATIENTS AND METHODS: Paclitaxel pharmacokinetics were studied in 55 courses of therapy in 30 patients. Paclitaxel was administered at 135 mg/m2 or 175 mg/m2 by either a 3- or a 24-hour infusion schedule to patients with advanced ovarian cancer (n = 15), or at 225 mg/m2 by 3-hour infusion to patients with advanced breast cancer (n = 15). Paclitaxel and 6 alpha-hydroxylpaclitaxel were quantified by high-performance liquid chromatography (HPLC). Pharmacokinetics were assessed by noncompartmental and model-dependent methods. Pharmacodynamic correlations were evaluated statistically and by regression models. RESULTS: Paclitaxel disposition is nonlinear in humans and, on the 3-hour schedule, 6 alpha-hydroxylpaclitaxel was identified in the plasma of all patients treated. The plasma disposition of paclitaxel and 6 alpha-hydroxylpaclitaxel was well described by a model that featured multiple nonlinear processes. Neutropenia was not related to the areas under the curves (AUCs) of paclitaxel or 6 alpha-hydroxylpaclitaxel, or to palitaxel peak concentrations (Cmax). Neutropenia was related to the duration that plasma concentrations were > or = 0.05 mumol/L, a relationship that is well described by a sigmoid maximum response (Emax) model. CONCLUSION: The disposition of paclitaxel in humans is nonlinear. Paclitaxel metabolism to 6 alpha-hydroxylpaclitaxel is likely an important detoxification pathway. Myelosuppression is related to the duration that plasma paclitaxel concentrations are > or = 0.05 mumol/L. Trials of new doses and schedules of paclitaxel should take into account its nonlinear disposition to rule out adverse clinical consequences, especially if the drug is administered by short infusion. Our pharmacokinetic model should prove to be a powerful tool in predicting paclitaxel disposition, regardless of dose and schedule, and should facilitate further pharmacodynamic investigations.

Clinical and pharmacologic study of the epirubicin and paclitaxel combination in women with metastatic breast cancer.

PURPOSE: A pharmacokinetic interaction may cause increased cardiotoxicity of paclitaxel (PTX) and high cumulative dose of doxorubicin. We tested antitumor activity, tolerability, and pharmacokinetics of the lesser cardiotoxic epirubicin (EPI) and PTX (ET combination). PATIENTS AND METHODS: Twenty-seven women with untreated metastatic breast cancer, median age of 56 years, and prominent visceral involvement (74%) were studied. Three-weekly EPI (90 mg/m(2)) and PTX (200 mg/m(2) over 3 hours) were given for a maximum nine cycles. EPI was administered 24 hours before PTX (E --> T) in cycle 1, and 15 minutes before PTX (ET) thereafter. EPI, epirubicinol (EOL), EPI-glucuronide (EPI-glu), EOL-glucuronide (EOL-glu), PTX, and 6alpha-OH-PTX were measured in plasma and urine in 14 women. RESULTS: Patients received 205 cycles of ET and a median EPI dose of 720 mg/m(2). Grade 4 neutropenia (49% of cycles) was the most frequent toxicity. Cardiac contractility was decreased in five patients. Mild congestive heart failure occurred in two (7.4%). Response rate was 76% (28% complete). Median overall survival was 29 months. On the basis of intrapatient comparison in the first 24 hours of E --> T and ET cycles, PTX did not affect EPI disposition, but significantly increased plasma exposure to EOL (by 137%), EPI-glu (threefold) and EOL-glu (twofold). Urinary excretion of EPI dose went from 8.2% in E --> T to 11.8% in ET cycles. Clearance of PTX was 30% slower in ET than E --> T. ET cycles caused lower neutrophil nadir than E --> T (644 +/- 327 v 195 +/- 91, P <.05) CONCLUSION: ET is feasible, devoid of excessive cardiac toxicity, and active. A reciprocal pharmacokinetic interference between the two drugs has pharmacodynamic consequences, and suggests a direct effect of PTX on EPI metabolism requiring ad hoc investigation.

The nutritional status of zooplankton in a tropical reservoir: effects of food quality and community structure.

The temporal variability of energetic reserves of zooplankton in the eutrophic Pampulha reservoir was investigated during two successive annual cycles. The effects of dominance of large filter-feeding cladocerans (Daphnia) and the occurrence of massive blooms of the cyanobacteria Microcystis on the energetic reserves of zooplankton were tested. This study showed that phytoplankton composition has a greater effect on energetic reserves of zooplankton. Some associations between lipid levels and the specific composition of zooplankton were also found. This study also demonstrated that the elementary composition of phosphorus in zooplankton can be used as an estimator of the nutritional status of zooplankton.