Epigenetic response in mice mastitis: Role of histone H3 acetylation and microRNA(s) in the regulation of host inflammatory gene expression during Staphylococcus aureus infection.
There is renewed interest towards understanding the host-pathogen interaction in the light of epigenetic modifications. Although epithelial tissue is the major site for host-pathogen interactions, there is handful of studies to show how epithelial cells respond to pathogens. Bacterial infection in the mammary gland parenchyma induces local and subsequently systemic inflammation that results in a complex disease called mastitis. Globally Staphylococcus aureus is the single largest mastitis pathogen and the infection can ultimately result in either subclinical or chronic and sometimes lifelong infection. In the present report we have addressed the differential inflammatory response in mice mammary tissue during intramammary infection and the altered epigenetic context induced by two closely related strains of S. aureus, isolated from field samples. Immunohistochemical and immunoblotting analysis showed strain specific hyperacetylation at histone H3K9 and H3K14 residues. Global gene expression analysis in S. aureus infected mice mammary tissue revealed a selective set of upregulated genes that significantly correlated with the promoter specific, histone H3K14 acetylation. Furthermore, we have identified several differentially expressed known miRNAs and 3 novel miRNAs in S. aureus infected mice mammary tissue by small RNA sequencing. By employing these gene expression data, an attempt has been made to delineate the gene regulatory networks in the strain specific inflammatory response. Apparently, one of the isolates of S. aureus activated the NF-κB signaling leading to drastic inflammatory response and induction of immune surveillance, which could possibly lead to rapid clearance of the pathogen. The other strain repressed most of the inflammatory response, which might help in its sustenance in the host tissue. Taken together, our studies shed substantial lights to understand the mechanisms of strain specific differential inflammatory response to S. aureus infection during mastitis. In a broader perspective this study also paves the way to understand how certain bacteria can evade the immune surveillance and cause sustained infection while others are rapidly cleared from the host body.


Late acyltransferase genes lpxX and lpxL jointly contribute to the biological activities of Moraxella catarrhalis.
Lipo-oligosaccharide (LOS) is a major surface component and virulence factor of the human respiratory pathogen Moraxella catarrhalis. Two late acyltransferase genes, lpxX and lpxL, have been identified involved in the incorporation of acyloxyacyl-linked secondary acyl chains into lipid A during M. catarrhalis LOS biosynthesis. In this study, a double mutant with a deletion of both the lpxX and lpxL genes in M. catarrhalis strain O35E was constructed and named O35ElpxXL. Structural analysis of lipid A showed that the O35ElpxXL mutant lacked two decanoic acids (10 : 0) and one dodecanoic (lauric) acid (12 : 0). In comparison with the O35E parental strain and the single mutants O35ElpxX and O35ElpxL, the double mutant O35ElpxXL displayed prominently decreased endotoxin content, reduced resistance to normal human serum and accelerated bacterial clearance at 0, 3 and 6 h after an aerosol challenge in a mouse model of bacterial pulmonary clearance. These results indicate that these two genes encoding late acyltransferases responsible for lipid A biosynthesis jointly contribute to the biological activities and pathogenicity of M. catarrhalis. The double mutant O35ElpxXL with dramatically reduced toxicity is proposed as a potential vaccine candidate against M. catarrhalis infections for further investigation.


A Review on the Pharmacokinetic Properties of Naringin and Its Therapeutic Efficacies in Respiratory Diseases.
Flavonoids are an important class of phytopharmaceuticals in plants. Naringin (naringenin- 7-O-rhamnoglucoside) is a flavanone glycoside isolated from folk herbal medicine Exocarpium Citri grandis (called Huajuhong in Chinese). Massive experimental works have been performed on naringin describing its phytochemical, pharmacokinetic, and bioactive properties. Naringin was found to possess multiple pharmacological activities in relieving inflammation, diabetes, neurodegeneration, cardiovascular disorders, and metabolic syndrome. Recently, it has been approved as a potential antitussive and expectorant for clinical trials. However, the pharmacokinetic aspects of naringin and its therapeutic potentials in respiratory diseases have not been comprehensively reviewed. The present review provides highlights of naringin with respect to its absorption, distribution, metabolism, excretion and its therapeutic effects on cough, phlegm, and pulmonary inflammation. This review would be helpful for the interpretation of pharmacokinetics and pharmacodynamics of naringin in clinical trials.


Directed Evolution of an Enhanced POU Reprogramming Factor for Cell Fate Engineering.
Transcription factor-driven cell fate engineering in pluripotency induction, transdifferentiation, and forward reprogramming requires efficiency, speed, and maturity for widespread adoption and clinical translation. Here, we used Oct4, Sox2, Klf4, and c-Myc driven pluripotency reprogramming to evaluate methods for enhancing and tailoring cell fate transitions, through directed evolution with iterative screening of pooled mutant libraries and phenotypic selection. We identified an artificially evolved and enhanced POU factor (ePOU) that substantially outperforms wild-type Oct4 in terms of reprogramming speed and efficiency. In contrast to Oct4, not only can ePOU induce pluripotency with Sox2 alone, but it can also do so in the absence of Sox2 in a three-factor ePOU/Klf4/c-Myc cocktail. Biochemical assays combined with genome-wide analyses showed that ePOU possesses a new preference to dimerize on palindromic DNA elements. Yet, the moderate capacity of Oct4 to function as a pioneer factor, its preference to bind octamer DNA and its capability to dimerize with Sox2 and Sox17 proteins remain unchanged in ePOU. Compared with Oct4, ePOU is thermodynamically stabilized and persists longer in reprogramming cells. In consequence, ePOU: 1) differentially activates several genes hitherto not implicated in reprogramming, 2) reveals an unappreciated role of thyrotropin-releasing hormone signaling, and 3) binds a distinct class of retrotransposons. Collectively, these features enable ePOU to accelerate the establishment of the pluripotency network. This demonstrates that the phenotypic selection of novel factor variants from mammalian cells with desired properties is key to advancing cell fate conversions with artificially evolved biomolecules.


Perioperative hypoxemia is common with horizontal positioning during general anesthesia and is associated with major adverse outcomes: a retrospective study of consecutive patients.
Reported perioperative pulmonary aspiration (POPA) rates have substantial variation. Perioperative hypoxemia (POH), a manifestation of POPA, has been infrequently studied beyond the PACU, for patients undergoing a diverse array of surgical procedures. Consecutive adult patients with ASA I-IV and pre-operative pulmonary stability who underwent a surgical procedure requiring general anesthesia were investigated. Using pulse oximetry, POH was documented in the operating room and during the 48 hours following PACU discharge. POPA was the presence of an acute pulmonary infiltrate with POH. The 500 consecutive, eligible patients had operative body-positions of prone 13%, decubitus 8%, sitting 1%, and supine/lithotomy 78%, with standard practice of horizontal recumbency. POH was found in 150 (30%) patients. Post-operative stay with POH was 3.7 ± 4.7 days and without POH was 1.7 ± 2.3 days (p < 0.0001). POH rate varied from 14% to 58% among 11 of 12 operative procedure-categories. Conditions independently associated with POH (p < 0.05) were acute trauma, BMI, ASA level, glycopyrrolate administration, and duration of surgery. POPA occurred in 24 (4.8%) patients with higher mortality (8.3%), when compared to no POPA (0.2%; p = 0.0065). Post-operative stay was greater with POPA (7.7 ± 5.7 days), when compared to no POPA (2.0 ± 2.9 days; p = 0.0001). Conditions independently associated with POPA (p < 0.05) were cranial procedure, ASA level, and duration of surgery. POPA, acute trauma, duration of surgery, and inability to extubate in the OR were independently associated with post-operative stay (p < 0.05). POH, gastric dysmotility, acute trauma, cranial procedure, emergency procedure, and duration of surgery had independent correlations with post-operative length of stay (p < 0.05). Adult surgical patients undergoing general anesthesia with horizontal recumbency have substantial POH and POPA rates. Hospital mortality was greater with POPA and post-operative stay was increased for POH and POPA. POH rates were noteworthy for virtually all categories of operative procedures and POH and POPA were independent predictors of post-operative length of stay. A study is needed to determine if modest reverse-Trendelenburg positioning during general anesthesia has a relationship with reduced POH and POPA rates.


Crystal structure of the Epstein-Barr virus (EBV) glycoprotein H/glycoprotein L (gH/gL) complex.
The Epstein-Barr virus (EBV) is a γ-herpesvirus that infects B cells and epithelial cells and that has been linked to malignancies in both cell types in vivo. EBV, like other herpesviruses, has three glycoproteins, glycoprotein B (gB), gH, and gL, that form the core membrane fusion machinery mediating viral penetration into the cell. The gH and gL proteins associate to form a heterodimeric complex, which is necessary for efficient membrane fusion and also implicated in direct binding to epithelial cell receptors required for viral entry. To gain insight into the mechanistic role of gH/gL, we determined the crystal structure of the EBV gH/gL complex. The structure is comprised of four domains organized along the longest axis of the molecule. Comparisons with homologous HSV-2 gH/gL and partial pseudorabies virus gH structures support the domain boundaries determined for the EBV gH/gL structure and illustrate significant differences in interdomain packing angles. The gL subunit and N-terminal residues of gH form a globular domain at one end of the structure, implicated in interactions with gB and activation of membrane fusion. The C-terminal domain of gH, proximal to the viral membrane, is also implicated in membrane fusion. The gH/gL structure locates an integrin binding motif, implicated in epithelial cell entry, on a prominent loop in the central region of the structure. Multiple regions of gH/gL, including its two extreme ends, are functionally important, consistent with the multiple roles of gH/gL in EBV entry.


TPP-based mitocans: a potent strategy for anticancer drug design.
Cancer is one of the most important problems that endanger human health. The number of cancer patients is increasing rapidly worldwide. Compared with normal cells, cancer cells exhibit abnormal metabolism (abnormal glycolysis and oxidative phosphorylation, high levels of reactive oxygen species, anti-apoptosis, high mitochondrial membrane potential, and so on), and specific targeting of these metabolic abnormalities would be a promising drug design direction. These physiological characteristics are closely related to tumorigenesis and development, which are mainly regulated by mitochondria. Therefore, mitochondria have become important anticancer drug targets, attracting much attention in recent years. In this review, we systematically summarize various mitochondrial anticancer drugs developed, especially mitocans based on triphenylphosphonium (TPP), and discuss the advantages of TPP in endowing mitochondrial targeting function.


Small molecules for interference with cell-cell-communication systems in Gram-negative bacteria.
Quorum sensing (QS) systems are bacterial cell-to-cell communication systems that use small molecules as signals. Since QS is involved in the regulation of virulence and biofilm formation in several pathogenic bacteria, it has been suggested as a new target for the development of novel antibacterial therapies. As such, interference with the signal receptors by using chemical compounds has been proposed as an alternative strategy for treatment of bacterial infections and has already shown promising results in combination with traditional antibiotic treatments. In Gram-negative bacteria, the best studied QS systems use N-acyl homoserine lactones (AHLs) as signal molecules. This review provides an overview of all new chemical structure types that inhibit AHL-mediated QS systems as reported during the last three years in scientific journals and in the patent literature. The compounds were classified into three main groups depending on their structure: AHL analogues, 2(5H)-furanones, and compounds that are not structurally related to AHLs. We discuss the biological assays used and the different strategies applied to discover these molecules, including new approaches such as molecular docking for in silico identification of lead structures and random high-throughput screening of large libraries of chemicals. Finally, we elaborate on structure-activity relationships and on the new insights in the mechanisms of action of the identified inhibitors, highlighting the potential of these small molecules in medicine.


Tunable Photodetectors via In Situ Thermal Conversion of TiS3 to TiO2.
In two-dimensional materials research, oxidation is usually considered as a common source for the degradation of electronic and optoelectronic devices or even device failure. However, in some cases a controlled oxidation can open the possibility to widely tune the band structure of 2D materials. In particular, we demonstrate the controlled oxidation of titanium trisulfide (TiS3), a layered semicon-ductor that has attracted much attention recently thanks to its quasi-1D electronic and optoelectron-ic properties and its direct bandgap of 1.1 eV. Heating TiS3 in air above 300 °C gradually converts it into TiO2, a semiconductor with a wide bandgap of 3.2 eV with applications in photo-electrochemistry and catalysis. In this work, we investigate the controlled thermal oxidation of indi-vidual TiS3 nanoribbons and its influence on the optoelectronic properties of TiS3-based photodetec-tors. We observe a step-wise change in the cut-off wavelength from its pristine value ~1000 nm to 450 nm after subjecting the TiS3 devices to subsequent thermal treatment cycles. Ab-initio and many-body calculations confirm an increase in the bandgap of titanium oxysulfide (TiO2-xSx) when in-creasing the amount of oxygen and reducing the amount of sulfur.


Development of an instrument for collective assessment of fluency and comprehension of reading in elementary school students II.
The study presents the process of developing of an instrument for collective evaluation of reading fluency and comprehension of secondary elementary school students in grades 6-9 and verifies the effect of schooling on performance in the instrument. 100 students regularly enrolled in grades 6-9 in secondary elementary public schools participated in the study. The construction of the instrument involved seven steps, with the participation of two judges. The instrument was composed of narrative text appropriate for secondary elementary school students and for 10 multiple choice questions, which five were literal questions and five were inferential questions. The results showed a better performance for the participants with higher schooling in fluency and in reading comprehension. The reading fluency presented positive and moderate correlations with the reading comprehension. The instrument is easy to apply and analyze, and can be used in clinical, educational and research context to measure the performance of students in grades 6-9 in fluency and reading comprehension.


[Modes of subjectivity production of nurses for decision making].
This study aimed to analyze the nurse's ways of production of subjectivity in decision-making processes that involve nursing care. We adopted qualitative research design. The investigative scenario was a philanthropic hospital in the State of Rio Grande do Sul. The study participants were twelve nurses working in this institution. For data collection, it was used the technique of focus group, with three focal meetings conducted in December 2011. From discussions among nurses, it was found that the nurse's ways of production of subjectivity oscillate between two extremes: a relationship of alienation and oppression, resulting in a self-protection, redundancy and domination behavior, or an expression and creation ratio, resulting in proactive behavior of nurses. Nurse's ways of production of subjectivity can condition and define behaviors that prevail in the decision making in care practice.


A multifunctional mechanical testing stage for micro x-ray computed tomography.
An existing open and modular designed micro X-Ray Computed Tomography (μXRCT) system is extended by a test rig in order to combine mechanical and hydro-mechanical experiments with μXRCT characterization. The aim of the system is to cover the complete resolution range of the underlying μXRCT system in combination with a broad load capacity range. A characteristic feature of the developed setup is that it consists mainly of standard components. This makes the shown test rig potentially interesting for other researchers considering extending an existing μXRCT system with an apparatus for mechanical and hydro-mechanical in situ testing. For the load frame, an uniaxial 10 kN universal testing machine with a digital control system was employed, which was extended by two aligned rotational stages. The uniaxial load capacity is ±3.1 kN and can be combined with torque moments of up to ±15 N m both limited by the used rotational stages. The setup is designed in such a way that different x-ray transparent cells (flow cells, oedometer cells, triaxial cells, etc.) can be integrated to generate three-dimensional stress/strain states as required for porous media research. Three applications demonstrate the possible versatile use of the system. As part of these examples, we show how corresponding x-ray transparent cells are designed and implemented. Finally, we discuss the presented approach's technical advantages and disadvantages and suggest improvements.


Emergency department personnel accuracy at estimating patient satisfaction.
We investigated the congruence between emergency department (ED) personnel estimations of patient satisfaction and actual patient satisfaction. Actual patient satisfaction was assessed via telephone interview. Before the findings were disseminated, ED personnel were asked to "predict" the results by estimating the average rating they believed patients gave for each of the 22 indicators. Surveys were obtained from 478 of 1,139 (42.1%) patients and 59 of 76 (77.6%) ED personnel. ED personnel consistently estimated average satisfaction scores to be significantly lower than what patients actually reported across 19 of the 22 indicators. ED personnel grossly overestimated average patient length of stay (LOS) (5.7 h vs. 3.5 h actual). Results suggest that ED personnel consistently estimated patients to be less satisfied than they actually were across multiple domains. Moreover, ED personnel estimated average LOS to be more than 2 h longer than patients' actual LOS. Hypotheses for why this bias occurred are discussed.


On principal features of organic electrolyte molecules in lithium ion battery performance.
The lithium (Li) complexes of organic electrolyte solvents are theoretically investigated using the long-range correction for density functional theory in order to figure out the cause for the high performance of cyclic carbonate electrolytes in lithium ion batteries (LIBs). Calculations of the Li complexes with ethylene carbonate solvent molecules prove that ten ligand molecules should be incorporated to obtain near-degenerate four- and five-coordination optimum structures and dramatically improved orbital energies. The geometry optimizations of the Li complexes with thirteen types of organic solvent molecules give four-coordination neutral and five-coordination cation complexes for many solvent molecules. The five-coordination Li complexes are considered to use Berry pseudorotation to approach the electrodes from the Li atom. The calculated Koopmans, vertical and adiabatic ionization potentials and electron affinities show that near-degeneracy and structural deformation effects play significant roles in the electronic states of the Li complexes. Mulliken charge and dipole moment analyses indicate that the Li complexes of cyclic carbonates construct a deep electric double layer near electrodes due to the electron-donating ability of the ligand molecules. Molecular orbital analyses also explain that the Li complexes of cyclic carbonates easily construct a solid electrolyte interface, which contributes to Li ion conductance, by localizing the accepted electron to one ligand molecule. In conclusion, the Li complexes of cyclic carbonates have three main features: preference of five-coordination structures, high electron-donating ability of ligand molecules, and localization of the accepted electron to one ligand molecule.


Autophagy pathway: Cellular and molecular mechanisms.
Macroautophagy/autophagy is an essential, conserved self-eating process that cells perform to allow degradation of intracellular components, including soluble proteins, aggregated proteins, organelles, macromolecular complexes, and foreign bodies. The process requires formation of a double-membrane structure containing the sequestered cytoplasmic material, the autophagosome, that ultimately fuses with the lysosome. This review will define this process and the cellular pathways required, from the formation of the double membrane to the fusion with lysosomes in molecular terms, and in particular highlight the recent progress in our understanding of this complex process.


True chemical composition profiles at polymer interphases: recovery from measurements distorted by instrumental broadening.
The determination of chemical composition profiles at polymer interphases is an important issue at the moment of elucidating the physical mechanisms that operate in polymer diffusion processes and for calculating diffusion parameters. Several techniques are available to measure these profiles, the most common being forward recoil spectroscopy, Rutherford backscattering spectrometry, nuclear reaction analysis, confocal Raman microspectroscopy (CRM), and scanning infrared microscopy. However, all these techniques are affected by the limited resolution of the experimental setup, which in practice produces a rounding effect on the sharp corners of the composition profile; this may lead to incorrect conclusions regarding the measurements. In this work an inverse technique is proposed to correct this undesirable effect in the profiles. The inversion is performed on a model of the measuring process, which includes the instrumental broadening function, a quantitative representation of the limited resolution. The proposed methodology was tested using numerically generated experiments and genuine experimental runs obtained from CRM measurements at interphases of polymer bilayers. In all cases, the recovered profiles were close to the expected ones. In the truly experimental results diffusion tails are observed behind and ahead of the diffusion front before the numerical treatment of the data. These tails may be caused by a genuine mass diffusion or by an artifact. After the numerical treatment the tails disappear and a sharp interphase is recovered, a result one expects for the polymer pairs under study.


Memantine therapy of behavioral symptoms in community-dwelling patients with moderate to severe Alzheimer's disease.
Memantine is a moderate-affinity, uncompetitive antagonist of N-methyl-D-aspartate receptors, approved for the treatment of moderate to severe Alzheimer's disease (AD). Available data suggest that, in addition to its benefits on cognition, function, and global status, memantine treatment may also help alleviate behavioral symptoms. This article provides an overview of the prevalence, assessment, and treatment of behavioral disturbances in AD, and summarizes current knowledge regarding the effects of memantine on the behavior of community-dwelling patients. We searched EMBASE and PubMed (January 1992 to October 2008) for reports on memantine trials that involved outpatients with moderate to severe AD. All previously unpublished data were obtained from Forest Laboratories, Inc. Behavioral outcomes were assessed in three completed, double-blind, placebo-controlled trials.Overall, patients who received memantine performed better on behavioral measures than those treated with placebo. Post-hoc analyses suggest that memantine treatment was associated with a reduced severity or emergence of specific symptoms, particularly agitation and aggression. Prospective, well-designed trials are warranted to evaluate the efficacy of memantine in patients with significant behavioral symptoms.


Capsule enlargement in Cryptococcus neoformans confers resistance to oxidative stress suggesting a mechanism for intracellular survival.
Cryptococcus neoformans is a facultative intracellular pathogen. The most distinctive feature of C. neoformans is a polysaccharide capsule that enlarges depending on environmental stimuli. The mechanism by which C. neoformans avoids killing during phagocytosis is unknown. We hypothesized that capsule growth conferred resistance to microbicidal molecules produced by the host during infection, particularly during phagocytosis. We observed that capsule enlargement conferred resistance to reactive oxygen species produced by H(2)O(2) that was not associated with a higher catalase activity, suggesting a new function for the capsule as a scavenger of reactive oxidative intermediates. Soluble capsular polysaccharide protected C. neoformans and Saccharomyces cerevisiae from killing by H(2)O(2). Acapsular mutants had higher susceptibility to free radicals. Capsular polysaccharide acted as an antioxidant in the nitroblue tetrazolium (NBT) reduction coupled to beta-nicotinamide adenine dinucleotide (NADH)/phenazine methosulfate (PMS) assay. Capsule enlargement conferred resistance to antimicrobial peptides and the antifungal drug Amphotericin B. Interestingly, the capsule had no effect on susceptibility to azoles and increased susceptibility to fluconazole. Capsule enlargement reduced phagocytosis by environmental predators, although we also noticed that in this system, starvation of C. neoformans cells produced resistance to phagocytosis. Our results suggest that capsular enlargement is a mechanism that enhances C. neoformans survival when ingested by phagocytic cells.


Relationship between discharge practices and intensive care unit in-hospital mortality performance: evidence of a discharge bias.
Current intensive care unit performance measures include in-hospital mortality after intensive care unit admission. This measure does not account for deaths occurring after transfer to another hospital or soon after discharge and therefore, may be biased. Determine how transfer rates to other acute care hospitals and early post-discharge mortality rates impact hospital performance assessments using an in-hospital mortality model. Data were retrospectively collected on 10,502 eligible intensive care unit patients across 35 California hospitals between 2001 and 2004. We calculated the rates of acute care hospital transfers and early post-discharge mortality (30-day overall mortality-30-day in-hospital mortality) for each hospital. We assessed hospital performance with standardized mortality ratios (SMRs) using the Mortality Probability Model III. Using regression models, we explored the relationship between in-hospital SMRs and the rates of hospital transfers or early post-discharge mortality. We explored the same relationship using a 30-day SMR. In multivariable models, for each 1% increase in patients transferred to another acute care hospital, there was an in-hospital SMR reduction of -0.021 (-0.040-0.001). Additionally, a 1% increase in early post-discharge mortality was associated with an in-hospital SMR reduction of -0.049 (-0.142-0.045). Assessing hospital performance based upon 30-day mortality end point resulted in SMRs closer to 1.0 for hospitals at high and low ends of in-hospital mortality performance. Variations in transfer rates and potentially discharge timing appear to bias in-hospital SMR calculations. A 30-day mortality model is a potential alternative that may limit this bias.


Measuring diffuse metabolic activity on FDG-PET/CT: new method for evaluating Langerhans cell histiocytosis activity in pulmonary parenchyma.
Pulmonary Langerhans cell histiocytosis (PLCH) is a rare cause of interstitial lung disease characterized by formation of nodules in the active phase of the disease that evolve into nonactive cystic lesions later on. To evaluate PLCH activity in patients, we developed a new method for measuring diffuse metabolic activity on fluorine-18-fluorodeoxyglucose positron emission tomography/computed tomography (FDG-PET/CT) using a lung-to-liver activity ratio. We retrospectively studied a series of 4 FDG-PET and 23 FDG-PET/CT scans from 7 patients with PLCH and analyzed a sample of 100 randomly chosen FDG-PET/CT studies free from any known lung or hepatic diseases. Maximum standardized uptake value (SUVmax) in a spherical volume (6-8 cm in diameter) in the right lung was put into relation with SUVmax in a spherical volume (9-10 cm in diameter) in the reference liver parenchyma to set up the SUVmaxPULMO/SUVmaxHEPAR index. The index values were compared to the disease course in each patient. In patients with PLCH, a close correlation between the index value and the disease course was found in all seven subjects, where the increasing index values indicated disease activity, while decreasing index values were observed after therapy administration. In the group of 100 healthy control subjects, we found index values lower than 0.3 in 80% and lower than 0.4 in 96% [range: 0.14-0.43; 0.24±0.07 (100)]. Measuring SUVmaxPULMO/SUVmaxHEPAR values and their time-trend monitoring represent simple, noninvasive screening tools allowing an early diagnosis and treatment response follow-up assessment in patients with PLCH.


Genomewide expression profiling of cryptolepine-induced toxicity in Saccharomyces cerevisiae.
We have used the budding yeast Saccharomyces cerevisiae to identify genes that may confer sensitivity in vivo to the antimalarial and cytotoxic agent cryptolepine. Five S. cerevisiae strains, with different genetic backgrounds in cell permeability and DNA damage repair mechanisms, were exposed to several concentrations of cryptolepine. Cryptolepine showed a relatively mild toxicity for wild-type strains, which was augmented by either increasing cell permeability (Deltaerg6 or ISE2 strains) or disrupting DNA damage repair (Deltarad52 strains). These results are compatible with the ability of cryptolepine to intercalate into DNA and thus promote DNA lesions. The effects of low concentrations of cryptolepine (20% and 40% inhibitory concentrations [IC(20) and IC(40)]) were analyzed by comparing the gene expression profiles of treated and untreated Deltaerg6 yeast cells. Significant changes in expression levels were observed for 349 genes (117 upregulated and 232 downregulated). General stress-related genes constituted the only recognizable functional cluster whose expression was increased upon cryptolepine treatment, making up about 20% of upregulated genes. In contrast, analysis of the characteristics of downregulated genes revealed a specific effect of cryptolepine on genes related to iron transport or acid phosphatases, as well as a significant proportion of genes related to cell wall components. The effects of cryptolepine on the transcription of iron transport-related genes were consistent with a loss of function of the iron sensor Aft1p, indicating a possible disruption of iron metabolism in S. cerevisiae. Since the interference of cryptolepine with iron metabolism is considered one of its putative antimalarial targets, this finding supports the utility of S. cerevisiae in drug-developing schemes.


GAGA protein is required for multiple aspects of Drosophila oogenesis and female fertility.
Investigation of Drosophila oogenesis provides the opportunity to understand conservative genetic mechanisms underlying fertile female gamete development. In this study, we showed that the Drosophila DNA-binding protein GAGA factor (GAF) had a multifunctional role in oogenesis and it is involved in the regulation of this process genetic program. We studied the influence on Drosophila oogenesis of a number of mutations in the 5' region of the Trl gene that encodes GAF. We found that our originally generated Trl mutations lead to a decrease in transcriptional gene activity and levels of GAF expression in both germline and follicular cells. Cytological (fluorescence and electron microscopy) analysis showed that GAF loss resulted in multiple oogenesis defects. Mutations affected the actin cytoskeleton, leading to decrease of cytoplasmic filaments in nurse cells and basal actin in follicular cells. GAF depletion also leads to abnormal follicular cells migration, both border and centripetal. In addition, mutant ovaries demonstrated abnormalities in germ cells, including mitochondria, endoplasmic reticulum, karyosome organization, yolk granule formation and selective transport. Loss of GAF also promoted excessive cell death and egg chamber degradation. In sum, these defects caused very high or full female sterility. Since one of the main GAF activities is regulation of transcription, the complex phenotypes of the Trl mutants might be the consequence of its multiple target genes misexpression.


Provider choice and use of mental health care: implications for gatekeeper models.
To examine the ways in which the costs of nonresidential mental health care depend on (1) the type of provider who initiates the treatment episode and (2) the level of cost sharing imposed on the patient. The 1987 National Medical Expenditure Survey, a national probability sample of the U.S. civilian, noninstitutionalized population. Data were collected during four personal interviews conducted during 1987 and 1988. Key variables include the type of provider contacted at the beginning of treatment (psychiatrist, other physician, nonmedical mental health care specialist) and the cost (total actual payments from all sources) for the treatment episode. An episodic model of demand for mental health care is estimated using a two-step procedure. Multinomial probit analysis is first used to determine the factors that influence the choice of initial provider type. Right-censored Tobit analysis is used to determine the factors that affect the costs of care, including the type of provider who initiates the care episode. Results indicate that out-of-pocket price does significantly (p < .05) affect the patient's initial choice of provider type but that, after controlling for the endogeneity of provider choice, price is no longer significant in explaining overall treatment costs. After controlling for selection effects, care episodes initiated by nonspecialist physicians are found to be as expensive as those initiated by psychiatrists and significantly more expensive than episodes initiated by nonphysicians. The results suggest that nonmedical mental health care specialists may be more effective than physicians in controlling costs when used as case managers in the care of persons with mental illnesses.


Second primary cancer after surgery for colorectal cancer without lymph node metastasis.
Colorectal cancer patients without lymph node metastasis usually show a favorable prognosis with low recurrence rates; however, there is an increased risk of the development of a second primary cancer. Understanding the features of a second primary cancer is important to establish an effective postoperative follow-up program for colorectal cancer without lymph node metastasis. The clinicopathological data on 801 patients with Dukes' A and Dukes' B colorectal cancer were examined in respect to second primary cancer. In patients with Dukes' A cancer, the incidences of recurrence and second primary cancer were similar. When tumor invasion was limited within subserosa in Dukes' B patients, the incidence of a second primary cancer was almost two-thirds that of recurrence. More than half of the second primary cancers again developed from the colorectum, followed by stomach and lung. When colorectal cancer patients without lymph node metastasis show tumor invasion limited within the subserosa, postoperative follow-up should monitor a balance of recurrence with a second primary cancer.


Expression profiling of the ovarian surface kinome reveals candidate genes for early neoplastic changes.
We tested the hypothesis that co-coordinated up-regulation or down-regulation of several ovarian cell surface kinases may provide clues for better understanding of the disease and help in rational design of therapeutic targets. We compared the expression signature of 69 surface kinases in normal ovarian surface epithelial cells (OSE), with OSE from patients at high risk and with ovarian cancer. Seven surface kinases, ALK, EPHA5, EPHB1, ERBB4, INSRR, PTK, and TGFbetaR1 displayed a distinctive linear trend in expression from normal, highrisk, and malignant epithelium. We confirmed these results using semiquantitative reverse transcription-polymerase chain reaction and tissue array of 202 ovarian cancer samples. A strong correlate was shown between disease-free survival and the expression of ERBB4. DNA sequencing revealed two novel mutations in ERBB4 in two cancer samples. A distinct subset of the ovarian surface kinome is altered in the transition from high risk to invasive cancer and genetic mutation is not a dominant mechanism for these modifications. These results have significant implications for early detection and targeted therapeutic approaches for women at high risk of developing ovarian cancer.


Increased enzymatic activity of renin and hyperlipidemia.
We have previously reported that the in vitro enzymatic activity of exogenous renin, plasma renin reactivity (PRR), is increased in plasma of patients with chronic renal failure, possibly due to the deficiency of a renin inhibitor. To determine whether increases PRR is related to renal failure per se or to hyperlipidemia, PRR was measured in 10 control subjects, 10 patients with renal failure, and 10 hyperlipidemic patients with normal renal function. Compared to that in control subjects (52.6 ng angiotensin I generated per ml/h +/- 3.8 SE) PRR was increased (P < 0.05) in plasma of uremic patients (65.1 +/- 4.3) and hyperlipidemic patients (71.4 +/- 10.7). Renin substrate concentration did not differ among groups, and after denaturation of endogenous substrate by acidification of plasma, PRR was still increased. A "protein-free" extract of plasma from normal subjects inhibited renin, whereas little or no inhibition occurred with a comparable extract from uremic patients and hyperlipidemic patients. Thus, alterations in lipid metabolism may account for the increased enzymatic activity of renin in uremic plasma. Increased PRR may be related to the deficiency of a normally occurring renin inhibitor.


[Risk and crisis management by anesthesiologists regarding 'Guidelines for Actions Against Intraoperative Critical Hemorrhage' published by the Japanese Society of Anesthesiologists and the Japan Society of Transfusion Medicine and Cell Therapy].
According to a survey of anesthesia-related critical incidents by the Japanese Society of Anesthesiologists, hemorrhage was the major cause of cardiac arrest developing in the operating room. To deal with critical hemorrhage swiftly, not only cooperation between anesthesiologists and surgeons but also the linkage of operating rooms with transfusion management divisions and the blood center is important. It is desirable for the hospital transfusion committee to prepare hospital regulations on 'actions to be taken to manage critical hemorrhage', and practice the implementation of these guidelines by simulated drills. When critical hemorrhage occurs, a person in charge is appointed, and an emergency is declared (call for manpower and notification of the emergency to the transfusion management divisions). A person in charge comprehensively assesses the hemostatic condition, hemodynamics, laboratory data, and blood product supply system, and consults the operator regarding the continuation of surgery or changing surgical procedures. When time is short, the cross-matching test is omitted, and the ABO-identical blood is used. When a supply of the identical ABO-type blood is not available, compatible blood type is used. The evolving concept of hemostatic resuscitation seems to be important to prevent coagulopathy, which easily develops during massive hemorrhage. Anesthesiologists should be aware of the risk of such an emergency transfusion and procedures to be taken to switch to transfusion of the ABO-identical blood. Establishment of a hospital emergency transfusion system depends on the overall capability of the critical and crisis management systems of the hospital.


The effects of vasoconstrictor on transmission of impulses in the peripheral nerve.
The effects of extraneural and intramuscular applications of l-adrenaline on peripheral nerve function were examined. Change in the compound action potential was monitored until a complete conduction block was obtained if it occurred within 100 min. Extraneural application of 0.5 ml l-adrenaline to an isolated segment of the sciatic nerve of the rat in three concentrations of 1:100,000; 1:10,000, and 1:2,000 resulted in complete conduction block within 8 min. However, intramuscular injections of similar concentrations resulted in concentration dependent neural blockades with the highest concentration causing conduction blockade in 27.1 +/- 8.0 min.


Isoproterenol stimulates rapid extrusion of sodium from isolated smooth muscle cells.
beta-Agonists cause an inhibition of contractility and a transient stimulation of Na+/K+ pumping in smooth muscle cells of the stomach from the toad Bufo marinus. To determine if the stimulation of Na+/K+ pumping causes changes in intracellular [Na+] ([Na+]i) that might link Na+ pump stimulation to decrease Ca2+ availability for contraction, [Na+]i was measured in these cells with SBFI, a Na(+)-sensitive fluorescent indicator. Basal [Na+]i was 12.8 +/- 4.2 mM (n = 32) and was uniform throughout the cell. In response to isoproterenol, [Na+]i decreased an average of 7.1 +/- 1.1 mM in 3 sec. Since this decrease in [Na+]i could be completely blocked by inhibition of the Na+ pump, or by blockade of the beta-receptor, [Na+]i reduction is the result of occupation of the beta-receptor by isoproterenol and subsequent stimulation of the Na+ pump. 8-Bromoadenosine 3',5'-cyclic monophosphate and forskolin mimicked the effect of isoproterenol, indicating that the sequence of events linking beta-receptor occupation to Na+ pump stimulation most likely includes activation of adenylate cyclase, production of cAMP, and stimulation of cAMP-dependent protein kinase. The decrease in [Na+]i is sufficiently large and fast that it is expected to stimulate turnover of the Na+/Ca2+ exchanger in the Ca2+ extrusion mode, thereby accounting for the observed linkage between stimulation of the Na+/K+ pump and inhibition of contractility in response to beta-adrenergic agonists.


The discovery of insulin revisited: lessons for the modern era.
2021 to 2022 marks the one hundredth anniversary of ground-breaking research in Toronto that changed the course of what was, then, a universally fatal disease: type 1 diabetes. Some would argue that insulin's discovery by Banting, Best, Macleod, and Collip was the greatest scientific advance of the 20th century, being one of the first instances in which modern medical science was able to provide lifesaving therapy. As with all scientific discoveries, the work in Toronto built upon important advances of many researchers over the preceding decades. Furthermore, the Toronto work ushered in a century of discovery of the purification, isolation, structural characterization, and genetic sequencing of insulin, all of which influenced ongoing improvements in therapeutic insulin formulations. Here we discuss the body of knowledge prior to 1921 localizing insulin to the pancreas and establishing insulin's role in glucoregulation, and provide our views as to why researchers in Toronto ultimately achieved the purification of pancreatic extracts as a therapy. We discuss the pharmaceutical industry's role in the early days of insulin production and distribution and provide insights into why the discoverers chose not to profit financially from the discovery. This fascinating story of bench-to-beside discovery provides useful considerations for scientists now and in the future.


Application of a rapid and efficient h.p.l.c. method to measure bilirubin and its conjugates from native bile and in model bile systems. Potential use as a tool for kinetic reactions and as an aid in diagnosis of hepatobiliary disease.
We have developed an extremely rapid and efficient reverse-phase h.p.l.c. method for the measurement of bilirubin and its conjugates in human bile and in model bile systems. Our method involves the use of a Perkin-Elmer 3 mu C18 column and a methanol/sodium acetate/aq. ammonium acetate buffer system. Three isomers of bilirubin diglucuronide (BDG), two isomers of bilirubin monoglucuronide (BMG), three isomers of unconjugated bilirubin (UCB) and minor conjugates containing glucose and xylose were separated in 12 min. Initial quantification of BDG and BMG was based on the use of the ethyl anthranilate azo derivative of bilirubin (AZO UCB); however, the standard curves for BDG, BMG and UCB were similar enough to permit quantification to be later based on the UCB standard curve only, thereby simplifying the quantification process. Routine direct injection of 6 or 10 microliter of crude undiluted or diluted (1:1) bile sample was sufficient for analysis. The method was helpful in diagnosing biliary-tract obstruction in a newborn and a partial deficiency state of bilirubin conjugation (Crigler-Najjar syndrome) in a 10-year-old male. When the method was applied to biles of patients both with and without gallstones, levels of UCB were less than 2% of total pigment, consistent with previous reports. Because of its speed and efficiency, this method has the potential for a broad range of applications including enzymic, kinetic and bile sample analyses.


Modeling cross-modal enhancement and modality-specific suppression in multisensory neurons.
Cross-modal enhancement (CME) occurs when the neural response to a stimulus of one modality is augmented by another stimulus of a different modality. Paired stimuli of the same modality never produce supra-additive enhancement but may produce modality-specific suppression (MSS), in which the response to a stimulus of one modality is diminished by another stimulus of the same modality. Both CME and MSS have been described for neurons in the deep layers of the superior colliculus (DSC), but their neural mechanisms remain unknown. Previous investigators have suggested that CME involves a multiplicative amplifier, perhaps mediated by N-methyl D-aspartate (NMDA) receptors, which is engaged by cross-modal but not modality-specific input. We previously postulated that DSC neurons use multisensory input to compute the posterior probability of a target using Bayes' rule. The Bayes' rule model reproduces the major features of CME. Here we use simple neural implementations of our model to simulate both CME and MSS and to argue that multiplicative processes are not needed for CME, but may be needed to represent input variance and covariance. Producing CME requires only weighted summation of inputs and the threshold and saturation properties of simple models of biological neurons. Multiplicative nodes allow accurate computation of posterior target probabilities when the spontaneous and driven inputs have unequal variances and covariances. Neural implementations of the Bayes' rule model account better than the multiplicative amplifier hypothesis for the effects of pharmacological blockade of NMDA receptors on the multisensory responses of DSC neurons. The neural implementations also account for MSS, given only the added hypothesis that input channels of the same modality have more spontaneous covariance than those of different modalities.


A Standardized Arabic Reading Acuity Chart: The Balsam Alabdulkader-Leat Chart.
The aim of this study was to develop and validate the first standardized Arabic continuous text near-visual-acuity chart, the Balsam Alabdulkader-Leat (BAL) chart. Three versions of the BAL chart were created from previously validated sentences. Reading acuity (RA) and reading speed in standard-length words per minute (SLWPM) were measured for three versions of the BAL chart and three English charts (MNREAD, Colenbrander, and Radner) for 86 bilingual adults with normal vision aged 15 to 59 years. The RA and SLWPM were compared using analysis of variance. To analyze agreement between the charts, Bland-Altman plots were used. Normal visual acuity (0.00 logMAR [log minimum angle of resolution]) was calibrated for the BAL chart with linear regression analysis. Average RAs for BAL1, BAL2, and BAL3 were 0.62, 0.64 and 0.65 log-point print, respectively, which were statistically significantly different (repeated-measures analysis of variance, P < .05), but not considered clinically significant. The coefficients of agreement for RA between the BAL charts were 0.054 (between 1 and 2), 0.061 (between 2 and 3), and 0.059 (between 1 and 3). Linear regression between the average RA for the BAL chart and the MNREAD and Radner charts showed that 0.7 log-point size at 40 cm is equivalent to 0.00 logMAR, and the new BAL chart was labeled accordingly. Mean SLWPM for the BAL charts was 201, 195, and 195 SLWPM, respectively, and for the Colenbrander, MNREAD, and Radner charts was 146, 171, and 146, respectively. The coefficients of agreement for log-SLWPM between BAL1 and BAL2, BAL2 and BAL3, and BAL1 and BAL3 were 0.063, 0.064, and 0.057 log-SLWPM, respectively. The BAL chart showed high interchart agreement. It is recommended for accurate near performance measures in Arabic for both research and clinical settings.


Ginkgo biloba leaf extract (EGb 761®) and its specific acylated flavonol constituents increase dopamine and acetylcholine levels in the rat medial prefrontal cortex: possible implications for the cognitive enhancing properties of EGb 761®.
Experimental and clinical data suggest that the Ginkgo biloba standardized extract EGb 761® exerts beneficial effects in conditions which are associated with impaired cognitive function. However, the neurochemical correlates of these memory enhancing effects are not yet fully clarified. The aim of this study was to examine the effect of repeated oral administration of EGb 761® and some of its characteristic constituents on extracellular levels of dopamine (DA), noradrenaline (NA), serotonin (5-HT), acetylcholine (ACh) and the metabolites 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) in the medial prefrontal cortex (mPFC) of awake rats by use of in vivo microdialysis technique. Subacute (14 days, once daily), but not acute, oral treatment with EGb 761® (100 and 300 mg/kg) or the flavonoid fraction, which represents about 24% of the whole extract caused a significant and dose-dependent increase in extracellular DA levels in the mPFC. Repeated administration of EGb 761® also caused a modest but significant increase in the NA levels, whereas the concentrations of 5-HT and those of the metabolites DOPAC, HVA and 5-HIAA were not affected. The same treatment regimen was used in a subsequent study with the aim of investigating the effects of two Ginkgo-specific acylated flavonols, 3-O-(2''-O-(6'''-O-(p-hydroxy-trans-cinnamoyl)-β-D-glucosyl)-α-L-rhamnosyl)quercetin (Q-ag) and 3-O-(2''-O-(6'''-O-(p-hydroxy-trans-cinnamoyl)-β-D-glucosyl)-α-L-rhamnosyl)kaempferol (K-ag). Both compounds together represent about 4.5% of the whole extract. Repeated oral treatment with Q-ag (10 mg/kg) for 14 days caused a significant increase in extracellular DA levels of 159% and extracellular acetylcholine (ACh) levels of 151% compared to controls. Similarly, administration of K-ag (10 mg/kg) induced a significant rise of DA levels to 142% and ACh levels to 165% of controls, whereas treatment with isorhamnetin, an O-methylated aglycon component of EGb 761® flavonol glycosides had no effect. None of the tested flavonoids had a significant effect on extracellular DOPAC and HVA levels. The present findings provide evidence that the subacute treatment with EGb 761® and its flavonol constituents increases DA and ACh release in the rat mPFC, and suggest that the two Ginkgo-specific acylated flavonol glycosides Q-ag and K-ag are active constituents contributing to these effects. As seen for isorhamnetin, the effect on neurotransmitter levels seems not to be a general effect of flavonols but rather to be a specific action of acylated flavonol glycosides which are present in EGb 761®. The direct involvement of these two flavonol derivatives in the increase of dopaminergic and cholinergic neurotransmission in the prefrontal cortex may be one of the underlying mechanisms behind the reported effects of EGb 761® on the improvement of cognitive function.


Aryl hydrocarbon hydroxylase induction in human leukocytes.
A method for determining aryl hydrocarbon hydroxylase induction in human leukocytes is described. Leukocytes from healthy volunteers were cultured in the presence of phytohemagglutinin, a mitogen. Addition of 3-methylcholanthrene to 72-hour cultures induced a fourfold increase in aryl hydroxylase activity. In the absence of a mitogenic agent, 3-methylcholanthrene stimulation of increased enzymatic activity did not occur.


Self-efficacy and outcome expectations for quitting among adolescent smokers.
Relatively little is known about smoking cessation self-efficacy and outcome expectations for quitting smoking in adolescent smokers. In this study, we created measures of these two constructs and conducted factor analyses with data from a diverse sample of 1126 adolescent smokers. Results yielded a two-factor solution for the self-efficacy measure, and a four-factor solution for the outcome expectations scale. In a subset of the original sample (n=515), we re-administered the measures one year later and also examined the longitudinal associations between the baseline sub-scale scores and cigarettes smoked per week at follow-up. Results revealed significant relationships between the negative affect sub-scales of the self-efficacy and outcome expectations measures and weekly smoking level. These associations remained when adjusting for baseline smoking level and other sub-scales. The findings lend support for the possible role of affect regulation in smoking reduction in adolescents.


Myrica faya: a new source of antioxidant phytochemicals.
Myrica faya is a fruit tree endemic of the Macaronesia (Azores, Madeira, and Canary Island), and its edible fruits are known as "amorinhos" (little loves), bright red to purple berries, used fresh and in jams and liquors. The phenolic composition and antioxidant capacity of leaves and berries from M. faya are presented here for the first time. The screening of phytochemical compounds was carried out using high-performance liquid chromatography with online UV and electrospray ionization mass spectrometric detection (HPLC-DAD-ESI-MS(n)). There were 55 compounds characterized, mostly galloyl esters of flavonoids and phenolic acids; 26 of the identified compounds (anthocyanins, isoflavonoids, lignans, terpenes, fatty acids, and phenylethanoids) have not been reported in Myrica genus so far. From the data presented here, it can be concluded that faya berries represent a rich source of cyanidin-3-glucoside, flavonoids, and vitamin C. In fact, higher antioxidant activity than that of the well-known Myrica rubra berries (Chinese bayberry) has been observed.


Identification of protein ligands in complex biological samples using intensity-fading MALDI-TOF mass spectrometry.
The easy detection of biomolecular interactions in complex mixtures using a minimum amount of material is of prime interest in molecular and cellular biology research. In this work, a mass spectrometry MALDI-TOF based approach, which we call intensity-fading (IF MALDI-TOFMS), and which was designed for just such a purpose, is reported. This methodology is based on the use of the MALDI ion intensities to detect quickly the formation of complexes between nonimmobilized biomolecules in which a protein is one of the partners (protein-protein, protein-peptide, protein-organic molecule, and protein-nucleic acid complexes). The complex is detected through the decrease (fading) of the molecular ion intensities of the partners as directly compared to the MALDI mass spectrum of the mixture (problem and control molecules) following the addition of the target molecule. The potential of the approach is examined in several examples of model interactions, mainly involving small nonprotein and protein inhibitors of proteases, at both the qualitative and semiquantitative levels. Using this method, different protein ligands of proteolytic enzymes in total extracts of invertebrate organisms have been identified in a simple way. The proposed procedure should be easily applied to the high-throughput screening of biomolecules, opening a new experimental strategy in functional proteomics.


Conformational changes in the C terminus of Shaker K+ channel bound to the rat Kvbeta2-subunit.
We studied the structure of the C terminus of the Shaker potassium channel. The 3D structures of the full-length and a C-terminal deletion (Delta C) mutant of Shaker were determined by electron microscopy and single-particle analysis. The difference map between the full-length and the truncated channels clearly shows a compact density, located on the sides of the T1 domain, that corresponds to a large part of the C terminus. We also expressed and purified both WT and Delta C Shaker, assembled with the rat KvBeta2-subunit. By using a difference map between the full-length and truncated Shaker alpha-beta complexes, a conformational change was identified that shifts a large part of the C terminus away from the membrane domain and into close contact with the Beta-subunit. This conformational change, induced by the binding of the KvBeta2-subunit, suggests a possible mechanism for the modulation of the K+ voltage-gated channel function by its Beta-subunit.


Factors affecting the growth of Pseudomonas fluorescens in liquid egg white.
Pseudomonas fluorescens grew rapidly in fresh egg albumen diluted with water. Growth of the bacteria in egg albumen was stimulated by the addition of carbohydrate and ovomucoid-rich egg exudate. Polyacrylamide-gel electrophoresis for residual egg albumen revealed extensive proteolysis of albumen inoculated with the organism. A fluorescent compound with absorption maximum at 408 nm was isolated from a defined salt medium inoculated with P. fluorescens. It shortened the lag phase and increased the final cell yield of the organism when added to the salt medium (100 mug/ml).


Capillary Electrophoresis Assessment of Plasma Protein Changes in an African Penguin (Spheniscus demersus) With Aspergillosis.
A decrease of avian biodiversity in the African continent has been the result of anthropogenic pressure in the region. This has resulted in the African penguin (Spheniscus demersus) being placed on the endangered species list and requires conservation efforts to maintain its free-ranging population and placement under managed care. In the latter environment, infection by Aspergillus fumigatus can be common. The diagnosis and treatment of this fungal disease in birds has presented with many difficulties, largely due to the diversity and limited knowledge that exists about this species. In this study, we implement a high-resolution capillary electrophoresis system for the fractionation of African penguin plasma, followed by mass spectrometry analysis for the identification of proteins associated with aspergillosis. Several protein differences were revealed, including changes in acute phase proteins and lipid metabolism. In addition, our results demonstrated that fibrinogen β chain is a protein largely present during the inflammatory process in an African penguin infected with A. fumigatus. These findings present a new avenue for the measurement of plasma proteins as a potential method for identifying important biomarkers to aid in monitoring African penguin health.


Lumbar spine loads during the lifting of extremely heavy weights.
The reaction moments at the knee, hip, and L4/L5 joints, and the compressive and shearing forces on L4/L5 are documented in powerlifters competing in a national powerlifting championship. Analyses were made of 13 female and 44 male competitors. The joint moments and forces were estimated from a linked segment model (WATBAK) that incorporated functional low back extensor musculature with a moment arm of 6 cm and a line action that was oriented 5 degrees posteriorly to the L4/L5 compression axis. This oblique orientation of the extensor muscles reduced the anterior shearing load on the vertebral motion unit. Average compressive loads on L4/L5 were estimated up to 17,192 N while the highest average L4/L5 and hip moments were 988 and 1047 N.m, respectively. The sumo deadlift style resulted in a 10% reduction in the joint moment and 8% reduction in the load shear force at the L4/L5 level when compared with the conventional lifting style. Formulation of linear regression equations to predict the load lifted using reaction joint moments yielded substantial unexplained variability, though significant relationships were found. This analysis suggested that there is large variability in the pattern of loading joints among national class powerlifters.


Bleaching effect of ozone on pigmented teeth.
There have been numerous researches on ozone application in dentistry; yet the data regarding its whitening effect is very limited. The present study compares the bleaching effect of ozone with office bleaching. In this experimental study, 15 maxillary premolar teeth were selected and sectioned mesio-distally and bucco-lingually. The sections were then placed in tea for 1 week according to the Sulieman method and were divided into three groups each comprised of 15 sections. The samples were bleached as followed; Group I: Bleached with 35% hydrogen peroxide in three intervals of 8 min each, Group II: Underwent ozone treatment using Ozotop unite for 4 min and Group III: Bleached with a combination of both methods. The color indices of the samples, i.e., (a) green-red pigment, (b) blue-yellow pigment, (L) brightness, (ΔE) overall color change, were evaluated pre- and post-bleaching utilizing a digital camera, Photoshop software and CIE lab index. The color changes of specimens then were calculated and analyzed through randomized analysis of variance and Tukey tests. P < 0.001 was considered to be significant. The color change (ΔE) in Group II was significantly lower than those in the two other groups (P < 0.001). There was no significant difference between the color change of Groups I and III (P = 0.639). In addition, the results of L, a and b brought forth a similar pattern to the findings obtained from ΔE. The hydrogen peroxide gel has a more powerful whitening effect than ozone; in addition, ozone has no synergistic effect when is used simultaneously with hydrogen peroxide.


Preservation of comet assay slides: comparison with fresh slides.
The single cell gel electrophoresis assay (comet assay) is an inexpensive, rapid and highly sensitive method for the determination of DNA damage, crosslinks, and alkaline-labile lesions in individual cells. A limitation of the procedure is that the microelectrophoretic gels must be scored rapidly as the comet configuration deteriorates on storage due to dehydration of the agarose and diffusion of DNA. The objectives of this study were firstly to evaluate drying regimes as rapid and simple methods of preservation of the microgels as close to their original fresh state as possible, and secondly to examine the effects of storage of the slides. Human hepatoma (HepG2) cells challenged for 30 min with hydrogen peroxide (H(2)O(2)) were used in the study. Microgel slides were prepared and evaluated immediately, or after drying with or without a methanol fixation step. Microgels that were dried at a variety of temperatures (22-50 degrees C) and re-hydrated did not differ in the values obtained for H(2)O(2)-induced DNA damage when compared to fresh samples. Samples could also be continually dried and re-hydrated over a period of up to 3 months with no obvious loss of information. In conclusion, drying of microgels represents a simple and inexpensive method of preserving comet assay slides.


Influence of combat blast-related mild traumatic brain injury acute symptoms on mental health and service discharge outcomes.
Assessment of acute mild traumatic brain injury (mTBI) symptoms after a combat blast could aid diagnosis and guide follow-up care. Our objective was to document acute mTBI symptoms following a combat blast and to examine associations between acute symptoms and mental health and service discharge outcomes. A retrospective cohort study was conducted with 1656 service personnel who experienced a combat blast-related mTBI in Iraq. Acute mTBI symptoms were ascertained from point-of-injury medical records. The associations between acute symptoms and posttraumatic stress disorder (PTSD), postconcussion syndrome (PCS), and type of service discharge were examined. Disability discharge occurred in 11% of patients, while 36% had a non-disability discharge and 52% had no recorded discharge. A PTSD and PCS diagnosis was made in 19% and 15% of the sample, respectively. The most common acute mTBI symptoms were headache (62.8%), loss of consciousness (LOC) (34.5%), and tinnitus (33.2%). LOC was predictive of PTSD (odds ratio [OR] 1.54; 95% confidence interval [CI] 1.18, 2.00) and PCS (OR 2.08; 95% CI 1.56, 2.77), while altered mental status (OR 1.53; 95% CI 1.07, 2.17) and previous blast history (OR 1.83; 95% CI 1.15, 2.90) also were predictive of PCS. While no acute mTBI symptoms were associated with discharge outcomes, injury severity was associated with disability discharge. LOC after blast-related mTBI was associated with PTSD and PCS, and injury severity was predictive of disability discharge. The assessment of cognitive status immediately after a blast could assist in diagnosing mTBI and indicate a need for follow-up care.


RET-mediated modulation of tumor microenvironment and immune response in multiple endocrine neoplasia type 2 (MEN2).
Medullary thyroid carcinomas (MTC) arise from thyroid parafollicular, calcitonin-producing C-cells and can occur either as sporadic or as hereditary diseases in the context of familial syndromes, including multiple endocrine neoplasia 2A (MEN2A), multiple endocrine neoplasia 2B (MEN2B) and familial MTC (FMTC). In a large fraction of sporadic cases, and virtually in all inherited cases of MTC, activating point mutations of the RET proto-oncogene are found. RET encodes for a receptor tyrosine kinase protein endowed with transforming potential on thyroid parafollicular cells. As in other cancer types, microenvironmental factors play a critical role in MTC. Tumor-associated extracellular matrix, stromal cells and immune cells interact and influence the behavior of cancer cells both in a tumor-promoting and in a tumor-suppressing manner. Several studies have shown that, besides the neoplastic transformation of thyroid C-cells, a profound modification of tumor microenvironment has been associated to the RET FMTC/MEN2-associated oncoproteins. They influence the surrounding stroma, activating cancer-associated fibroblasts (CAFs), promoting cancer-associated inflammation and suppressing anti-cancer immune response. These mechanisms might be exploited to develop innovative anti-cancer therapies and novel prognostic tools in the context of familial, RET-associated MTC.


Adipogenic Activity of Chemicals Used in Plastic Consumer Products.
Bisphenols and phthalates, chemicals frequently used in plastic products, promote obesity in cell and animal models. However, these well-known metabolism-disrupting chemicals (MDCs) represent only a minute fraction of all compounds found in plastics. To gain a comprehensive understanding of plastics as a source of exposure to MDCs, we characterized the chemicals present in 34 everyday products using nontarget high-resolution mass spectrometry and analyzed their joint adipogenic activities by high-content imaging. We detected 55,300 chemical features and tentatively identified 629 unique compounds, including 11 known MDCs. Importantly, the chemicals extracted from one-third of the products caused murine 3T3-L1 preadipocytes to proliferate, and differentiate into adipocytes, which were larger and contained more triglycerides than those treated with the reference compound rosiglitazone. Because the majority of plastic extracts did not activate the peroxisome proliferator-activated receptor γ and the glucocorticoid receptor, the adipogenic effects are mediated via other mechanisms and, thus, likely to be caused by unknown MDCs. Our study demonstrates that daily-use plastics contain potent mixtures of MDCs and can, therefore, be a relevant yet underestimated environmental factor contributing to obesity.


Are Level of Education and Employment Related to Symptoms of Common Mental Disorders in Current and Retired Professional Footballers?
Mental disorders have become a topic of increasing interest in research due to their serious consequences for quality of life and functioning. The objective of this study was to explore the relationship of level of education, employment status and working hours with symptoms of common mental disorders (distress, anxiety/depression, sleep disturbance, adverse alcohol behaviour, smoking, adverse nutritional behaviour) among current and retired professional footballers. Cross-sectional analyses were conducted on baseline questionnaires from an ongoing prospective cohort study among current and retired professional footballers. Based on validated scales, an electronic questionnaire was set up and distributed by players' unions in 11 countries across three continents. A total of 607 current professional footballers (mean age of 27 years) and 219 retired professional footballers (mean age of 35 years) were involved in the study. Among retired professional footballers, statistically significant negative correlations were found between employment status and symptoms of distress and anxiety/depression (P < 0.05), as well as between number of working hours and symptoms of anxiety/depression (P < 0.05). No other statistically significant associations were found among retired players. Among current professional footballers, level of education was not associated with symptoms of common mental disorders. Among retired professional footballers, employment status as well as a higher number of working hours was weakly correlated to symptoms of distress and anxiety/depression. Combining a football career with sustainable attention for educational and career planning might be important and of high priority.


Glycine as an irrigant during microsurgical hemostasis. A microsurgeon's observations.
The "drip-stop-coagulate" technique used during microsurgery is tedious and, at times, frustrating when electrolyte-rich solutions are used as an irrigant. Glycine, a nonelectrolyte solution, allows the gynecologist to apply electrosurgical energy in a fluid medium; lactated Ringer's solution, the irrigant frequently used by microsurgeons, will not. When, during microsurgery, glycine is used in place of lactated Ringer's solution, the origin of the bleeding site can be easily isolated and coagulated during irrigation, assisting the surgeon in precise and complete hemostasis.


A task-specific deep-learning-based denoising approach for myocardial perfusion SPECT.
Deep-learning (DL)-based methods have shown significant promise in denoising myocardial perfusion SPECT images acquired at low dose. For clinical application of these methods, evaluation on clinical tasks is crucial. Typically, these methods are designed to minimize some fidelity-based criterion between the predicted denoised image and some reference normal-dose image. However, while promising, studies have shown that these methods may have limited impact on the performance of clinical tasks in SPECT. To address this issue, we use concepts from the literature on model observers and our understanding of the human visual system to propose a DL-based denoising approach designed to preserve observer-related information for detection tasks. The proposed method was objectively evaluated on the task of detecting perfusion defect in myocardial perfusion SPECT images using a retrospective study with anonymized clinical data. Our results demonstrate that the proposed method yields improved performance on this detection task compared to using low-dose images. The results show that by preserving task-specific information, DL may provide a mechanism to improve observer performance in low-dose myocardial perfusion SPECT.


Efficient replication of over 180 genetic associations with self-reported medical data.
While the cost and speed of generating genomic data have come down dramatically in recent years, the slow pace of collecting medical data for large cohorts continues to hamper genetic research. Here we evaluate a novel online framework for obtaining large amounts of medical information from a recontactable cohort by assessing our ability to replicate genetic associations using these data. Using web-based questionnaires, we gathered self-reported data on 50 medical phenotypes from a generally unselected cohort of over 20,000 genotyped individuals. Of a list of genetic associations curated by NHGRI, we successfully replicated about 75% of the associations that we expected to (based on the number of cases in our cohort and reported odds ratios, and excluding a set of associations with contradictory published evidence). Altogether we replicated over 180 previously reported associations, including many for type 2 diabetes, prostate cancer, cholesterol levels, and multiple sclerosis. We found significant variation across categories of conditions in the percentage of expected associations that we were able to replicate, which may reflect systematic inflation of the effects in some initial reports, or differences across diseases in the likelihood of misdiagnosis or misreport. We also demonstrated that we could improve replication success by taking advantage of our recontactable cohort, offering more in-depth questions to refine self-reported diagnoses. Our data suggest that online collection of self-reported data from a recontactable cohort may be a viable method for both broad and deep phenotyping in large populations.


A role for replication repair in the genesis of templated mutations.
Replication repair mediates error-free bypass of DNA damage in a series of steps that include regression of the replication fork, primer-terminus switching to use the other daughter strand as an undamaged template, primer extension, primer switching back to its cognate template with the primer terminus now having bypassed the damage, and fork rearrangement to a normal configuration. By both genetic and biochemical criteria, bacteriophage T4 catalyzes replication repair with two alternative sets of proteins, one including the gp32 SSB and the gp41 DNA helicase and the other including the UvsX recombinase. In each pathway, synthesis is conducted by the gp43 DNA polymerase. Here we show that defects in gp32, gp41 or UvsX that impair replication repair also increase mutation rates generally, but especially for templated mutations. Such templated mutations are associated with palindromic or direct repeats that are either perfect or imperfect. Models of templated mutagenesis require that the primer terminus switches to an ectopic template, but one that yields mutations instead of error-free bypass. We suggest that the proteins that conduct replication repair normally direct a blocked primer strand specifically to the other daughter strand with considerable accuracy, but that strand switching becomes promiscuous when these proteins are mutationally impaired, thus promoting templated mutations.


Physicochemical stability of cefiderocol, a novel siderophore cephalosporin, in syringes at 62.5 mg/mL for continuous administration in intensive care units.
Cefiderocol is a new siderophore time-dependent antibiotic of last resort. The manufacturer reports a stability of 6 hours for the infusion solution diluted in normal saline (NS) or dextrose 5% in water (D5W) for a concentration between 7.5 and 20 mg/mL. Optimising its effectiveness by continuous infusion is crucial. The aim of this work was to study the physicochemical stability of cefiderocol diluted in NS or D5W in polypropylene syringes for 48 hours at a concentration of 62.5 mg/mL stored at room temperature, protected or not from light. Three preparations for each condition were performed. At each time of the analysis, one sample for each preparation was analysed in triplicate by a validated high performance liquid chromatography method coupled to a photodiode array detector at 260 nm. Particle contamination, absorbance measurement, visual inspection and pH measurement were assessed. The limit of stability was set at 90% of the initial concentration, without physical modification. The linearity was validated with an R² of 0.9999. The coefficients of variation for repeatability and intermediate precision were less than 2%. In NS and D5W, cefiderocol retained more than 90% of the initial concentration after 12 hours in syringes, exposed or not to light. Two degradation products (nos 2 and 11, observed during forced degradation) were detected during the stability study. The absorbance at 410 nm increased progressively, regardless of the storage conditions. The particulate contamination test met the specifications of the container. pH values were all between 5.22 and 5.32. No visual changes were detected. In polypropylene syringes, cefiderocol 62.5 mg/mL (3 g in 48 mL) diluted in NS or D5W was stable for 12 hours at room temperature. These new data allow the use of cefiderocol in continuous infusion.


From proteomic analysis to clinical significance: overexpression of cytokeratin 19 correlates with hepatocellular carcinoma metastasis.
To better understand the mechanism underlying the hepatocellular carcinoma (HCC) metastasis and to search potential markers for HCC prognosis, differential proteomic analysis on two well-established HCC cell strains with high and low metastatic potentials, MHCC97-H and MHCC97-L, was conducted using two-dimensional gel electrophoresis followed by matrix-assisted laser desorption/time-of-flight mass spectrometry. Cytokeratin 19 (CK19) was identified and found to be overexpressed in MHCC97-H as compared with MHCC97-L. This result was further confirmed by two-dimensional Western blot analysis and immunofluorescence assay. Furthermore, one-dimensional Western blot analysis showed consistently increased CK19 expression in progressively more metastatic cells. Immunohistochemical study on 102 human HCC specimens revealed that more patients in the CK19-positive group had overt intrahepatic metastases (satellite nodules, p < 0.05; vascular tumor emboli, p < 0.001; tumor node metastatis staging, p < 0.001). CK19 fragment CYFRA 21-1 levels measured in sera from nude mice model of human HCC metastasis with radioimmunoassay increased in parallel with tumor progression and rose remarkably when pulmonary metastases occurred. The results demonstrated that overexpression of CK19 in HCC cells is related to metastatic behavior. Serum CK19 level might reflect the pathological progression in some HCC and may be a useful marker for predicting tumor metastasis and a therapeutic target for the treatment of HCC patients with metastases.


Combined Erdheim-Chester disease and Langerhans cell histiocytosis of skin are both monoclonal: a rare case with human androgen-receptor gene analysis.
Erdheim-Chester disease (ECD) is a rare xanthogranulomatous histiocytic disorder. Langerhans cell histiocytosis (LCH) is a proliferative disorder of histiocytes with a phenotype similar to dendritic Langerhans cells. Both are derived from myeloid stem cells in the bone marrow and, thus, can overlap. We report a rare case of hybrid LCH and ECD of the skin with systemic ECD. Pathologic examinations and human androgen-receptor gene assay were used to study this case. A 34-year-old woman presented with recurrent ulcerative skin lesions on both thighs associated with polydipsia and polyuria since childhood. Radiography revealed osteosclerosis of bilateral distal tibias and soft tissue masses of bilateral chest walls and ankles. Pathologically, the chest wall lesions showed dense aggregates of lipid-laden histiocytes, which were CD68(+)/CD163(+)/S100(-)/CD1a(-). Combined with the clinical and radiographic findings, this xanthogranulomatous infiltrate was consistent with ECD. However, thigh skin showed discrete foci of a xanthogranulomatous infiltrate and S100(+)/CD1a(+) Langerhans cells with eosinophils. In addition, Birbeck granules were found. Dissected tissues from both ECD and LCH were monoclonal, supporting their neoplastic nature. Single case report is a limitation. ECD and LCH may have a close association with divergent differentiation from the same stem cells under different microenvironmental conditions.


Interaction between specific forms of childhood maltreatment and the serotonin transporter gene (5-HTT) in recurrent depressive disorder.
There is inconsistent evidence of interaction between stressful events and a serotonin transporter promoter polymorphism (5-HTTLPR) in depression. Recent studies have indicated that the moderating effect of 5-HTTLPR may be strongest when adverse experiences have occurred in childhood and the depressive symptoms persist over time. However, it is unknown whether this gene-environment interaction is present for recurrent depressive disorder and different forms of maltreatment. Therefore, patients with recurrent clinically diagnosed depression and controls screened for the absence of depression were utilised to examine the moderating effect of 5-HTTLPR on associations between specific forms of childhood adversity and recurrent depression. A sample of 227 recurrent unipolar depression cases and 228 never psychiatrically ill controls completed the Childhood Trauma Questionnaire to assess exposure to sexual, physical and emotional abuse, physical and emotional neglect in childhood. DNA extracted from blood or cheek swabs was genotyped for the short (s) and long (l) alleles of 5-HTTLPR. All forms of childhood maltreatment were reported as more severe by cases than controls. There was no direct association between 5-HTTLPR and depression. Significant interactions with additive and recessive 5-HTTLPR genetic models were found for overall severity of maltreatment, sexual abuse and to a lesser degree for physical neglect, but not other maltreatment types. The cross-sectional design limits causal inference. Retrospective report of childhood adversity may have reduced the accuracy of the findings. This study provides support for the role of interplay between 5-HTTLPR and a specific early environmental risk in recurrent depressive disorder.


Ligand migration in the apolar tunnel of Cerebratulus lacteus mini-hemoglobin.
The large apolar tunnel traversing the mini-hemoglobin from Cerebratulus lacteus (CerHb) has been examined by x-ray crystallography, ligand binding kinetics, and molecular dynamic simulations. The addition of 10 atm of xenon causes loss of diffraction in wild-type (wt) CerHbO(2) crystals, but Leu-86(G12)Ala CerHbO(2), which has an increased tunnel volume, stably accommodates two discrete xenon atoms: one adjacent to Leu-86(G12) and another near Ala-55(E18). Molecular dynamics simulations of ligand migration in wt CerHb show a low energy pathway through the apolar tunnel when Leu or Ala, but not Phe or Trp, is present at the 86(G12) position. The addition of 10-15 atm of xenon to solutions of wt CerHbCO and L86A CerHbCO causes 2-3-fold increases in the fraction of geminate ligand recombination, indicating that the bound xenon blocks CO escape. This idea was confirmed by L86F and L86W mutations, which cause even larger increases in the fraction of geminate CO rebinding, 2-5-fold decreases in the bimolecular rate constants for ligand entry, and large increases in the computed energy barriers for ligand movement through the apolar tunnel. Both the addition of xenon to the L86A mutant and oxidation of wt CerHb heme iron cause the appearance of an out Gln-44(E7) conformer, in which the amide side chain points out toward the solvent and appears to lower the barrier for ligand escape through the E7 gate. However, the observed kinetics suggest little entry and escape (≤ 25%) through the E7 pathway, presumably because the in Gln-44(E7) conformer is thermodynamically favored.


Corralling a black swan: natural range of variation in a forest landscape driven by rare, extreme events.
The natural range of variation (NRV) is an important reference for ecosystem management, but has been scarcely quantified for forest landscapes driven by infrequent, severe disturbances. Extreme events such as large, stand-replacing wildfires at multi-century intervals are typical for these regimes; however, data on their characteristics are inherently scarce, and, for land management, these events are commonly considered too large and unpredictable to integrate into planning efforts (the proverbial "Black Swan"). Here, we estimate the NRV of late-seral (mature/old-growth) and early-seral (post-disturbance, pre-canopy-closure) conditions in a forest landscape driven by episodic, large, stand-replacing wildfires: the Western Cascade Range of Washington, USA (2.7 million ha). These two seral stages are focal points for conservation and restoration objectives in many regions. Using a state-and-transition simulation approach incorporating uncertainty, we assess the degree to which NRV estimates differ under a broad range of literature-derived inputs regarding (1) overall fire rotations and (2) how fire area is distributed through time, as relatively frequent smaller events (less episodic), or fewer but larger events (more episodic). All combinations of literature-derived fire rotations and temporal distributions (i.e., "scenarios") indicate that the largest wildfire events (or episodes) burned up to 105 -106 ha. Under most scenarios, wildfire dynamics produced 5th-95th percentile ranges for late-seral forests of ~47-90% of the region (median 70%), with structurally complex early-seral conditions composing ~1-30% (median 6%). Fire rotation was the main determinant of NRV, but temporal distribution was also important, with more episodic (temporally clustered) fire yielding wider NRV. In smaller landscapes (20,000 ha; typical of conservation reserves and management districts), ranges were 0-100% because fires commonly exceeded the landscape size. Current conditions are outside the estimated NRV, with the majority of the region instead covered by dense mid-seral forests (i.e., a regional landscape with no historical analog). Broad consistency in NRV estimates among widely varied fire regime parameters suggests these ranges are likely relevant even under changing climatic conditions, both historical and future. These results indicate management-relevant NRV estimates can be derived for seral stages of interest in extreme-event landscapes, even when incorporating inherent uncertainties in disturbance regimes.


Effects of Fusarium toxin-contaminated wheat grain on nutrient turnover, microbial protein synthesis and metabolism of deoxynivalenol and zearalenone in the rumen of dairy cows.
The aim of the present study was to examine the effects of feeding Fusarium toxin-contaminated wheat to dairy cows on nutrient utilization in the rumen and on duodenal flow of deoxynivalenol (DON), zearalenone (ZON) and their metabolites. Six dairy cows fitted with a large rumen cannula and a simple T-shaped cannula at the proximal duodenum was used in two experiments. The experiments included a control period in which the uncontaminated control wheat was fed and a period in which the control wheat was replaced by the Fusarium toxin-contaminated wheat (8.05 and 7.15 mg DON/kg and 0.26 and 0.1 mg ZON/kg in Expts 1 and 2 respectively). The wheat portion of the daily ration amounted to 50% on a dry matter (DM) basis and rations were completed with hay or grass silage. Five of the six cows were non-lactating and the total daily DM-intake ranged between 4 and 12 kg. The pH-values and the concentration of volatile fatty acids in ruminal fluid were not significantly influenced by feeding the contaminated wheat. In contrast, the postprandial ammonia concentration was consistently higher when the mycotoxin-contaminated wheat was fed. Moreover, the flow of microbial protein and utilizable protein at the duodenum were reduced at the same time. The concentrations of DON and ZON and of their metabolites in freeze-dried duodenal digesta were either not detectable or negligible during the control periods whereas distinct concentrations were measured during the periods where the contaminated wheat was fed. DON was nearly completely metabolized to de-epoxy-DON and the flow at the duodenum ranged between 4% and 28% of DON-intake. The ZON metabolites alpha-zearalenol (ZOL) and beta-ZOL were recovered at the duodenum beside the parent toxin ZON. Their recovery as a percentage of ZON-intake ranged between 43% and 132%. In conclusion, feeding of Fusarium toxin-contaminated wheat altered the ruminal protein utilization. The question of whether this effect was a result of the mycotoxin being present in the rumen or of Fusarium growth-related structural (cell wall) changes of the wheat grain needs to be clarified. The low recovery of DON at the duodenum would indicate either a nearly complete degradation of the molecule in the rumen or an absorption by the mucosa of the rumen, whereas the higher ZON recovery would suggest a lower degradation of the parent toxin in the rumen and/or recovery of some bile-originating entero-hepatic cycling ZON/metabolites.


Laboratory investigation of platelet function in patients with thalassaemia.
The aim of this study was to investigate platelet function in patients with thalassaemia and to detect any relation to chelation treatment (deferasirox or deferiprone/deferiprone plus desferioxamine). Thirty-three transfusion-dependent patients with thalassaemia were included. The investigation consisted of aggregation testing of platelet-rich plasma by light transmission aggregometry (LTA) with the use of 5 agonists as well as the global test of haemostasis by means of the PFA-100 platelet function analyser. In 66.67% of the patients, there was reduced LTA to at least one agonist and in 18.18% there was reduced LTA to two or more agonists. The PFA-100 test was prolonged in 60.6% of the cases. An abnormal LTA and a prolonged PFA-100 time were recorded in 33.3% of the patients and 27.4% had a normal aggregation and PFA-100 test. No correlation between chelation regimen and either LTA or PFA-100 test was found. The abnormal LTA can be explained either by the release of ADP from the haemolysed red blood cells, which leads to defective platelet aggregation, or by the presence of two platelet populations. An in vitro effect without an in vivo impact could be an alternative explanation. In patients with thalassaemia, the reduced LTA and the prolonged PFA-100 closure time could be an in vitro effect and has a close correlation to the bleeding phenotype of each patient.


Enzymatic oxidation of NADP+ to its 4-oxo derivative is a side-reaction displayed only by the adrenodoxin reductase type of ferredoxin-NADP+ reductases.
We have previously shown that Mycobacterium tuberculosis FprA, an NADPH-ferredoxin reductase homologous to mammalian adrenodoxin reductase, promotes the oxidation of NADP(+) to its 4-oxo derivative 3-carboxamide-4-pyridone adenine dinucleotide phosphate [Bossi RT, Aliverti A, Raimondi D, Fischer F, Zanetti G, Ferrari D, Tahallah N, Maier CS, Heck AJ, Rizzi M et al. (2002) Biochemistry41, 8807-8818]. Here, we provide a detailed study of this unusual enzyme reaction, showing that it occurs at a very slow rate (0.14 h(-1)), requires the participation of the enzyme-bound FAD, and is regiospecific in affecting only the C4 of the NADP nicotinamide ring. By protein engineering, we excluded the involvement in catalysis of residues Glu214 and His57, previously suggested to be implicated on the basis of their localization in the three-dimensional structure of the enzyme. Our results substantiate a catalytic mechanism for 3-carboxamide-4-pyridone adenine dinucleotide phosphate formation in which the initial and rate-determining step is the nucleophilic attack of the nicotinamide moiety by an active site water molecule. Whereas plant-type ferredoxin reductases were unable to oxidize NADP(+), the mammalian adrenodoxin reductase also catalyzed this unusual reaction. Thus, the 3-carboxamide-4-pyridone adenine dinucleotide phosphate formation reaction seems to be a peculiar feature of the mitochondrial type of ferredoxin reductases, possibly reflecting conserved properties of their active sites. Furthermore, we showed that 3-carboxamide-4-pyridone adenine dinucleotide phosphate is good ligand and a competitive inhibitor of various dehydrogenases, making this nucleotide analog a useful tool for the characterization of the cosubstrate-binding site of NADPH-dependent enzymes.


An in vitro study of the effects of lead on an epithelioid cell line.
The effects of a growth-inhibiting dose of lead on the cell surface, mitochondria, and vacuoles of RLC-GAI cells, an epithelioid cell line, were examined. The cells were found to begin to retract from the culture dish, becoming rounded in shape as early as 6 hours after lead addition. The rounded cells were found to be more susceptible to osmotic shock than normal cells. Studies with cytochalasin B showed that microfilaments were able to polymerize in the lead-treated rounded cells and microvilli were normal. Calcium did not prevent cell rounding by lead. Lead-induced detachment was reversed when cells were placed in normal medium. Mitochondrial and vacuolar alterations became apparent after several additional days. There was a decrease in the fraction of cytoplasm containing mitochondria and in the amount of cristae surface area in mitochondria. In cells incubated in mainly particulate lead increased numbers of lead-containing vacuoles were also seen.


Production of 3,4-dihydroxy L-phenylalanine by a newly isolated Aspergillus niger and parameter significance analysis by Plackett-Burman design.
The amino acid derivative 3,4-dihydroxy L-phenylalanine (L-dopa) is gaining interest as a drug of choice for Parkinson's disease. Aspergillus oryzae is commonly used for L-dopa production; however, a slower growth rate and relatively lower tyrosinase activity of mycelia have led to an increasing interest in exploiting alternative fungal cultures. In the present investigation, we report on the microbiological transformation of L-tyrosine to L-dopa accomplished by a newly isolated filamentous fungus Aspergillus niger. The culture A. niger (isolate GCBT-8) was propagated in 500 ml Erlenmeyer flasks and the pre-grown mycelia (48 h old) were used in the reaction mixture as a source of enzyme tyrosinase. Grinded mycelia gave 1.26 fold higher L-dopa production compared to the intact at 6% glucose (pH 5.5). The rate of L-tyrosine consumption was improved from 0.198 to 0.281 mg/ml. Among the various nitrogen sources, 1.5% peptone, 1% yeast extract and 0.2% ammonium chloride were optimized. The maximal L-dopa was produced (0.365 mg/ml) at 0.3% potassium dihydrogen phosphate with L-tyrosine consumption of 0.403 mg/ml. Over ~73% yield was achieved (degree of freedom 3) when the process parameters were identified using 2k-Plackett-Burman experimental design. The results are highly significant (p ≤ 0.05) and mark the commercial utility (LSD 0.016) of the mould culture which is perhaps the first ever report on L-dopa production from A. niger.


Aberrant localization of beta-catenin correlates with overexpression of its target gene in human papillary thyroid cancer.
Alterations of the Wnt/beta-catenin signaling pathway are known to occur in mutations of the component genes such as APC, Axin, and beta-catenin, and play a pathogenetic role in tumorigenesis. Activated Wnt signaling stabilizes beta-catenin, which associates with T cell factor, resulting in transactivation of the downstream target genes including c-myc and cyclin D1. To investigate the involvement of Wnt/beta-catenin signaling pathway in thyroid tumorigenesis, we analyzed its activation and localization in 5 human thyroid cancer cell lines and 132 thyroid tumor tissue samples. Dislocalization of beta-catenin was observed in all cell lines. Constitutive activation of T cell factor in two of four thyroid cancer cell lines was observed using reporter gene assay. Furthermore, high expression levels of c-Myc and cyclin D1 were observed in cell lines that showed cytoplasmic or nuclear accumulation of beta-catenin. In 132 paraffin-embedded thyroid carcinoma tissue samples, cytoplasmic beta-catenin was immunohistochemically observed in 52 out of 78 (67%) papillary thyroid cancers, but only in 3 of 34 (9%) follicular adenomas and 5 of 20 (25%) follicular cancers. Cytoplasmic localization of beta-catenin significantly correlated with overexpression of cyclin D1 in papillary carcinomas. Our results suggest that aberrant activation of Wnt/beta-catenin signaling is strongly involved in thyroid tumorigenesis.