Poor achievement of guidelines-recommended targets in type 2 diabetes: findings from a contemporary prospective cohort study.
To prospectively evaluate diabetes management in the primary care setting and explore factors related to guideline-recommended triple target achievement [blood pressure (BP) ≤ 130/80 mmHg, A1C ≤ 7% and low-density lipoprotein (LDL)-cholesterol < 2.5 mmol/l]. Baseline, 6 and 12 month data on clinical and laboratory parameters were measured in 3002 patients with type 2 diabetes enrolled as part of a prospective quality enhancement research initiative in Canada. A generalised estimating equation model was fitted to assess variables associated with triple target achievement. At baseline, 54%, 53% and 64% of patients, respectively, had BP, A1C and LDL-cholesterol at target; all three goals were met by 19% of patients. The percentage of individuals achieving these targets significantly increased during the study [60%, 57%, 76% and 26%, respectively, at the final visit, p < 0.0001 except for A1C, p = 0.27]. A much smaller proportion of patients had adequate control during the entire study period [30%, 39%, 53% and 7%, respectively]. In multivariable analysis, women, patients younger than 65 years and patients of Afro-Canadian origin were less likely to achieve the triple target. As part of a quality enhancement research initiative, we observed important improvements in the attainment of guidelines-recommended targets in patients with type 2 diabetes followed for a 12-month period in the primary care setting; however, many individuals still failed to achieve and especially maintain optimal goals for therapy, particularly the triple target. Results of the multivariable analysis reinforce the need to address barriers to improve diabetes care, particularly in more susceptible groups.


Non-essential roles of cysteine residues in functional expression and redox regulatory pathways for canine glutamate/aspartate transporter based on mutagenic analysis.
A redox regulatory mechanism and a molecular link between oxidative and excitotoxic neurodegeneration have been postulated for high-affinity Na(+)-dependent glutamate transporters. In the present study, mutations were introduced at three cysteine residues in canine glutamate/aspartate transporter (GLAST) to investigate the functional significance of thiol groups in response to oxidation. Cys(-) GLAST, in which all cysteines were replaced by other amino acids, as well as other mutants with disruption of one of three cysteine residues, showed insoluble oligomer formation, which was considered to be due to spontaneous and excessive oxidation as observed in wild-type GLAST. The mutant transporters also showed plasma-membrane localization and glutamate-transport kinetics that were very similar to those of wild-type GLAST. Glutamate-transport activities in COS-7 cells transfected with wild-type and Cys(-) GLAST were inhibited to the same degree when cells were exposed to Hg(2+) and were recovered by the addition of thiol-specific reductant dithiothreitol. These findings suggest that cysteine residues are not critical in functional expression of GLAST and the redox-sensing pathway via glutamate transporters.


Something old, something new: challenges and developments in Aspergillus niger biotechnology.
The filamentous ascomycete fungus Aspergillus niger is a prolific secretor of organic acids, proteins, enzymes and secondary metabolites. Throughout the last century, biotechnologists have developed A. niger into a multipurpose cell factory with a product portfolio worth billions of dollars each year. Recent technological advances, from genome editing to other molecular and omics tools, promise to revolutionize our understanding of A. niger biology, ultimately to increase efficiency of existing industrial applications or even to make entirely new products. However, various challenges to this biotechnological vision, many several decades old, still limit applications of this fungus. These include an inability to tightly control A. niger growth for optimal productivity, and a lack of high-throughput cultivation conditions for mutant screening. In this mini-review, we summarize the current state-of-the-art for A. niger biotechnology with special focus on organic acids (citric acid, malic acid, gluconic acid and itaconic acid), secreted proteins and secondary metabolites, and discuss how new technological developments can be applied to comprehensively address a variety of old and persistent challenges.


The influence of hemochromatosis mutations on iron overload of thalassemia major.
Hemochromatosis is a genetic form of iron overload due to a defective HFE gene. Secondary iron overload is the main complication in transfusion-dependent thalassemia patients. In this work we have examined the prevalence of HFE mutations in thalassemia major and evaluated the degree of iron overload of patients with and without HFE mutations. HFE mutations were studied in 71 Italian thalassemic patients and in 189 normal controls, using PCR and restriction enzyme analysis. The degree of iron overload, assessed by serum ferritin and liver iron concentration (LIC), was compared in 17 patients with mutations in the HFE gene, and in 17 subjects with wild type HFE genotype. The two groups of patients had comparable globin gene mutations, were matched for age and were homogeneous for transfusion and chelation history. In all cases the iron balance calculated on the basis of transfusion regimen and iron excreted by chelation was available. The allele frequencies of C282Y and H63D were respectively 1.4% and 12.7% in patients and 1.1% and 11.4% in controls. No case of C282Y homozygosity was recorded among patients. No significant difference was found in terms of serum ferritin, LIC, or the age at chelation start between patients with and without HFE mutations. The single patient with H63D homozygosity was severely iron-loaded. Our data suggest that the presence of a single mutation in the HFE gene does not influence the severity of iron loading in thalassemia patients following a regular transfusion and chelation program.


RSF-1 siRNA Enhances Tumor Radiosensitivity in Cervical Cancer via Enhanced DNA Damage, Cell Cycle Redistribution, and Promotion of Apoptosis.
Remodeling and spacing factor-1 (RSF-1) is an identified tumor biomarker that is overexpressed in a variety of human cancers, but its effect on radiotherapy remains unclear. In this study, we aimed to explore the effect of RSF-1 siRNA on sensitizing cervical cancer cells to radiation and its underlying mechanism. The mRNA and protein expression of RSF-1 in tissue and cells were measured by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting. Cell counting kit-8 (CCK-8) and colony formation assay were used to examine cell proliferation. Flow cytometry was used to analyzed the cell cycle and cell apoptosis. DNA damage was examined by the comet assay. ATM, ATR, CHK1, CHK2, H2AX, γH2AX and phosphorylated ATM, ATR, CHK1 and CHK2 were detected by Western blotting. γH2AX foci were demonstrated by immunofluorescence staining. RSF-1 was upregulated in cervical cancer tissue and decreased after effective treatment. RSF-1 siRNA in combination with radiation suppressed cell viability, redistributed cell cycles and also induced cell apoptosis in HeLa and SiHa cell lines. Further, knockdown of RSF-1 induced DNA damage by attenuating DNA repair capability, thereby sensitizing cervical cancer cells to radiation. These data demonstrate that RSF-1 siRNA enhanced the sensitivity of radiotherapy, and targeting RSF-1 may be a promising approach for the development of novel radiosensitizing agents for the treatment of cervical cancer.


Isolation, biochemical characterization and ultrastructural analysis of the limbic system-associated membrane protein (LAMP), a protein expressed by neurons comprising functional neural circuits.
The limbic system-associated membrane protein (LAMP) is a cell surface glycoprotein expressed by cortical and subcortical regions of the mammalian CNS that comprise or receive direct projections from limbic system structures. The early and restricted expression of LAMP has led to its postulated role in neural development. Purification and biochemical characterization of LAMP was performed in order to ascertain its relationship to other, well-defined cell surface proteins in the nervous system. Subcellular fractionation, immunoaffinity chromatography, and Western blots of rodent and bovine hippocampus revealed that LAMP is an integral membrane protein with a molecular mass of 64-68 kDa and a pI of 5.2-5.5. Deglycosylation of LAMP indicates that it contains N-linked high mannose or hybrid sugars and a minor amount of sialic acid. The LAMP protein exhibits an identical molecular mass in developing hippocampus and in several different brain regions in the adult. No cross-reactivity was obtained using the monoclonal antibody that recognizes the HNK-1 carbohydrate epitope, a complex sulfated moiety expressed on members of a large family of glycoproteins. Immunocytochemical analysis at the ultrastructural level reveals that LAMP immunoreactivity is exhibited by neurons in a stereotyped pattern throughout limbic system areas. Glial cells are not immunoreactive. In the adult, LAMP-immunoreactive membrane patches are present exclusively postsynaptically on neuronal somata and dendrites. Myelinated and unmyelinated axons are not stained in any brain region examined. Analysis of LAMP expression in the developing CNS during synaptogenesis demonstrates that LAMP is located on growing axons and both pre- and postsynaptically at forming terminal complexes. Double-labeling studies of the hippocampal neurons grown in vitro reveal that the LAMP epitope is extracellular and is expressed on neurofilament- and microtubule-associated protein 2-positive neurites. Cells expressing glial fibrillary acidic protein are not LAMP-immunoreactive. These results demonstrate that in the adult brain, LAMP is expressed almost exclusively by the postsynaptic (target) elements in limbic circuits, but that during development, all components of the surface of the growing neuron contain LAMP. The stereotyped anatomical pattern of expression of LAMP in the developing and mature brain and its biochemical characteristics suggest that LAMP is a unique, system-associated membrane glycoprotein that is distinct from previously identified, developmentally important cell surface proteins.


Cross Talk Between TGF-β and JAK Expressions and Nepherotoxicity Induced by Tetrachloromethane: Role of Phytotherapy.
The aim of the current study is to assess the effectiveness of milk thistle seeds (Mth) in combination with Taraxacum officinale (Tof) and/or Camellia sinensis (Csin) against tetrachloromethane (Tcm) renotoxicity in rats. Tetrachloromethane was injected in a single dose, followed by 1-month treatments with Mth, Tof, and Csin alone or in combination. Serum urea, uric acid, and creatinine levels were significantly increased matched with the control group. Masson trichrome stain revealed increase in the deposition of fibrous tissue in the interstitium between the tubules and the renal corpuscles. Immunohistochemical analysis of kidney tissues revealed that Tcm induced an increase in the immune response of tumor growth factor β (TGF-β) and Janus kinase (JAK) protein expressions and cysteine-aspartic acid protease 3 (caspase 3), while B-cell lymphoma 2 (Bcl2) was downregulated. Treatment with the antioxidants in question either alone or in combination ameliorated all kidney function parameters and showed mild immune reactivity toward TGF-β and JAK protein expressions in blood vessels and glomeruli in the kidney tissues and downregulated caspase 3 and activated Bcl2 protein expression. The combination regimen of the 3 antioxidants showed the most significant renoprotective effect. This was also confirmed histopathologically. It was concluded that the antioxidant mixture is considered as a promising candidate toward renal dysfunction and immune reactivity induced by Tcm and other toxicants.


Overnight polysomnography and the recording of sleep and sleep-related respiration in orchestra musicians - possible protective effects of wind instruments on respiration.
Our study is the first to objectively assess sleep and sleep-related respiration in orchestra musicians. We hypothesized low sleep quality due to high work demands and irregular work-sleep schedules, and a better respiration for wind instrument (WI) players than string instrument (SI) players due to habitual upper airway muscles training. We recorded overnight polysomnography with 29 professional orchestra musicians (21 men, 14 WI/ 15 SI). The musicians presented a sleep efficiency of 88% (IQR 82-92%) with WI having a significant higher sleep efficiency than SI (89%, 85-93% vs. 85%, 74-89%; p = 0.029). The group had a total sleep time around 6 hours (377min, 340-421min) with signs of increased NREM 1 (light sleep) and decreased REM (dream sleep). The musicians displayed an apnea-hypopnea-index of 2.1events/hour (0.7-5.5) and an oxygen saturation of 98% (97-100%). While SI player exhibited declining sleep-related respiration with age (breathing events: r = 0.774, p = 0.001, oxygen: r = -0.647, p = 0.009), WI player showed improved respiration with age (breathing events: r = -0.548, p = 0.043; oxygen: r = 0.610, p = 0.020). Our study is the first objective investigation of sleep pattern and respiration during sleep with overnight polysomnography in professional orchestra musicians. While sleep and respiration were unexpectedly good, our results revealed possible signs of sleep deprivation and an interesting age-related pattern on respiration depending on instrument. While sample size was small and results modest, these findings present first objective evidence towards the assumption that habitual playing of a WI-and training of the upper airway muscles-may have a protective effect on respiration.


Sequence-selective DNA recognition with peptide-bisbenzamidine conjugates.
Transcription factors (TFs) are specialized proteins that play a key role in the regulation of genetic expression. Their mechanism of action involves the interaction with specific DNA sequences, which usually takes place through specialized domains of the protein. However, achieving an efficient binding usually requires the presence of the full protein. This is the case for bZIP and zinc finger TF families, which cannot interact with their target sites when the DNA binding fragments are presented as isolated monomers. Herein it is demonstrated that the DNA binding of these monomeric peptides can be restored when conjugated to aza-bisbenzamidines, which are readily accessible molecules that interact with A/T-rich sites by insertion into their minor groove. Importantly, the fluorogenic properties of the aza-benzamidine unit provide details of the DNA interaction that are eluded in electrophoresis mobility shift assays (EMSA). The hybrids based on the GCN4 bZIP protein preferentially bind to composite sequences containing tandem bisbenzamidine-GCN4 binding sites (TCAT⋅AAATT). Fluorescence reverse titrations show an interesting multiphasic profile consistent with the formation of competitive nonspecific complexes at low DNA/peptide ratios. On the other hand, the conjugate with the DNA binding domain of the zinc finger protein GAGA binds with high affinity (KD≈12 nM) and specificity to a composite AATTT⋅GAGA sequence containing both the bisbenzamidine and the TF consensus binding sites.


Down syndrome screening in women under 35 with maternal serum hCG.
Human chorionic gonadotropin and maternal serum alpha-fetoprotein (MSAFP) concentrations were measured in frozen maternal serum samples from 16 pregnancies with Down syndrome and from 614 unaffected pregnancies in women younger than 35. By using only maternal age and MSAFP level to determine Down syndrome risk, four of 16 (25%) Down syndrome pregnancies were identified, using risk estimates as a screening variable. This detection rate required performing amniocentesis on 4.8% of pregnancies with a normal outcome. By adding hCG level as a third risk parameter, ten of 16 (62.5%) Down syndrome pregnancies were detected. To achieve this detection rate, 4.7% of women under 35 would be recommended for amniocentesis. These results indicate that the estimation of risk for Down syndrome based on the addition of maternal hCG level to MSAFP level and maternal age will substantially improve the detection rate for Down syndrome, with no change in the amniocentesis rate. These findings are in agreement with other studies that suggest that hCG is a valuable addition to screening programs for Down syndrome.


A closed-form field analysis of a broad-band annular array.
A closed-form transient field response under the paraxial approximation for an impulse wave excited by a circularly symmetric ultrasonic transducer is derived. The analysis is based on linear transform techniques to obtain the impulse response of a planar or a lensed transducer such as a disk or an annulus in the whole field of view. A closed-form solution is obtained for the focal plane response of a nonuniform aperture with an apodizing function represented by a polynomial of the radial distance on the transducer surface. Moreover, a closed-form impulse response for a planar annular is derived without the paraxial approximation and taking into account the obliquity factor. For a phased annular array, the impulse responses of array elements are convolved with the delayed excitation pulse and then summed to get the resultant field disturbance of the array. These results provide an effective means of studying the focusing characteristics of a phased annular array, including its focusing delay and apodizing quantization error effects.


Sympathetic nerves protect against stroke in stroke-prone hypertensive rats. A preliminary report.
Studies were performed to determine whether sympathetic nerves protect against stroke in hypertensive rats. The superior cervical ganglion was removed on one side in 28 stroke-prone spontaneously hypertensive rats (SHRSP) when the rats were 4 weeks old. The rats were fed Japanese rat chow and 1% saline drinking water. When the rats were 19 weeks old, systolic pressure was 206 +/- 4 mm Hg (mean +/- SE). All rats died between 19 and 23 weeks of age. Microscopic and histological examination demonstrated cerebral hemorrhage in seven rats. All the hemorrhages occurred in the denervated hemispheres. Ischemic cerebral infarctions were found in 13 rats; in 10 rats, the infarcts were only in the denervated hemisphere. Pathological changes of cerebral arteries (hyalinosis, fibrinoid changes, and thrombus formation) were observed primarily in denervated hemispheres. Wall-to-lumen ratio was less in arteries of the denervated hemisphere than in arteries of the innervated hemisphere. These preliminary observations suggest that denervation of cerebral vessels increases susceptibility to stroke and inhibits development of cerebral vascular hypertrophy in SHRSP.


Association of sociodemographic factors and internet query data with pertussis infections in Shandong, China.
This study explored how internet queries vary in facilitating monitoring of pertussis, and the effects of sociodemographic characteristics on such variation by city in Shandong province, China. We collected weekly pertussis notifications, Baidu Index (BI) data and yearly sociodemographic data at the city level between 1 January 2009 and 31 December 2017. Spearman's correlation was performed for temporal risk indices, generalised linear models and regression tree models were developed to identify the hierarchical effects and the threshold between sociodemographic factors and internet query data with pertussis surveillance. The BI was correlated with pertussis notifications, with a strongly spatial variation among cities in temporal risk indices (composite temporal risk metric (CTRM) range: 0.59-1.24). The percentage of urban population (relative risk (RR): 1.05, 95% confidence interval (CI) 1.03-1.07), the proportion of highly educated population (RR: 1.27, 95% CI 1.16-1.39) and the internet access rate (RR: 1.04, 95% CI 1.02-1.05) were correlated with CTRM. Higher RRs in the three identified sociodemographic factors were associated with higher stratified CTRM. The percentage of highly educated population was the most important determinant in the BI with pertussis surveillance. The findings may lead to spatially-specific criteria to inform development of an early warning system of pertussis infections using internet query data.


A microfluidic oligonucleotide synthesizer.
De novo gene and genome synthesis enables the design of any sequence without the requirement of a pre-existing template as in traditional genetic engineering methods. The ability to mass produce synthetic genes holds great potential for biological research, but widespread availability of de novo DNA constructs is currently hampered by their high cost. In this work, we describe a microfluidic platform for parallel solid phase synthesis of oligonucleotides that can greatly reduce the cost of gene synthesis by reducing reagent consumption (by 100-fold) while maintaining a approximately 100 pmol synthesis scale so there is no need for amplification before assembly. Sixteen oligonucleotides were synthesized in parallel on this platform and then successfully used in a ligation-mediated assembly method to generate DNA constructs approximately 200 bp in length.


Conception and pilot testing of a self-management health application for patients with pollen-related allergic rhinitis and allergic asthma-the APOLLO app.
It has been shown that pollen information services are an important self-management tool for patients with pollen-related allergic rhinitis (AR) and allergic asthma (AA). This study aimed to design an online application for patients with AR and AA, which supports patients to better manage their disease as well as to evaluate the app and present the first results of the pilot study. The pollen data were obtained from the electronic pollen information network of Bavaria, Germany. Participants were asked to fill in their allergy-related complaints in the app over a 60-day period. Subsequently, the app was evaluated. Indices and diagrams visualized the participants' individual complaints as well as the daily pollen concentration in the air. In order to motivate participants to complete the app on a daily basis, we used elements of gamification. Two thirds of the participants (N = 46) reported feeling better informed about pollen counts and their allergy when using the app. The app's simple and comprehensible design was rated positively. More than 80% of the participants would recommend the app to their family and friends. The app can be a tool for patients with AR and AA to better understand their disease.


Enhanced Biogenic Sulfidation of Zero-Valent Iron in Columns: Implications for Promoting Dechlorination in Permeable Reactive Barriers.
Biogenic sulfidation of zero-valent iron (ZVI) using sulfate reducing bacteria (SRB) has shown enhanced dechlorination rates comparable to those produced by chemical sulfidation. However, controlling and sustaining biogenic sulfidation to enhance in situ dechlorination are poorly understood. Detailed interactions between SRB and ZVI were examined for 4 months in column experiments under enhanced biogenic sulfidation conditions. SRB proliferation and changes in ZVI surface properties were characterized along the flow paths. The results show that ZVI can stimulate SRB activity by removing excessive free sulfide (S2-), in addition to lowering reduction potential. ZVI also hinders downgradient movement of SRB via electrostatic repulsion, restricting SRB presence near the upgradient interface. Dissolved organic carbon (e.g., >2.2 mM) was essential for intense biogenic sulfidation in ZVI columns. The presence of SRB in the upgradient zone appeared to promote the formation of iron polysulfides. Biogenic FeSx deposition increased the S content on ZVI surfaces ∼3-fold, corresponding to 3-fold and 2-fold improvements in the trichloroethylene degradation rate and electron efficiency in batch tests. Elucidation of SRB and ZVI interactions enhances sustained sulfidation in ZVI permeable reactive barrier.


Pharmacokinetics and pharmacodynamics of anti-tuberculosis drugs: An evaluation of in vitro, in vivo methodologies and human studies.
There has been an increased interest in pharmacokinetics and pharmacodynamics (PKPD) of anti-tuberculosis drugs. A better understanding of the relationship between drug exposure, antimicrobial kill and acquired drug resistance is essential not only to optimize current treatment regimens but also to design appropriately dosed regimens with new anti-tuberculosis drugs. Although the interest in PKPD has resulted in an increased number of studies, the actual bench-to-bedside translation is somewhat limited. One of the reasons could be differences in methodologies and outcome assessments that makes it difficult to compare the studies. In this paper we summarize most relevant in vitro, in vivo, in silico and human PKPD studies performed to optimize the drug dose and regimens for treatment of tuberculosis. The in vitro assessment focuses on MIC determination, static time-kill kinetics, and dynamic hollow fibre infection models to investigate acquisition of resistance and killing of Mycobacterium tuberculosis populations in various metabolic states. The in vivo assessment focuses on the various animal models, routes of infection, PK at the site of infection, PD read-outs, biomarkers and differences in treatment outcome evaluation (relapse and death). For human PKPD we focus on early bactericidal activity studies and inclusion of PK and therapeutic drug monitoring in clinical trials. Modelling and simulation approaches that are used to evaluate and link the different data types will be discussed. We also describe the concept of different studies, study design, importance of uniform reporting including microbiological and clinical outcome assessments, and modelling approaches. We aim to encourage researchers to consider methods of assessing and reporting PKPD of anti-tuberculosis drugs when designing studies. This will improve appropriate comparison between studies and accelerate the progress in the field.


Three-dimensional modeling of mechanical forces in the extracellular matrix during epithelial lumen formation.
Mechanical interactions between cells and extracellular matrix (ECM) mediate epithelial cyst formation. This work relies on the combination of numerical modeling with live cell imaging, to piece together a novel nonintrusive method for determining three-dimensional (3D) mechanical forces caused by shape changes of a multicellular aggregate at the early stages of epithelial cyst formation. We analyzed the evolution of Madin-Darby canine kidney cells in 3D cultures using time-lapse microscopy, with type I collagen gel forming the ECM. The evolving 3D interface between the ECM and the cell aggregate was obtained from microscopy images, and the stress on the surface of a proliferating aggregate and in the surrounding ECM was calculated using the finite element method. The viscoelastic properties of the ECM (a needed input for the finite element method solver) were obtained through oscillatory shear flow experiments on a rheometer. For validation purpose, the forces exerted by an aggregate on a force-sensor array were measured and compared against the computational results.


User-centred clinical decision support to implement emergency department-initiated buprenorphine for opioid use disorder: protocol for the pragmatic group randomised EMBED trial.
The goal of this trial is to determine whether implementation of a user-centred clinical decision support (CDS) system can increase adoption of initiation of buprenorphine (BUP) into the routine emergency care of individuals with opioid use disorder (OUD). A pragmatic cluster randomised trial is planned to be carried out in 20 emergency departments (EDs) across five healthcare systems over 18 months. The intervention consists of a user-centred CDS integrated into ED clinician electronic workflow and available for guidance to: (1) determine whether patients presenting to the ED meet criteria for OUD, (2) assess withdrawal symptoms and (3) ascertain and motivate patient willingness to initiate treatment. The CDS guides the ED clinician to initiate BUP and facilitate follow-up. The primary outcome is the rate of BUP initiated in the ED. Secondary outcomes are: (1) rates of receiving a referral, (2) fidelity with the CDS and (3) rates of clinicians providing any ED-initiated BUP, referral for ongoing treatment and receiving Drug Addiction Act of 2000 training. Primary and secondary outcomes will be analysed using generalised linear mixed models, with fixed effects for intervention status (CDS vs usual care), prespecified site and patient characteristics, and random effects for study site. The protocol has been approved by the Western Institutional Review Board. No identifiable private information will be collected from patients. A waiver of informed consent was obtained for the collection of data for clinician prescribing and other activities. As a minimal risk implementation study of established best practices, an Independent Study Monitor will be utilised in place of a Data Safety Monitoring Board. Results will be reported in ClinicalTrials.gov and published in open-access, peer-reviewed journals, presented at national meetings and shared with the clinicians at participating sites via a broadcast email notification of publications. NCT03658642; Pre-results.


Zinc-, copper- and cadmium-binding protein in Ehrlich ascites tumour cells.
The properties of Ehrlich ascites tumour cells exposed in vivo to cadmium were investigated as a function of the zinc status of the host animals. Tumour-cell growth was inhibited by cadmium in both zinc-sufficient and zinc-deficient animals. However, cells in zinc-sufficient tumours accumulate much less cadmium than those in deficient tumours. The subcellular distributions of cadmium and zinc do not depend on zinc status. Cadmium and zinc are bound to a low-molecular-weight protein with properties similar to metallothionein. Without exposure to cadmium, a zinc- and copper-binding protein is still present that behaves like a metallothionein. This protein can rapidly bind cadmium added to Ehrlich cells in vitro. It is shown that the zinc- and copper-binding protein contains free thiol groups. Ehrlich cells isolated from cadmium-treated animals are viable and show normal incorporation of uridine into RNA, but the cellular uptake of thymidine and its incorporation into DNA are inhibited.


[Serotypes of adeno-associated viruses].
Cancer, monogenic and cardiovascular diseases are the main targets of clinical gene therapy. For the most part, the research is making use of virus gene carriers based on genome of well characterized adenoviruses and retroviruses. There is an intensive research being done on cloning vectors that would successfully deliver the therapeutic genes into the cells of interest without causing side effects. Experimental research and first clinical trials emphasize therapeutic significance of recombinant adeno-associated viruses (AAV). The presence of AAV serotypes and possibility of their cloning in vitro allow conducting clinical trials of gene therapy using gene products introducing therapeutic genes into targeted cells, tissues, organs. In the work twelve known serotypes of AAV are described. The cell tropism as well as biological activity were discussed. Research shows that AAV vectors mostly are able to infect skeletal muscles (AAV1, 8, 9), heart (AAV9), liver (AAV8) and sense organs (AAV3, 4, 5).


Complete loss of PTEN protein expression correlates with shorter time to brain metastasis and survival in stage IIIB/C melanoma patients with BRAFV600 mutations.
Loss of function of PTEN is a frequent event in melanoma, particularly in tumors with BRAF(V600) mutations. The prevalence, pathologic features, and clinical outcomes associated with PTEN loss in patients with stage IIIB/C melanoma were interrogated to improve our understanding of the clinical significance of this molecular event. Archival tissue from lymphadenectomy specimens among patients (n = 136) with stage IIIB or IIIC melanoma was assessed by DNA sequencing for activating BRAF and NRAS mutations, and by immunohistochemistry for the expression of PTEN protein. Associations of these molecular aberrations with demographics, tumor characteristics, and clinical outcomes were determined. The prevalence of BRAF(V600) mutations (40% overall), NRAS mutations (10%), and PTEN loss (25%) did not vary by pathologic substage. BRAF/NRAS mutation status did not correlate with distant disease-free survival (DDFS) or overall survival (OS). Complete loss of PTEN expression correlated with shorter OS but not DDFS. When stratified by specific sites of distant metastasis, PTEN loss was associated with significantly shorter time to melanoma brain metastasis (MBM), but not to liver, lung, or bone metastasis. Analysis of PTEN in mutationally defined subsets showed that PTEN loss was significantly associated with OS and time to MBM in patients with BRAF(V600) mutations. Loss of PTEN protein expression correlates significantly with decreased OS and time to MBM in stage IIIB/C melanoma patients with BRAF(V600) mutations. The findings add to evidence supporting a significant role for PTEN loss and the PI3K-AKT pathway in melanoma.


Ice-sheet acceleration driven by melt supply variability.
Increased ice velocities in Greenland are contributing significantly to eustatic sea level rise. Faster ice flow has been associated with ice-ocean interactions in water-terminating outlet glaciers and with increased surface meltwater supply to the ice-sheet bed inland. Observed correlations between surface melt and ice acceleration have raised the possibility of a positive feedback in which surface melting and accelerated dynamic thinning reinforce one another, suggesting that overall warming could lead to accelerated mass loss. Here I show that it is not simply mean surface melt but an increase in water input variability that drives faster ice flow. Glacier sliding responds to melt indirectly through changes in basal water pressure, with observations showing that water under glaciers drains through channels at low pressure or through interconnected cavities at high pressure. Using a model that captures the dynamic switching between channel and cavity drainage modes, I show that channelization and glacier deceleration rather than acceleration occur above a critical rate of water flow. Higher rates of steady water supply can therefore suppress rather than enhance dynamic thinning, indicating that the melt/dynamic thinning feedback is not universally operational. Short-term increases in water input are, however, accommodated by the drainage system through temporary spikes in water pressure. It is these spikes that lead to ice acceleration, which is therefore driven by strong diurnal melt cycles and an increase in rain and surface lake drainage events rather than an increase in mean melt supply.


Screening for lung cancer.
Lung cancer is the commonest cause of death from cancer in both men and women, with approximately 152,000 new cases and 139,000 deaths in 1988. The incidence and mortality rates are increasing rapidly in women. Two main tests have been used to screen for lung cancer: chest roentgenography and sputum cytology. Four recent controlled trials and one case-control study failed, however, to show that screening reduces lung cancer mortality even in high-risk persons (smokers). In the Mayo Lung Project, for example, the lung cancer death rate in high-risk men offered sputum cytology and chest roentgenogram every 4 months was 3.1 per 1000 person-years, compared with 3.0 per 1000 person-years in a control group. Chest roentgenograms and sputum cytology lead to false-positive test results in smokers of approximately 5% and 0.5%, respectively. Because of the lack of evidence of benefit and because of its potential harms and costs, screening for lung cancer is not recommended.


A novel tool to analyze MRI recurrence patterns in glioblastoma.
At least 10% of glioblastoma relapses occur at distant and even contralateral locations. This disseminated growth limits surgical intervention and contributes to neurological morbidity. Preclinical data pointed toward a role for temozolomide (TMZ) in reducing radiotherapy-induced glioma cell invasiveness. Our objective was to develop and validate a new analysis tool of MRI data to examine the clinical recurrence pattern of glioblastomas. MRIcro software was used to map the location and extent of initial preoperative and recurrent tumors on MRI of 63 patients in the European Organisation for Research and Treatment of Cancer (EORTC) 26981/22981/National Cancer Institute of Canada (NCIC) CE.3 study into the same stereotaxic space. This allowed us to examine changes of site and distance between the initial and the recurrent tumor on the group level. Thirty of the 63 patients were treated using radiotherapy, while the other patients completed a radiotherapy-plus-TMZ treatment. Baseline characteristics (median age, KPS) and outcome data (progression-free survival, overall survival) of the patients included in this analysis resemble those of the general study cohort. The patient groups did not differ in the promoter methylation status of methyl guanine methyltransferase (MGMT). Overall frequency of distant recurrences was 20%. Analysis of recurrence patterns revealed no difference between the groups in the size of the recurrent tumor or in the differential effect on the distance of the recurrences from the preoperative tumor location. The data show the feasibility of groupwise recurrence pattern analysis. An effect of TMZ treatment on the recurrence pattern in the EORTC 26981/22981/NCIC CE.3 study could not be demonstrated.


Conventional and organic soil management as divergent drivers of resident and active fractions of major soil food web constituents.
Conventional agricultural production systems, typified by large inputs of mineral fertilizers and pesticides, reduce soil biodiversity and may negatively affect ecosystem services such as carbon fixation, nutrient cycling and disease suppressiveness. Organic soil management is thought to contribute to a more diverse and stable soil food web, but data detailing this effect are sparse and fragmented. We set out to map both the resident (rDNA) and the active (rRNA) fractions of bacterial, fungal, protozoan and metazoan communities under various soil management regimes in two distinct soil types with barley as the main crop. Contrasts between resident and active communities explained 22%, 14%, 21% and 25% of the variance within the bacterial, fungal, protozoan, and metazoan communities. As the active fractions of organismal groups define the actual ecological functioning of soils, our findings underline the relevance of characterizing both resident and active pools. All four major organismal groups were affected by soil management (p < 0.01), and most taxa showed both an increased presence and an enlarged activity under the organic regime. Hence, a prolonged organic soil management not only impacts the primary decomposers, bacteria and fungi, but also major representatives of the next trophic level, protists and metazoa.


Transjugular intrahepatic portosystemic shunt creation in children: initial clinical experience.
To assess an initial clinical experience with the creation of a transjugular intrahepatic portosystemic shunt (TIPS) in children. Twelve consecutive patients with complications of portal hypertension underwent TIPS creation at our institution between July 1993 and September 1996. There were six boys and six girls aged 2 years 5 months to 16 years 10 months (median, 9 years 2 months) who weighed 13.9-80.9 kg (median, 27.65 kg). A standard radiographic technique was used. Thirteen procedures were performed to achieve 12 successful TIPS creations. One child with a reduced-size liver transplant had to undergo two procedures for a successful TIPS creation. No major procedural complications or morbidity occurred. In 10 children, TIPS patency was documented by means of pathologic inspection at orthotopic liver transplantation (median shunt duration, 53 days). Shunt stenosis developed in one child at 186 days but was treated successfully. Two children had functional shunts at 301 and 357 days, respectively. No episodes of repeat variceal hemorrhage were noted. One child developed postprocedural encephalopathy, which responded to medical therapy. This initial clinical experience suggests that TIPS creation is technically feasible and is as safe in children as in adults. TIPS creation can aid in the management of portal hypertension in children, especially in those needing temporary relief before liver transplantation.


An Uncertainty-Guided Deep Learning Method Facilitates Rapid Screening of CYP3A4 Inhibitors.
Cytochrome P450 3A4 (CYP3A4), a prominent member of the P450 enzyme superfamily, plays a crucial role in metabolizing various xenobiotics, including over 50% of clinically significant drugs. Evaluating CYP3A4 inhibition before drug approval is essential to avoiding potentially harmful pharmacokinetic drug-drug interactions (DDIs) and adverse drug reactions (ADRs). Despite the development of several CYP inhibitor prediction models, the primary approach for screening CYP inhibitors still relies on experimental methods. This might stem from the limitations of existing models, which only provide deterministic classification outcomes instead of precise inhibition intensity (e.g., IC50) and often suffer from inadequate prediction reliability. To address this challenge, we propose an uncertainty-guided regression model to accurately predict the IC50 values of anti-CYP3A4 activities. First, a comprehensive data set of CYP3A4 inhibitors was compiled, consisting of 27,045 compounds with classification labels, including 4395 compounds with explicit IC50 values. Second, by integrating the predictions of the classification model trained on a larger data set and introducing an evidential uncertainty method to rank prediction confidence, we obtained a high-precision and reliable regression model. Finally, we use the evidential uncertainty values as a trustworthy indicator to perform a virtual screening of an in-house compound set. The in vitro experiment results revealed that this new indicator significantly improved the hit ratio and reduced false positives among the top-ranked compounds. Specifically, among the top 20 compounds ranked with uncertainty, 15 compounds were identified as novel CYP3A4 inhibitors, and three of them exhibited activities less than 1 μM. In summary, our findings highlight the effectiveness of incorporating uncertainty in compound screening, providing a promising strategy for drug discovery and development.


MRI for measuring therapy efficiency after revascularisation in ST-segment elevation myocardial infarction: a systematic review and meta-regression analysis.
To summarise existing data on the relation between the time from symptom onset until revascularisation (time to reperfusion) and the myocardial salvage index (MSI) calculated as proportion of non-necrotic myocardium inside oedematous myocardium on T2-weighted and T1-weighted late gadolinium enhancement MRI after ST-segment elevation myocardial infarction (STEMI). Studies including patients with revascularised STEMI and stating both the time to reperfusion and the MSI measured by T2-weighted and T1-weighted late gadolinium enhancement MRI were searched in MEDLINE, EMBASE and ISI Web of Science until 16 May 2020. A mixed effects model was used to evaluate the relation between the time to reperfusion and the MSI. The gender distribution and mean age in included patient groups, the timing of MRI, used MRI sequences and image interpretation methodology were included in the mixed effects model to explore between-study heterogeneity. We included 38 studies with 5106 patients. The pooled MSI was 42.6% (95% CI: 38.1 to 47.1). The pooled time to reperfusion was 3.8 hours (95% CI: 3.5 to 4.0). Every hour of delay in reperfusion was associated with an absolute decrease of 13.1% (95% CI: 11.5 to 14.6; p<0.001) in the MSI. Between-study heterogeneity was considerable (σ2=167.8). Differences in the gender distribution, timing of MRI and image interpretation among studies explained 45.2% of the between-study heterogeneity. The MSI on T2-weighted and T1-weighted late gadolinium enhancement MRI correlates inversely with the time to reperfusion, which indicates that cardioprotection achieved by minimising the time to reperfusion leads to a higher MSI. The analysis revealed considerable heterogeneity between studies. The heterogeneity could partly be explained by differences in the gender distribution, timing and interpretation of MRI suggesting that the MRI-assessed MSI is not only influenced by cardioprotective therapy but also by patient characteristics and MRI parameters.


Stevens-johnson syndrome induced by carbamazepine treatment in a patient who previously had carbamazepine induced pruritus - a case report -.
Stevens-Johnson syndrome (SJS) is a rare but life-threatening skin reaction disease and carbamazepine is one of its most common causes. We report a case of SJS secondary to carbamazepine in a patient with previous pruritus due to carbamazepine which was given for treatment of trigeminal neuralgia. We would like to caution all providers that carbamazepine readministration should be avoided in the patient with a previous history of SJS or adverse skin reaction. In addition, we strongly recommend gradual titration when initiating treatment with carbamazepine.


Acute myelitis after asthma attacks with onset after puberty.
A poliomyelitis-like illness after asthma attacks has been found and is called asthmatic amyotrophy (Hopkins' syndrome). All of the previously reported cases were under 13 years of age. Three patients are described who developed acute myelitis after asthma attacks at 15, 22, and 73 years of age. All of them showed acute flaccid monoparesis, and needle EMG disclosed denervation potentials in the relevant muscles. In addition, in the two adult patients the sensory or pyramidal tracts were involved, and evoked potential studies confirmed an involvement of the pyramidal tracts in one of them. This 22 year old patient showed a second episode of monoparesis in the other limb after another asthma attack. All three patients had no significant changes in their antiviral antibody titres, whereas every patient had hyperIgEaemia and allergen specific IgE. These findings suggest that asthmatic amyotrophy can develop after puberty and that patients who develop this disease in adulthood seem to show both a widespread involvement of the spinal cord and a more varied course.


Histologic characterization of renal tumors (nephroblastomas) induced transplacentally in IIIVO/J and WH/J rabbits by N-ethylnitrosourea.
The histologic features of 63 renal tumors induced in 39 rabbits of two partially inbred strains, IIIVO/J and WH/J, by transplacental exposure to N-ethylnitrosourea (ENU) were analyzed. All tumors in the series conformed to nephroblastoma, permitting the establishment of histologic standards for this neoplasm in the rabbit as well as observations on tumor progression. Essentially, nephroblastoma proved to be predominantly an epithelial tumor identifying with metanephrogenic blastema, which was presumed to be the tissue of origin during fetal development. The outstanding features comprised clusters or sheets of undifferentiated blastemalike tissue and differentiation along the epithelial pathway into tubular profiles and structures suggestive of primitive, nonvascularized glomeruli. The latter were frequently of a complex nature, with a papillary configuration. On the other hand, no definitive evidence of bipotential differentiation into malignant secondary mesenchyme was found, there being no recognizable areas of fibrosarcomatous elements or specialized connective tissue such as smooth or striated muscle, adipose tissue, cartilage, or osteoid. Mesenchymelike fascicular disposition of neoplastic cells between blastemal clusters was an acquired feature seen in advanced tumors but not in small early lesions. By light microscopy alone it was not possible to determine whether this represented a conformational change of tumor cells or true bipotential differentiation into neoplastic secondary mesenchyme. However, the reticulin pattern was not characteristic of sarcoma. A conspicuous feature accompanying the growth and development of tumors was the magnitude of host fibrous reaction discernible only as a simple ramifying stroma in the earliest lesions but attaining impressive proportions both within and around the tumor with advancing age. Increasing collagen formation appeared to be associated with ischemic necrosis of tumor tissue. Other features of advanced tumors were the presence of discrete foci of differentiated tubular structures suggestive of mature medullary elements and small islands of squamoid differentiation. Metastases occurred only in rabbits of strain IIIVO/J, which had been subjected to a single dose of the carcinogen, representing an incidence in this subgroup of 25%. Nephroblastomas resulting from transplacental induction in IIIVO/J rabbits, particularly by single, high doses of ENU, appear to provide a suitable model for the predominant histologic form of the Wilms' tumor complex in man.


RNF20/40-mediated eEF1BδL monoubiquitylation stimulates transcription of heat shock-responsive genes.
RNF20/40 E3 ubiquitin ligase-mediated histone H2B monoubiquitylation plays important roles in many cellular processes, including transcriptional regulation. However, the multiple defects observed in RNF20-depleted cells suggest additional ubiquitylation targets of RNF20/40 beyond histone H2B. Here, using biochemically defined assays employing purified factors and cell-based analyses, we demonstrate that RNF20/40, in conjunction with its cognate E2 ubiquitin-conjugating enzyme RAD6, monoubiquitylates lysine 381 of eEF1BδL, a heat shock transcription factor. Notably, monoubiquitylation of eEF1BδL increases eEF1BδL accumulation and potentiates recruitment of p-TEFb to the promoter regions of heat shock-responsive genes, leading to enhanced transcription of these genes. We further demonstrate that cooperative physical interactions among eEF1BδL, RNF20/40, and HSF1 synergistically promote expression of heat shock-responsive genes. In addition to identifying eEF1BδL as a novel ubiquitylation target of RNF20/40 and elucidating its function, we provide a molecular mechanism for the cooperative function of distinct transcription factors in heat shock-responsive gene transcription.


Putting the Pieces Together: Completing the Mechanism of Action Jigsaw for Sipuleucel-T.
Sipuleucel-T is an autologous cellular immunotherapy that induces an immune response targeted against prostatic acid phosphatase (PAP) to treat asymptomatic or minimally symptomatic metastatic castration-resistant prostate cancer. In the phase III IMPACT study, sipuleucel-T was associated with a statistically significantly increased overall survival (OS) (median = 4.1 months) vs placebo. Patients with baseline prostate-specific antigen levels in the lowest quartile (≤22.1 ng/mL) exhibited a 13-month improvement in OS with sipuleucel-T. Together, this led sipuleucel-T to be approved and recommended as first-line therapy in various guidelines for treatment of metastatic castration-resistant prostate cancer. This review discusses the varied findings about the mechanisms of action of sipuleucel-T, bringing them together to form a more coherent picture. These pieces include inducing a statistically significant increase in antigen-presenting cell activation; inducing a peripheral immune response specific to the target (PAP) and/or immunizing (PA2024) antigens; stimulating systemic cytotoxic T-lymphocyte activity; and mediating antigen spread (ie, increased antibody responses to secondary proteins in addition to PAP and PA2024). Each of these pieces individually correlates with OS. Sipuleucel-T also traffics T cells to the prostate and is associated with long-term immune memory such that a second course of treatment induces an anamnestic immune response. Prostate cancer does not have a strongly inflamed microenvironment, thus its response to immune checkpoint inhibitors is limited. Because sipuleucel-T is able to traffic T cells to the tumor, it may be an ideal combination partner with immunotherapies including immune checkpoint inhibitors or with radiation therapy.


Screening for cancers of the head and neck: addressing the problem.
More than 90% of upper aerodigestive tract (UADT) cancers occur in people with specific lifestyle risks, including tobacco and alcohol use. More than 90% of tumors occur in easily examined parts of the head and neck, therefore, there is the possibility of identifying the vast majority of patients through selective screening. Physicians should keep in mind that the mucosa's sojourn from visually suspicious (and possibly malignant) tissue is most likely less than two years, and frequent examination of asymptomatic patients is necessary. When patients wait to bring symptoms to medical attention, their cancers will be advanced 60% of the time when the chance of cure is less than 30%. Given the difficulty of implementing regular examinations in a poorly compliant, high risk population, genetic and molecular screening tools may allow very high risk individuals to be identified.


Desmutagenic effect of alpha-dicarbonyl and alpha-hydroxycarbonyl compounds against mutagenic heterocyclic amines.
The desmutagenic effects of alpha-hydroxycarbonyl compounds, such as glyceraldehyde, glycolaldehyde, dihydroxyacetone, furfural, 5-hydroxymethylfurfural, maltol, acetol and acetoin and alpha-dicarbonyl compounds, such as diacetyl, glyoxal, methyl glyoxal and 2,3-pentanedione were investigated against the mutagenic heterocyclic amines, such as Trp-P-1, Trp-P-2, Glu-P-1, Glu-P-2 and IQ. Most of the carbonyl compounds suppressed the mutagenicity of heterocyclic amines for S. typhimurium TA98, alpha-dicarbonyl compounds showing a higher desmutagenic effect than alpha-hydroxycarbonyl compounds. Among the alpha-hydroxycarbonyl compounds, glyceraldehyde, glycolaldehyde and dihydroxyacetone showed more effective desmutagenicity, and diacetyl among the alpha-dicarbonyl compounds had the highest desmutagenic effect. These carbonyl compounds alone also showed mutagenicity to S. typhimurium TA100 without S9 mix. The reaction of carbonyl compounds with mutagenic heterocyclic amines also eliminated the mutagenicity of the former for S. typhimurium TA100.


ABCB5 expression and cancer stem cell hypothesis in oral squamous cell carcinoma.
The vast majority of oral cancers are squamous cell carcinomas (OSCC). The effectiveness of adjuvant cytostatic chemotherapy for OSCC is frequently restricted due to an inducible cellular mechanism called multidrug resistance (MDR) and a putative cancer stem cell (CSC) compartment in human carcinogenesis expressing multidrug efflux pumps. The novel human ATP-binding cassette (ABC) transporter ABCB5 [subfamily B (MDR/TAP) member 5] acts as an energy-dependent drug efflux transporter and marks tumour cells of a putative CSC compartment. However, to date, there is no link between ABCB5 expression and OSCC. Expression of ABCB5 was analysed in OSCC specimen (n=191) and cancer cell lines (BICR3, BICR56) by immunohistochemistry, real-time polymerase chain reaction (RT-PCR) analysis and western blotting. Scanned images were digitally analysed using ImageJ and the immunomembrane plug-in. ABCB5 expression on protein level was correlated with clinical characteristics and impact on survival. ABCB5 was co-labelled with CD44 in immunohistochemical and immunofluorescence double labelling experiments. Expression subgroups were identified by receiver operating characteristics (ROC) analysis. High ABCB5 expression was significantly associated with tumour progression and recurrence of the tumour. Multivariate analysis demonstrated high ABCB5 expression as an independent prognostic factor (p=0.0004). Immunohistochemical and immunofluorescence double labelling experiments revealed ABCB5 expression by CD44+ cancer cells. ABCB5 specificity was confirmed by western blot and RT-PCR analysis. For the first time, this study provides evidence that ABCB5 expression in OSCC might be associated with tumour formation, metastasis and a putative CSC compartment. One of the principal mechanisms for protecting putative cancer stem cells is through the expression of multifunctional efflux transporters from the ABC gene family, like ABCB5. This provides one mechanism in which putative cancer stem cells could survive and may lead to tumour relapse. Knowledge of expression profiles of ABC transporters and other genes involved in MDR will likely help therapeutic optimisation for cancer patients in clinic. However, this hypothesis requires further in vitro and in vivo studies.


Emerging role of kinase-targeted strategies in chronic lymphocytic leukemia.
Chronic lymphocytic leukemia (CLL) is a malignancy of mature B cells that depend on host factors in the tissue microenvironment for survival and proliferation. In vitro, CLL cells rapidly undergo apoptosis unless microenvironmental factors are provided that support their survival. Signaling pathways activated in the microenvironment in vivo include the B-cell receptor (BCR) and NF-κB pathways. Thus, CLL is a disease "addicted to the host" and is dependent on pathways that promote normal B-cell development, expansion, and survival; this is particularly true in the case of the BCR signaling cascade. Small-molecule inhibitors of kinases that are essential for BCR signal transduction abrogate the stimulating effects of the microenvironment on CLL cells. The orally administered tyrosine kinase inhibitors fostamatinib and ibrutinib and the phosphatidylinositol 3-kinase inhibitor GS-1101 have induced impressive responses in relapsed and refractory CLL patients, mostly with moderate side effects. Reductions in lymphadenopathy and splenomegaly are seen within weeks and are frequently accompanied by a transient rise in absolute lymphocyte count that is asymptomatic and probably the result of changes in CLL cell trafficking. This review discusses the biologic basis for kinase inhibitors as targeted therapy of CLL and summarizes the exciting early clinical experience with these agents.


Presynaptic beta-adrenoceptors.
The existence of facilitatory presynaptic beta-adrenoceptors has been shown in approximately 30 tissues of 6 different species including human. A positive feed back loop for further release of the transmitter appears to be activated by an endogenous agonist, epinephrine, taken up and released as a cotransmitter with norepinephrine rather than norepinephrine itself released from peripheral noradrenergic nerve terminals. Presynaptic beta-adrenoceptors are mainly of a beta 2-subtype. Some beta 1-subtype receptors are also suggested. There coexist presynaptic beta 1- and beta 2-adrenoceptors in cat and rat hypothalamus. Higher sensitivity of peripheral presynaptic beta-adrenoceptors to isoproterenol may be implicated in the early development of hypertension in SHR. Epinephrine taken up and released initiates the development of hypertension in rats via activation of these receptors. Increased activation of these receptors by epinephrine may play a role in the development of essential hypertension. The antihypertensive action of beta-antagonists may be in part due to blockade of these facilitatory presynaptic beta-adrenoceptors.


Plasma arginine vasopressin in hyponatraemic patients receiving diuretics.
Random estimations of plasma arginine vasopressin concentration were undertaken in 6 non-oedematous patients receiving diuretic therapy for hypertension, who were admitted to hospital with severe hyponatraemia. Hyponatraemia resolved within 2 weeks of discontinuing the diuretic. Measurable amounts of plasma arginine vasopressin were detected in all 6 patients. Sequential biochemical measurements in one patient, performed when plasma sodium concentration and osmolality were returning to the normal range, disclosed that urine osmolality remained higher than plasma osmolality during the first 5 days, when urine volume and sodium excretion were low. Thus the rise in plasma sodium was not initially related to water diuresis. The ability to excrete a water load was severely limited on the fifth day, but improved progressively by the tenth and seventeenth days. Diuretic-induced hyponatraemia is associated with incomplete suppression of anti-diuretic hormone secretion arising from non-osmotic stimulation, in conjunction with transient impairment of renal diluting ability which could be due to net sodium deficit.


South China karst aquifer storm-scale hydrochemistry.
The peak cluster and peak forest karst regions of Southeast Asia form one of the earth's most extensive karst regions. Although there exists a rich, descriptive tradition of geomorphic work performed there, little quantitative study has been made of carbonate hydrochemistry and related aquifer/landscape behavior and evolution. In this paper, high-resolution measurements of ground water carbonate chemistry and flow were made and analyzed at two adjacent locations within the subtropical peak cluster karst of the Guilin Karst Experimental Site in Guangxi Province, China. While waters from a large, perennial spring represent the exit for the 2 km2 catchment's conduit flow, a nearby well (within 5 m) measures water in the conduit-adjacent, fractured media. Results indicate that within peak cluster karst aquifer flow systems, spatially heterogeneous flow conditions can exist with respect to timing, magnitude, and, in some cases, direction of responses, as different controls can operate in the different flow system components. Storm-scale chemical responses are controlled by dilution from rapid infiltration of rain water, CO2 gas sources and sinks, and water-carbonate rock interactions. At this particular location, there is also an influence from high pH recharge, apparently buffered by atmospheric limestone dust. An example of the varying controls on storm-scale responses within the flow system is that within the fractured medium, variations in the ground water calcite saturation index, a key parameter influencing rates of aquifer/landscape evolution, are small and controlled by CO2 gas, while in the conduit they are more significant and dominated instead by dilution with rain water.


Soymilk fermentation by Enterococcus faecalis VB43 leads to reduction in the immunoreactivity of allergenic proteins β-conglycinin (7S) and glycinin (11S).
Food allergies represent a serious problem affecting human health and soy proteins rank among the most allergenic proteins from food origin. The proteolytic enzymes produced by lactic acid bacteria (LAB) can hydrolyse the major allergens present in soybean, reducing their immunoreactivity. Many studies have reported the ability of LAB to ferment soy-based products; while the majority of them focus on the improvement of the sensory characteristics and functionality of soy proteins, a lack of information about the role of lactic fermentation in the reduction of immunoreactivity of these proteins exists. The aim of the present study was to evaluate the capability of the proteolytic strain Enterococcus faecalis VB43 to hydrolyse the main allergenic proteins present in soymilk and to determine the immunoreactivity of the obtained hydrolysates. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) results of fermented soymilk demonstrated complete hydrolysis of the β-subunit from β-conglycinin and the acidic polypeptide from glycinin. Reversed phase high performance liquid chromatography (RP-HPLC) analysis of the peptides released after hydrolysis revealed the appearance of new peptides and the disappearance of non-hydrolysed proteins, indicating extensive hydrolysis of the substrate. Results from competitive enzyme-linked immunosorbent assay (ELISA) tests clearly indicated a reduction in the immunoreactivity (more than one logarithmic unit) in the fermented sample as compared to the non-fermented control. Our results suggest that the soymilk fermented by E. faecalis VB43 may induce lower allergic responses in sensitive individuals. The strain E. faecalis VB43 may be considered as an excellent candidate to efficiently reduce the immunoreactivity of soymilk proteins.


[Premature ejaculation: diagnosis, classification, algorithm of the patients' examination and results of their application].
We have analysed literature data and own experience with management of premature ejaculation (PE) in 3200 patients. This gave grounds for formulation of this phenomenon, creation of the classification and the examination algorithm. We applied the proposed methods in the treatment of 258 PE patients and made the conclusion about their adequacy and efficacy. PE is a polyetiological phenomenon and a multidisciplinary problem. Urological examination must be used as the first stage of the diagnostic process.


Mapping the active site of the Haemophilus influenzae methionyl-tRNA formyltransferase: residues important for catalysis and tRNA binding.
Formylation of the initiator methionyl-tRNA by methionyl-tRNA formyltransferase (MTF) is an essential step in initiation of protein synthesis in eubacteria. Here, site-directed mutagenesis was used to identify active site residues of the Haemophilus influenzae MTF. Of the nine residues investigated, only Arg-41, Asn-107, His-109 and Asp-145 were important for the function of the H. influenzae MTF. Replacement of these residues with Ala resulted in a significant reduction in the efficiency of catalysis. Intrinsic fluorescence analysis indicated that this was not due to a defect in N10-formyltetrahydrofolate (fTHF) binding. The Asp-145 and Arg-41 mutations reduced the affinity of the enzyme for the initiator tRNA, whereas the Asn-107 and His-109 mutations affected catalysis but not tRNA binding. Replacement of Arg-41, His-109 and Asp-145 with functionally similar residues also affected the activity of the enzyme. The data suggest that Asn-107, His-109 and Asp-145 are catalytic residues, whereas Arg-41 is involved in tRNA recognition. In the Escherichia coli glycinamide ribonucleotide formyltransferase, which also uses fTHF as the formyl donor, Asn-106, His-108 and Asp-144 participate in the catalytic step. Together, these observations imply that this group of enzymes uses the same basic mechanism in formylating their substrates.


Curbing potential radiation-induced cancer risks in oncologic imaging: perspectives from the 'image gently' and 'image wisely' campaigns.
Medical imaging that uses ionizing radiation, such as CT, radiography, nuclear medicine, and fluoroscopy, is a cornerstone of the care of oncology patients and provides great benefit. Ionizing radiation at high doses is a known carcinogen.The exact degree of the risk of carcinogenesis from the lower doses of ionizing radiation used in medical imaging is less clear. The purpose of this review is to provide the oncology community with knowledge about the doses used in medical imaging, radiation-induced cancer risks from imaging, considerations to keep in mind when balancing imaging benefits and risks in pediatric and adult oncologic settings, dose reduction strategies, and the "Image Gently" and "Image Wisely" campaigns; the latter campaigns facilitate the translation of existing evidence into best practices for providers and patients.


Using individual and group reflection in and on assessment as a tool for effective learning.
This paper outlines the background to the current United Kingdom educational scene for post-registration nurse education, highlighting the influence of the English National Board (ENB) framework and higher award. The study reported was undertaken with the help of two student cohorts who were registered consecutively on a 6 month ENB course at diploma level. Formal reflection using both individual written and group discussion was introduced focusing on the two summative course assessments. This kind of formal reflection was seen as a learning strategy to influence positively students' educational outcomes and professional practice via six key factors: (a) academic self-concept; (b) task awareness; (c) views of knowledge; (d) the influence of knowledge on behaviour; (e) writing as a learning activity; and (f) generating knowledge by reflecting in and on assessment with discussion. The findings, reported under the six factor-headings, fully support the use of reflection in this way for these purposes.


Fingerprinting the Excited-State Dynamics in Methyl Ester and Methyl Ether Anions of Deprotonated para-Coumaric Acid.
Chromophores based on the para-hydroxycinnamate moiety are widespread in the natural world, including as the photoswitching unit in photoactive yellow protein and as a sunscreen in the leaves of plants. Here, photodetachment action spectroscopy combined with frequency- and angle-resolved photoelectron imaging is used to fingerprint the excited-state dynamics over the first three bright action-absorption bands in the methyl ester anions (pCEs-) of deprotonated para-coumaric acid at a temperature of ∼300 K. The excited states associated with the action-absorption bands are classified as resonances because they are situated in the detachment continuum and are open to autodetachment. The frequency-resolved photoelectron spectrum for pCEs- indicates that all photon energies over the S1(ππ*) band lead to similar vibrational autodetachment dynamics. The S2(nπ*) band is Herzberg-Teller active and has comparable brightness to the higher lying 21(ππ*) band. The frequency-resolved photoelectron spectrum over the S2(nπ*) band indicates more efficient internal conversion to the S1(ππ*) state for photon energies resonant with the Franck-Condon modes (∼80%) compared with the Herzberg-Teller modes (∼60%). The third action-absorption band, which corresponds to excitation of the 21(ππ*) state, shows complex and photon energy-dependent dynamics, with 20-40% of photoexcited population internally converting to the S1(ππ*) state. There is also evidence for a mode-specific competition between prompt autodetachment and internal conversion on the red edge of the 21(ππ*) band. There is no evidence for recovery of the ground electronic state and statistical electron ejection (thermionic emission) following photoexcitation over any of the three action-absorption bands. The photoelectron spectra for the deprotonated methyl ether derivative (pCEt-) at photon energies over the S1(ππ*) and S2(nπ*) bands indicate diametrically opposed dynamics compared with pCEs-, namely, intense thermionic emission due to efficient recovery of the ground electronic state.


A conceptual overview and critique of functional neuroimaging techniques in humans: I. MRI/FMRI and PET.
A critical evaluation of current, noninvasive brain-mapping techniques indicates that no one technique in isolation can adequately address the varied questions of interest to basic researchers and clinicians. Consequently, an integrated analysis of anatomical and functional measurements has become the ultimate goal to better characterize the nature of neuronal and hemodynamic responses to sensory and cognitive stimulation. Such an analysis will optimize both spatial and temporal resolution, thereby enabling patterns of activation to be followed throughout the central nervous system, a prerequisite for examining stages of information processing. This review, written in two parts, provides a conceptual overview of three techniques (MRI/fMRI, PET, and MEG/EEG) and examines current efforts at integrating these measures. Each technique is initially evaluated in isolation by considering a set of primary issues (e.g., spatial resolution, temporal resolution, the nature of the signal, individual variability, repeatability, etc.). The first part compares and contrasts MRI/fMRI techniques with PET. Part II, which examines MEG/EEG techniques and current attempts at integrating techniques, will appear in a subsequent volume. This two-part review focuses on issues, not results per se, and it is assumed that the reader is not proficient in any of the techniques described.


Novel role of surfactant protein A in bacterial sinusitis.
Chronic rhinosinusitis (CRS) is a common inflammatory disorder of the upper airway characterized by chronic inflammation and significant sinonasal remodeling. CRS is comprised of 2 major subgroups, based on whether polyps are present or absent. In some cases, it is characterized by colonization with opportunistic pathogens such as Pseudomonas aeruginosa (PA), Staphylococcus aureus, and other bacteria. The innate immune system of the sinonasal epithelium is the first line of defense against inhaled pathogens. Surfactant protein A (SP-A) is a member of the collectin family secreted by the airway epithelia and plays a critical role in airway innate immunity, as it can aggregate bacteria. We hypothesized that SP-A plays a role in bacterial CRS. Air-liquid interface (ALI) cultures of nasal epithelial cells were derived from human ex-vivo healthy and CRS sinus tissues (n = 26) and challenged with PA. SP-A levels were measured with western blot and quantitative reverse transcript-polymerase chain reaction (qRT-PCR) in ALI and sinus tissues. We determined that SP-A: (i) mRNA and protein levels are increased significantly in CRS tissues compared with healthy sinuses; (ii) although primarily expressed in the lung, it is also synthesized and expressed in sinonasal epithelia; (ii) is expressed in the sinuses of an SP-A humanized transgenic mouse but not in SP-A knockout mice; (iv) mRNA levels are upregulated significantly during PA challenge, but protein levels are downregulated 4 hours postchallenge and upregulated at 12 hours. Our data suggest that SP-A is expressed in the sinuses and that it plays a role in the sinus innate immune responses during bacterial infections.


Head and neck injuries in college football: an eight-year analysis.
The present study documented head and neck injuries in a study group of 342 college football players at a single institution for a period of 8 years. All freshmen players were screened for evidence of: (1) past history of head and neck injuries, and (2) abnormalities of the cervical spine on physical examination and x-ray film. By recording all head injuries and those neck injuries with time loss, incidence rates and patterns of injury incurred in college competition were determined. A total of 175 head and neck injuries were sustained by 100 players over the 8 year period. Those players with abnormal findings on screening examination were twice as likely to have a head or neck injury at some point in their college careers as those players with a normal screening examination. The greater the degree of abnormality on freshman screening examination, the more severe the neck injury in college was likely to be. Twenty-nine percent of all players in the study group sustained a head or neck injury during their college careers. The probability of a subsequent head or neck injury escalated sharply following a single incident. The overall incidence of injury was found to have been dramatically reduced over the 8 years. Influential factors such as legislative rule changes, medical status of recruits, and general coaching philosophies are discussed with regard to injury reduction and prevention of head and neck injuries in college football.


[Medical reform in Brazil and the US: a comparison of two rhetorics].
The article examines the genesis of the medical education model proposed by A. Flexner in the United States and compares it with Antonio da Silva Mello's 1930s proposal to apply the German university model to Brazilian medical teaching. The heart of these medical reforms - which sought to introduce the teaching of biomedicine and to boost the esteem of the scientific career - did not depend solely on these two reformers' perfect understanding of the bases of the new model. Each rhetoric expressed a political arrangement wherein any expectations for change in the consolidated educational system depended upon its power structure and upon traditional career expectations.


Identification of an Spc110p-related protein in vertebrates.
Although varying in size and complexity, centrosomes have conserved functions throughout the evolutionary range of eukaryotes, and thus may display conserved components. In this work, we took advantage of the recent advances in the isolation of the budding yeast spindle pole body, the development of specific immunological probes and the molecular characterisation of genes involved in spindle pole body duplication or assembly. Screening a monoclonal antibody library against Saccharomyces cerevisiae spindle pole body components, we found that two monoclonal antibodies, directed against two different parts of the yeast Spc110p, decorate the centrosome from mammalian cells in an asymmetrical manner. Western blot experiments identified a 100 kDa protein specifically enriched in centrosome preparations from human cells. This protein is phosphorylated during mitosis and is tightly associated with the centrosome: only denaturing conditions such as 8 M urea were able to solubilise it. Purified immunoglobulins directed against Spc110p inhibit microtubule nucleation on isolated human centrosomes, using brain phosphocellulose-tubulin or Xenopus egg extract tubulin. This result suggested that the centrosomal 100 kDa protein could be involved in a microtubule nucleation complex. To test this hypothesis, we turned to Xenopus species, in which mAb anti-Spc110p decorated centrosomes from somatic cells and identified a 116 kDa protein in egg extract. We performed a partial purification of the gamma-tubulin-ring complex from egg extract. Sucrose gradient sedimentation, immunoprecipitation and native gels demonstrated that the Xenopus 116 kDa protein and gamma-tubulin were found in the same complex. Altogether, these results suggest the existence of an yeast Spc110-related protein in vertebrate centrosomes which is involved in microtubule nucleation.


Development and application of an enzyme-linked immunosorbent assay for the quantitation of alternative complement pathway activation in human serum.
We have developed a new, specific, and highly sensitive enzyme-linked immunosorbent assay (ELISA) which quantitates activation of the alternative pathway in human serum, plasma, or on the surface of activators. The ELISA detects the third component of complement (C3b), proteolytic fragment of complement Factor B (Bb), and properdin (P) complex or its derivative product, C3b,P. In the method, activator-plasma mixtures, plasma containing an activated alternative pathway, or other samples are added to the wells of microtitration plates precoated with antibody to P. C3b, Bb,P or C3b,P complexes which become bound are quantitated by subsequently added, enzyme-labeled, anti-C3. The resulting hydrolysis of the chromogenic substrate is expressed as nanograms of C3b by reference to a C3 standard curve. In addition to absolute specificity for activation of the pathway because of the nature of the complex detected by the assay, the ELISA is highly sensitive and able to reproducibly detect 10-20 ng/ml of C3b,P complexes in serum. This value corresponds to 0.0015% of the C3 in serum. In a series of studies to validate the parameters of the ELISA, reactivity was found to be dependent on the presence of alternative pathway proteins, the functional integrity of the pathway, and on the presence of magnesium. Sheep erythrocytes were converted to activators by treatment with neuraminidase. By using a variety of activators, the kinetics of activation and the numbers of bound C3b molecules quantitated by the ELISA were very similar to those measured by C3b deposition. The ELISA also detected identical activation kinetics when MgEGTA-serum and a mixture of the purified alternative pathway proteins were used as sources of the pathway. ELISA reaction kinetics also correlated with the restriction index, a measure of alternative pathway-activating ability. These studies cumulatively validate the ELISA as a direct and quantitative assay for alternative pathway activation. The sensitivity of the ELISA has permitted its use to detect direct alternative pathway activation by several viruses. The ELISA has also shown that certain classical pathway activators trigger the amplification loop of the alternative pathway while others do not. In addition, stable ELISA reactive complexes appeared in the supernatant of mixtures of serum with certain, but not other activators. The ability of the ELISA to detect activation which has already occurred and the stability of the reactive complexes permits studies of clinical sera. Normal human sera (20) contained low levels (5-20 ng/ml) of ELISA-reactive complexes. A proportion of sera from individuals with the adult respiratory distress syndrome (9-10), typhoid fever (8-10), malaria (3-5), gram-negative sepsis (9 of 47), acute trauma and shock (6 f 25), and systemic lupus erythematosus (3 of 29) showed elevated levels of complexes reactive in the alternative pathway ELISA. In contrast, nine sera from patients with circulating C3 nephritic factor were not reactive in the ELISA.


Distribution dynamics and functional importance of NHERF1 in regulation of Mrp-2 trafficking in hepatocytes.
Na(+)/H(+) exchanger regulatory factor 1 (NHERF1) is a multifunctional scaffolding protein that interacts with receptors and ion transporters in its PDZ domains and with the ezrin-radixin-moesin (ERM) family of proteins in its COOH terminus. The role of NHERF1 in hepatocyte function remains largely unknown. We examine the distribution and physiological significance of NHERF1 and multidrug resistance-associated protein 2 (Mrp-2) in hepatocytes. A WT radixin binding site mutant (F355R) and NHERF1 PDZ1 and PDZ2 domain adenoviral mutant constructs were tagged with yellow fluorescent protein and expressed in polarized hepatocytes to study localization and function of NHERF1. Cellular distribution of NHERF1 and radixin was visualized by fluorescence microscopy. A 5-chloromethylfluorescein diacetate (CMFDA) assay was used to characterize Mrp-2 function. Similar to Mrp-2, WT NHERF1 and the NHERF1 PDZ2 deletion mutant were localized to the canalicular membrane. In contrast, the radixin binding site mutant (F355R) and the NHERF1 PDZ1 deletion mutant, which interacts poorly with Mrp-2, were rarely associated with the canalicular membrane. Knockdown of NHERF1 led to dramatically impaired CMFDA secretory response. Use of CMFDA showed that the NHERF1 PDZ1 and F355R mutants were devoid of a secretory response, while WT NHERF1-infected cells exhibited increased secretion of glutathione-methylfluorescein. The data indicate that NHERF1 interacts with Mrp-2 via the PDZ1 domain of NHERF1 and, furthermore, that NHERF1 is essential for maintaining the localization and function of Mrp-2.


[Conservative surgery in ovarian cancer].
Surgery for ovarian cancer is always radical. In young patients with wish of pregnancy and in early stages, the conservative surgery can be contemplated. This type of surgery involves removing the tumoral ovary, leaving the healthy ovary and uterus. In these cases, the age, stage, histologic type, grade and biological factors must be considered. There is controversy about the surgical approach: laparoscopy or laparotomy, the indication to perform biopsy of the contralateral ovary and pelvic-paraaortic lymphadenectomy. In high-risk cases adjuvant chemotherapy must be considered. We present our experience in 330 common epithelial and germinal ovarian carcinomas. We performed conservative surgery in 13. Four patients got pregnant and 3 of them delivered a term newborn. In 13 of 52 borderline tumors a conservative surgery was performed and 3 term deliveries were obtained. All patients are alive and free of disease.


Viscoelastic properties of vimentin originate from nonequilibrium conformational changes.
Structure and dynamics of living matter rely on design principles fundamentally different from concepts of traditional material science. Specialized intracellular filaments in the cytoskeleton permit living systems to divide, migrate, and grow with a high degree of variability and durability. Among the three filament systems, microfilaments, microtubules, and intermediate filaments (IFs), the physical properties of IFs and their role in cellular mechanics are the least well understood. We use optical trapping of individual vimentin filaments to investigate energy dissipation, strain history dependence, and creep behavior of stretched filaments. By stochastic and numerical modeling, we link our experimental observations to the peculiar molecular architecture of IFs. We find that individual vimentin filaments display tensile memory and are able to dissipate more than 70% of the input energy. We attribute these phenomena to distinct nonequilibrium folding and unfolding of α helices in the vimentin monomers constituting the filaments.


Epitope mapping of the U1 small nuclear ribonucleoprotein particle in patients with systemic lupus erythematosus and mixed connective tissue disease.
Systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD) are autoimmune illnesses characterized by the presence of high titers of autoantibodies directed against a wide range of 'self ' antigens. Proteins of the U1 small nuclear ribonucleoprotein particle (U1 snRNP) are among the most immunogenic molecules in patients with SLE and MCTD. The recent release of a crystallized U1 snRNP provides a unique opportunity to evaluate the effects of tertiary and quaternary structures on autoantigenicity within the U1 snRNP. In the present study, an epitope map was created using the U1 snRNP crystal structure. A total of 15 peptides were tested in a cohort of 68 patients with SLE, 29 with MCTD and 26 healthy individuals and mapped onto the U1 snRNP structure. Antigenic sites were detected in a variety of structures and appear to include RNA binding domains, but mostly exclude regions necessary for protein-protein interactions. These data suggest that while some autoantibodies may target U1 snRNP proteins as monomers or apoptosis-induced, protease-digested fragments, others may recognize epitopes on assembled protein subcomplexes of the U1 snRNP. Although nearly all of the peptides are strong predictors of autoimmune illness, none were successful at distinguishing between SLE and MCTD. The antigenicity of some peptides significantly correlated with several clinical symptoms. This investigation implicitly highlights the complexities of autoimmune epitopes, and autoimmune illnesses in general, and demonstrates the variability of antigens in patient populations, all of which contribute to difficult clinical diagnoses.


Chronic Wasting Disease Drives Population Decline of White-Tailed Deer.
Chronic wasting disease (CWD) is an invariably fatal transmissible spongiform encephalopathy of white-tailed deer, mule deer, elk, and moose. Despite a 100% fatality rate, areas of high prevalence, and increasingly expanding geographic endemic areas, little is known about the population-level effects of CWD in deer. To investigate these effects, we tested the null hypothesis that high prevalence CWD did not negatively impact white-tailed deer population sustainability. The specific objectives of the study were to monitor CWD-positive and CWD-negative white-tailed deer in a high-prevalence CWD area longitudinally via radio-telemetry and global positioning system (GPS) collars. For the two populations, we determined the following: a) demographic and disease indices, b) annual survival, and c) finite rate of population growth (λ). The CWD prevalence was higher in females (42%) than males (28.8%) and hunter harvest and clinical CWD were the most frequent causes of mortality, with CWD-positive deer over-represented in harvest and total mortalities. Survival was significantly lower for CWD-positive deer and separately by sex; CWD-positive deer were 4.5 times more likely to die annually than CWD-negative deer while bucks were 1.7 times more likely to die than does. Population λ was 0.896 (0.859-0.980), which indicated a 10.4% annual decline. We show that a chronic disease that becomes endemic in wildlife populations has the potential to be population-limiting and the strong population-level effects of CWD suggest affected populations are not sustainable at high disease prevalence under current harvest levels.


Phylogeography of Baryancistrus xanthellus (Siluriformes: Loricariidae), a rheophilic catfish endemic to the Xingu River basin in eastern Amazonia.
Baryancistrus xanthellus (Loricariidae) is an endemic fish species from the Xingu River basin with its life history in the shallow rapid waters flowing over bedrock substrates. In order to investigate the genetic diversity and demographic history of B. xanthellus we analyzed sequence data for one mitochondrial gene (Cyt b) and introns 1 and 5 of nuclear genes Prolactin (Prl) and Ribosomal Protein L3 (RPL3). The analyses contain 358 specimens of B. xanthellus from 39 localities distributed throughout its range. The number of genetically diverged groups was estimated using Bayesian inference on Cyt b haplotypes. Haplotype networks, AMOVA and pairwise fixation index was used to evaluate population structure and gene flow. Historical demography was inferred through neutrality tests and the Extended Bayesian Skyline Plot (EBSP) method. Five longitudinally distributed Cyt b haplogroups for B. xanthellus were identified in the Xingu River and its major tributaries, the Bacajá and Iriri. The demographic analysis suggests that rapids habitats have expanded in the Iriri and Lower Xingu rivers since 200 ka (thousand years) ago. This expansion is possibly related to an increase in water discharge as a consequence of higher rainfall across eastern Amazonia. Conversely, this climate shift also would have promoted zones of sediment trapping and reduction of rocky habitats in the Xingu River channel upstream of the Iriri River mouth. Populations of B. xanthellus showed strong genetic structure along the free-flowing river channels of the Xingu and its major tributaries, the Bacajá and Iriri. The recent impoundment of the Middle Xingu channel for the Belo Monte hydroelectric dam may isolate populations at the downstream limit of the species distribution. Therefore, future conservation plans must consider the genetic diversity of B. xanthellus throughout its range.


Bioisosteric approach in the design of new dopaminergic/serotonergic ligands.
Dopaminergic and serotonergic ligands are widely applied in the therapy of some severe diseases in humans connected to the malfunctioning of the corresponding membrane receptors within the CNS. However, no pharmaceuticals of this type with an ideal therapeutic index have been synthesized so far and there is a constant need of producing new dopaminergic/serotonergic ligands with improved properties especially with regard to undesirable side effects expressed after a prolonged therapy. Dopaminergic/serotonergic ratio turned out to be important for a fine tuning of pharmacological profile of new ligands. Employing a bioisosteric approach, we have synthesized numerous quinoxalinediones, benztriazoles, benzimidazoles and 2-substituted benzimidazoles as potential dopaminergic and/or mixed dopaminergic/serotonergic compounds. With this purpose, benzimidazole and its derivatives were incorporated into phenylethylamine, 3- and 4-substituted phenylethylpiperidine, 1-substituted 4-arylpiperazine and semirigid 2-aminotetralin frame and the resulting ligands were checked for the binding affinity at the D1 and D2 dopamine and 5-HT1A serotonin receptors in radioligand binding assays in vitro. Synaptosomal membranes prepared from bovine caudate nuclei and hippocampi served as a source of the dopamine and serotonin receptors, respectively. [3H]SCH 23390 (D1 receptor-selective), [3H]spiperone (D2 receptor-selective) and 8 OH [3H]DPAT (5-HT1A receptor-selective) were employed as radioligands in competition binding assays. Properties of substituents introduced into position 2 of benzimidazole ring, as well as the nature of the frame into which benzimidazole pharmacophore was incorporated have been shown to determine ligand binding affinity, mode of action and receptor preference, i.e. dopaminergic/serotonergic affinity ratio. Benzimidazolyl-2-thione and benztriazole derivatives were the most potent dopaminergic/serotonergic ligands. Molecular ab initio calculations of the electronic properties of pharmacophoric entities of the new ligands revealed different electron density distribution around the benzene ring in the active and inactive ligands. It can be assumed that this difference influences the properties of pi-pi interactions in a receptor-ligand complex. The results are discussed in comparison with the data of other authors working on similar topics.


Metal-mediated diradical tuning for DNA replication arrest via template strand scission.
A series of M(PyED)·X (X = 2Cl-, SO42-) pyridine-metalloenediyne complexes [M = Cu(II), Fe(II), or Zn(II)] and their independently synthesized, cyclized analogs have been prepared to investigate their potential as radical-generating DNA-damaging agents. All complexes possess a 1:1 metal-to-ligand stoichiometry as determined by electronic absorption spectroscopy and X-ray diffraction. Solution structural analysis reveals a pπ Cl [Formula: see text] Cu(II) LMCT (22,026 cm-1) for Cu(PyED)·2Cl, indicating three nitrogens and a chloride in the psuedo-equatorial plane with the remaining pyridine nitrogen and solvent in axial positions. EPR spectra of the Cu(II) complexes exhibit an axially elongated octahedron. This spectroscopic evidence, together with density functional theory computed geometries, suggest six-coordinate structures for Cu(II) and Fe(II) complexes and a five-coordinate environment for Zn(II) analogs. Bergman cyclization via thermal activation of these constructs yields benzannulated product indicative of diradical generation in all complexes within 3 h at 37 °C. A significant metal dependence on the rate of the reaction is observed [Cu(II) > Fe(II) > Zn(II)], which is mirrored in in vitro DNA-damaging outcomes. Whereas in situ chelation of PyED leads to considerable degradation in the presence of all metals within 1 h under hyperthermia conditions, Cu(II) activation produces >50% compromised DNA within 5 min. Additionally, Cu(II) chelated PyED outcompetes DNA polymerase I to successfully inhibit template strand extension. Exposure of HeLa cells to Cu(PyBD)·SO4 (IC50 = 10 μM) results in a G2/M arrest compared with untreated samples, indicating significant DNA damage. These results demonstrate metal-controlled radical generation for degradation of biopolymers under physiologically relevant temperatures on short timescales.


Protocol for a systematic review of randomized trials of knee arthroplasty decision aids and shared decision-making approaches.
Shared decision-making is an approach to making treatment-based decisions that rely on the patient encounter and clear discussions between the patient and the healthcare provider. Patients with arthritis of the knee frequently seek care, and depending on arthritis severity and impact on daily life, joint arthroplasty may be considered as a treatment option. We will conduct a systematic review of shared decision-making trials in knee arthroplasty to determine the types of shared decision-making approaches used and their impact on care received. Our systematic review will describe and critically appraise shared decision approaches used in randomized trials of patients undergoing knee arthroplasty, the types of outcomes reported, and the impact of these approaches on the patients' care. We will use the following databases: PubMed, Web of Science, Embase, CINAHL, PsycINFO, and the Cochrane Library, from inception through December 2018. Additionally, we will assess ongoing research by querying experts and searching trial registries. This study will characterize shared decision-making (SDM) approaches in knee arthroplasty randomized clinical trials and will summarize their effects of SDM on clinical and patient-reported outcomes. We anticipate this review will bring to light knowledge gaps and inform further research into the design and use of shared decision-making approaches in lower extremity arthroplasty. PROSPERO CRD42019123586.


Pumped flow biofilm reactors (PFBR) for treating municipal wastewater.
A novel laboratory bench-scale sequencing batch biofilm reactor (SBBR) system was developed for the treatment of synthetic domestic strength wastewater, comprising two side-by-side 18 l reactor tanks, each containing a plastic biofilm media module. Aerobic and anoxic conditions in the biofilms were effected by intermittent alternate pumping of wastewater between the two reactors. With a media surface area loading rate of 4.2 g chemical oxygen demand (COD)/m2.d, the average influent COD, total nitrogen (TN) and ammonium-nitrogen (NH4-N) concentrations of 1021 mg/l, 97 mg/l and 54 mg/l, respectively, reduced to average effluent concentrations of 72 mg COD/l, 17.8 mg TN/l, and 5.5 mg NH4-N /l. Using a similar alternating biofilm exposure arrangement, a 16 person equivalent pilot (PE) plant was constructed at a local village treatment works to remove organic carbon from highly variable settled municipal wastewater and comprised two reactors, one positioned above the other, each containing a module of cross-flow plastic media with a surface area of 100 m2. Two different pumping sequences (PS) in the aerobic phase were examined where the average influent COD concentrations were 220 and 237 mg/l for PS1 and PS2, respectively, and the final average effluent COD was consistently less than 125 mg/l--the European Urban Wastewater Treatment Directive limit--with the best performance occurring in PS1. Nitrification was evident during both PS1 and PS2 studies. A 300 PE package treatment plant was designed based on the bench-scale and pilot-scale studies, located at a local wastewater treatment works and treated municipal influent with average COD, suspended solids (SS) and TN concentrations of 295, 183 and 15 mg/l, respectively resulting in average effluent concentrations of 67 mg COD/l, 17 mg SS/l and 9 mg TN/l. The SBBR systems performed well, and were simple to construct and operate.