How to link ontologies and protein-protein interactions to literature: text-mining approaches and the BioCreative experience.
There is an increasing interest in developing ontologies and controlled vocabularies to improve the efficiency and consistency of manual literature curation, to enable more formal biocuration workflow results and ultimately to improve analysis of biological data. Two ontologies that have been successfully used for this purpose are the Gene Ontology (GO) for annotating aspects of gene products and the Molecular Interaction ontology (PSI-MI) used by databases that archive protein-protein interactions. The examination of protein interactions has proven to be extremely promising for the understanding of cellular processes. Manual mapping of information from the biomedical literature to bio-ontology terms is one of the most challenging components in the curation pipeline. It requires that expert curators interpret the natural language descriptions contained in articles and infer their semantic equivalents in the ontology (controlled vocabulary). Since manual curation is a time-consuming process, there is strong motivation to implement text-mining techniques to automatically extract annotations from free text. A range of text mining strategies has been devised to assist in the automated extraction of biological data. These strategies either recognize technical terms used recurrently in the literature and propose them as candidates for inclusion in ontologies, or retrieve passages that serve as evidential support for annotating an ontology term, e.g. from the PSI-MI or GO controlled vocabularies. Here, we provide a general overview of current text-mining methods to automatically extract annotations of GO and PSI-MI ontology terms in the context of the BioCreative (Critical Assessment of Information Extraction Systems in Biology) challenge. Special emphasis is given to protein-protein interaction data and PSI-MI terms referring to interaction detection methods.


Transcriptional orchestration of the regulated secretory pathway in neurons by the bHLH protein DIMM.
The Drosophila basic helix-loop-helix (bHLH) gene dimmed (dimm) promotes a neurosecretory/neuroendocrine phenotype in cells but is not associated with specific neuropeptides or neurohormones. Rather, it is expressed by those peptidergic neurons that project long axons and appear to produce large amounts of secretory peptides. Here, we genetically transform nonpeptidergic neurons in Drosophila to study DIMM's action mechanisms. Nonpeptidergic neurons normally fail to accumulate ectopic neuropeptides. We now show that they will do so when they are also forced to express ectopic DIMM. Furthermore, mass spectrometry shows that photoreceptors, which are normally nonpeptidergic, fail to process an ectopic neuropeptide precursor to make bioactive peptides but will do so efficiently when DIMM is co-misexpressed. Likewise, photoreceptors, which normally package the fast neurotransmitter histamine within small clear synaptic vesicles, produce numerous large dense-core vesicles (LDCVs) when they misexpress DIMM. These novel LDCVs accumulate ectopic neuropeptide when photoreceptors co-misexpress a neuropeptide transgene. DIMM-expressing photoreceptors no longer accumulate histamine and lose synaptic organelles critical to their normal physiology. These findings indicate that DIMM suppresses conventional fast neurotransmission and promotes peptidergic neurosecretory properties. We conclude that DIMM normally provides a comprehensive transcriptional control to direct the differentiation of dedicated neuroendocrine neurons.


Safety study of intraarticular injection of interleukin 1 receptor antagonist in patients with painful knee osteoarthritis: a multicenter study.
Interleukin 1 (IL-1) plays a pivotal role in the pathogenesis of osteoarthritis (OA). In animal models of OA, IL-1 blockade by IL-1 receptor antagonist (IL-1Ra) can slow the progression of disease. We examined the safety of intraarticular (IA) injections of recombinant human IL-1Ra in patients with knee OA. A prospective multicenter trial was conducted using the continual reassessment method. Six doses were considered, 0.05 mg up to 150 mg IL-1Ra, and the trial was double-blind regarding the dose administered. Patients with symptomatic knee OA and without synovial fluid effusion were included. Acute inflammatory reaction (the primary endpoint defining intolerance) was recorded if pain increase over 30 mm on 100 mm visual analog scale and synovial fluid effusion occurred within 72 h after the IA injection. As a secondary aim, efficacy was estimated (by total pain and Western Ontario and McMaster University OA functional index) until Month 3. One patient received 0.05 mg and 13 patients received 150 mg of IL-1Ra. No acute reaction occurred (one patient experienced postinjection joint swelling with no pain) and the 150 mg dose was considered the maximum tolerated dose (intolerance level 0%; confidence interval 0, 9.1%). A significant improvement was still observed until Month 3 in the 13 patients who received 150 mg IL-1Ra: pain improved by -20.4 +/- 23.3 mm (p = 0.008) and WOMAC global score by -19.5 +/- 20.1 (p = 0.005). IA injection of IL-1Ra in patients with knee OA was well tolerated and did not induce any acute inflammatory reaction. The feasibility of such IA injections of IL-1Ra opens a promising therapeutic perspective for patients with OA.


Practice development 'without walls' and the quandary of corporate practice.
The context of this study is a group of clinical nurse specialists from across a Trust seeking accreditation as a practice development unit. The university was asked to facilitate the accreditation process via 11 2-hour learning sessions (including a one-hour focus group). During initial discussions between the university and practice development unit, the overarching research question for this study was set as: 'what are the main roles and responsibilities of clinical nurse specialists?' Although there is no known study of a practice development unit based beyond a ward or speciality, the central tenet of the practice development unit literature is that units must demonstrate their worth if they are to survive and harness senior management support in doing so. Data gleaned from the transcribed audio tape-recordings of the learning sessions were studied at least three times to ensure transcription accuracy and produce detailed charts. Ethical approval was granted by the appropriate Local Research Ethics Committee and written informed consent obtained from clinical nurse specialists. The study lasted 30 months and ended in October 2004. The four crucial statements that give meaning to specialist practice are: quality care giver; expert; information giver and initiator of change. Further analysis reveals the area of corporate and political practice as being missing from this and other lists of clinical nurse specialist attributes found in the literature. Clinical nurse specialists characterize their relationship with the Trust in terms of dichotomy--differing agendas and perceptions of value. The specialist role requires professional development in the areas of corporate and political acumen and professional business management. While the findings of this study relate to one Trust and a group of 16 clinical nurse specialists, with careful application they may be transferable to other settings and groups of senior nurses.


Radiopacity measurements of direct and indirect resin composites at different thicknesses using digital image analysis.
Radiopacity is an important property of composite materials for clinical diagnosis. For seven direct composites (Aelite LS Posterior, Aelite All-Purpose Body, Quadrant Universal LC, Clearfil Majesty Posterior, Clearfil Majesty Esthetic, Filtek Ultimate Dentin, IPS Empress Direct Dentin) and six indirect composites (Ceromega, Epricord, Estenia C&B, Tescera, Signum Ceramis, Solidex), diskshaped specimens (N=260, n=10 per group) were fabricated for two thicknesses at 1 and 2 mm. Average radiographic density of each composite material was calculated. Radiopacity values of specimens were expressed in equivalent thickness of aluminum using the calibration curve. Data were analyzed using one-way and two-way ANOVA, followed by Tukey's HSD test (α=0.05). Radiopacity was significantly affected by resin composite type (p<0.05) and thickness (p<0.001). All composites, except Epricord (1.22-1.84), had higher radiopacity values than dentin (1.23-2.24). IPS Empress Direct (5.58-9.38) and Estenia C&B (5.49-9.16) showed significantly higher radiopacity (p<0.05) than the other materials including enamel and dentin.


Human immunodeficiency virus type 1 gp120 envelope glycoprotein regions important for association with the gp41 transmembrane glycoprotein.
Insertion of four amino acids into various locations within the amino-terminal halves of the human immunodeficiency virus type 1 gp120 or gp41 envelope glycoprotein disrupts the noncovalent association of these two envelope subunits (M. Kowalski, J. Potz, L. Basiripour, T. Dorfman, W. C. Goh, E. Terwilliger, A. Dayton, C. Rosen, W. A. Haseltine, and J. Sodroski, Science 237:1351-1355, 1987). To localize the determinants on the gp120 envelope glycoprotein important for subunit association, amino acids conserved among primate immunodeficiency viruses were changed. Substitution mutations affecting either of two highly conserved regions located at the amino (residues 36 to 45) and carboxyl (residues 491 to 501) ends of the mature gp120 molecule resulted in nearly complete dissociation of the envelope glycoprotein subunits. Partial dissociation phenotypes were observed for some changes affecting residues in the third and fourth conserved gp120 regions. These results suggest that hydrophobic regions at both ends of the gp120 glycoprotein contribute to noncovalent association with the gp41 transmembrane glycoprotein.


Association of ADHD with genetic variants in the 5'-region of the dopamine transporter gene: evidence for allelic heterogeneity.
Multiple studies have reported an association between attention deficit hyperactivity disorder (ADHD) and the 10-repeat allele of a variable number tandem repeat (VNTR) polymorphism in the 3'-untranslated region (3'UTR) of the dopamine transporter gene (DAT1). Yet, recent meta-analyses of available data find little or no evidence for this association; although there is strong evidence for heterogeneity between datasets. This pattern of findings could arise for several reasons including the presence of relatively rare risk alleles on common haplotype backgrounds or the functional interaction of two or more loci within the gene. We previously described the importance of a specific haplotype at the 3' end of DAT1, as well as the identification of associated single nucleotide polymorphisms (SNPs) within or close to 5' regulatory sequences. In this study we replicate the association of SNPs at the 5' end of the gene and identify a specific risk haplotype spanning the 5' and 3' markers. These findings indicate the presence of at least two loci associated with ADHD within the DAT1 gene and suggest that either additive or interaction effects of these two loci on the risk for ADHD. Overall these data provide further evidence that genetic variants of the dopamine transporter gene confer an increased risk for ADHD.


The epsomitic phototrophic microbial mat of Hot Lake, Washington: community structural responses to seasonal cycling.
Phototrophic microbial mats are compact ecosystems composed of highly interactive organisms in which energy and element cycling take place over millimeter-to-centimeter-scale distances. Although microbial mats are common in hypersaline environments, they have not been extensively characterized in systems dominated by divalent ions. Hot Lake is a meromictic, epsomitic lake that occupies a small, endorheic basin in north-central Washington. The lake harbors a benthic, phototrophic mat that assembles each spring, disassembles each fall, and is subject to greater than tenfold variation in salinity (primarily Mg(2+) and SO(2-) 4) and irradiation over the annual cycle. We examined spatiotemporal variation in the mat community at five time points throughout the annual cycle with respect to prevailing physicochemical parameters by amplicon sequencing of the V4 region of the 16S rRNA gene coupled to near-full-length 16S RNA clone sequences. The composition of these microbial communities was relatively stable over the seasonal cycle and included dominant populations of Cyanobacteria, primarily a group IV cyanobacterium (Leptolyngbya), and Alphaproteobacteria (specifically, members of Rhodobacteraceae and Geminicoccus). Members of Gammaproteobacteria (e.g., Thioalkalivibrio and Halochromatium) and Deltaproteobacteria (e.g., Desulfofustis) that are likely to be involved in sulfur cycling peaked in summer and declined significantly by mid-fall, mirroring larger trends in mat community richness and evenness. Phylogenetic turnover analysis of abundant phylotypes employing environmental metadata suggests that seasonal shifts in light variability exert a dominant influence on the composition of Hot Lake microbial mat communities. The seasonal development and organization of these structured microbial mats provide opportunities for analysis of the temporal and physical dynamics that feed back to community function.


The role of myeloid receptors on murine plasmacytoid dendritic cells in induction of type I interferon.
This study tested the hypothesis that a set of predominantly myeloid restricted receptors (F4/80, CD36, Dectin-1, CD200 receptor and mannan binding lectins) and the broadly expressed CD200 played a role in a key function of plasmacytoid DC (pDC), virally induced type I interferon (IFN) production. The Dectin-1 ligands zymosan, glucan phosphate and the anti-Dectin-1 monoclonal antibody (mAb) 2A11 had no effect on influenza virus induced IFNα/β production by murine splenic pDC. However, mannan, a broad blocking reagent against mannose specific receptors, inhibited IFNα/β production by pDC in response to inactivated influenza virus. Moreover, viral glycoproteins (influenza virus haemagglutinin and HIV-1 gp120) stimulated IFNα/β production by splenocytes in a mannan-inhibitable manner, implicating the function of a lectin in glycoprotein induced IFN production. Lastly, the effect of CD200 on IFN induction was investigated. CD200 knock-out macrophages produced more IFNα than wild-type macrophages in response to polyI:C, a MyD88-independent stimulus, consistent with CD200's known inhibitory effect on myeloid cells. In contrast, blocking CD200 with an anti-CD200 mAb resulted in reduced IFNα production by pDC-containing splenocytes in response to CpG and influenza virus (MyD88-dependent stimuli). This suggests there could be a differential effect of CD200 on MyD88 dependent and independent IFN induction pathways in pDC and macrophages. This study supports the hypothesis that a mannan-inhibitable lectin and CD200 are involved in virally induced type I IFN induction.


Microearthquakes at st. Augustine volcano, alaska, triggered by Earth tides.
Microearthquake activity at St. Augustine volcano, located at the mouth of Cook Inlet in the Aleutian Islands, has been monitored since August 1970. Both before and after minor eruptive activity on 7 October 1971, numerous shallow-foci microearthquake swarms were recorded. Plots of the hourly frequency of microearthquakes often show a diurnal peaking of activity. A cross correlation of this activity with the calculated magnitudes of tidal acceleration exhibited two prominent phase relationships. The first, and slightly more predominant, phase condition is a phase delay in the microearthquake activity of approximately 1 hour from the time of maximum tidal acceleration. This is thought to be a direct microearthquake-triggering effect caused by tidal stresses. The second is a phase delay in the microearthquake activity of approximately 5 hours, which correlates well with the time of maximum oceanic tidal loading. Correlation of the individual peaks of swarm activity with defined components of the tides suggests that it may be necessary for tidal stressing to have a preferential orientation in order to be an effective trigger of microearthquakes.


Drosophila neuroglian: a member of the immunoglobulin superfamily with extensive homology to the vertebrate neural adhesion molecule L1.
Drosophila neuroglian is an integral membrane glycoprotein that is expressed on a variety of cell types in the Drosophila embryo, including expression on a large subset of glial and neuronal cell bodies in the central and peripheral nervous systems and on the fasciculating axons that extend along them. Neuroglian cDNA clones were isolated by expression cloning. cDNA sequence analysis reveals that neuroglian is a member of the immunoglobulin superfamily. The extracellular portion of the protein consists of six immunoglobulin C2-type domains followed by five fibronectin type III domains. Neuroglian is closely related to the immunoglobulin-like vertebrate neural adhesion molecules and, among them, shows most extensive homology to mouse L1. Its homology to L1 and its embryonic localization suggest that neuroglian may play a role in neural and glial cell adhesion in the developing Drosophila embryo. We report here on the identification of a lethal mutation in the neuroglian gene.


Magnetic resonance-guided focused ultrasound for ablation of mesial temporal epilepsy circuits: modeling and theoretical feasibility of a novel noninvasive approach.
The authors tested the feasibility of magnetic resonance-guided focused ultrasound (MRgFUS) ablation of mesial temporal lobe epilepsy (MTLE) seizure circuits. Up to one-third of patients with mesial temporal sclerosis (MTS) suffer from medically refractory epilepsy requiring surgery. Because current options such as open resection, laser ablation, and Gamma Knife radiosurgery pose potential risks, such as infection, hemorrhage, and ionizing radiation, and because they often produce visual or neuropsychological deficits, the authors developed a noninvasive MRgFUS ablation strategy for mesial temporal disconnection to mitigate these risks. The authors retrospectively reviewed 3-T MRI scans obtained with diffusion tensor imaging (DTI). The study group included 10 patients with essential tremor (ET) who underwent pretreatment CT and MRI prior to MRgFUS, and 2 patients with MTS who underwent MRI. Fiber tracking of the fornix-fimbria pathway and inferior optic radiations was performed, ablation sites mimicking targets of open posterior hippocampal disconnection were modeled, and theoretical MRgFUS surgical plans were devised. Distances between the targets and optic radiations were measured, helmet angulations were prescribed, and the numbers of available MRgFUS array elements were calculated. Tractograms of fornix-fimbria and optic radiations were generated in all ET and MTS patients successfully. Of the 10 patients with both the CT and MRI data necessary for the analysis, 8 patients had adequate elements available to target the ablation site. A margin (mean 8.5 mm, range 6.5-9.8 mm) of separation was maintained between the target lesion and optic radiations. MRgFUS offers a noninvasive option for seizure tract disruption. DTI identifies fornix-fimbria and optic radiations to localize optimal ablation targets and critical surrounding structures, minimizing risk of postoperative visual field deficits. This theoretical modeling study provides the necessary groundwork for future clinical trials to apply this novel neurosurgical technique to patients with refractory MTLE and surgical contraindications, multiple prior surgeries, or other factors favoring noninvasive treatment.


Layers of regulation of cell-cycle gene expression in the budding yeast Saccharomyces cerevisiae.
In the budding yeast Saccharomyces cerevisiae, transcription factors (TFs) regulate the periodic expression of many genes during the cell cycle, including gene products required for progression through cell-cycle events. Experimental evidence coupled with quantitative models suggests that a network of interconnected TFs is capable of regulating periodic genes over the cell cycle. Importantly, these dynamical models were built on transcriptomics data and assumed that TF protein levels and activity are directly correlated with mRNA abundance. To ask whether TF transcripts match protein expression levels as cells progress through the cell cycle, we applied a multiplexed targeted mass spectrometry approach (parallel reaction monitoring) to synchronized populations of cells. We found that protein expression of many TFs and cell-cycle regulators closely followed their respective mRNA transcript dynamics in cycling wild-type cells. Discordant mRNA/protein expression dynamics was also observed for a subset of cell-cycle TFs and for proteins targeted for degradation by E3 ubiquitin ligase complexes such as SCF (Skp1/Cul1/F-box) and APC/C (anaphase-promoting complex/cyclosome). We further profiled mutant cells lacking B-type cyclin/CDK activity ( clb1-6) where oscillations in ubiquitin ligase activity, cyclin/CDKs, and cell-cycle progression are halted. We found that a number of proteins were no longer periodically degraded in clb1-6 mutants compared with wild type, highlighting the importance of posttranscriptional regulation. Finally, the TF complexes responsible for activating G1/S transcription (SBF and MBF) were more constitutively expressed at the protein level than at periodic mRNA expression levels in both wild-type and mutant cells. This comprehensive investigation of cell-cycle regulators reveals that multiple layers of regulation (transcription, protein stability, and proteasome targeting) affect protein expression dynamics during the cell cycle.


First Report of Laminated Root Rot on Sabina przewalskii Caused by Phellinus weirii sensu stricto in China.
Members of the Phellinus weirii complex cause laminated root rot of living conifers. The cedar type (P. weirii (Murrill) Gilb. sensu stricto) of the complex is usually found on species of the Cupressaceae family, especially Thuja plicata in western North America, and the Douglas-fir type (P. sulphurascens Pilát) is found on species of the Pinaceae family (1,2,3). Outside North America, P. weirii occurs on species of Juniperus in the Ural Mountains, and P. sulphurascens occurs on other conifers in eastern Asia, including China (1). During a field inventory of wood-decay fungi in western China in 2003, laminated root rot of Sabina przewalskii (synonym Juniperus przewalskii) was found in natural forests of the Qilian Mountains in Qinghai Province (37°36'N and 102°15'E). Trees were approximately 80 to 150 years old and occurred in pure stands. Distinct disease patches that were as much as one hectare consisted of dead-standing and symptomatic trees, suggesting that the fungus spread by root contact. Symptomatic trees showed slow growth, thin crowns, and chlorotic foliage. After cutting several of the symptomatic trees, cambial necrosis and wood decay were found, and the trees apparently died when the cambial necrosis girdled the base of the trees. The wood of infected trees was reddish brown at the early stages of decay and later had numerous small cavities and separated into sheets at the junction of annual rings. Perennial, poroid, resupinate, dark brown basidiocarps formed on the root surface of dead trees. The basidiocarps had a monomitic hyphal system, hyphae without clamp connections, trama tissue with hyphoid setae, and thin-walled, hyaline, smooth, ellipsoid basidiospores. The fungus was identified as P. weirii and distinguished from P. sulphurascens by its perennial basidiocarps, smaller pores (5 to 8 versus 4 to 5 per mm), and narrower hyphoid setae (4 to 6 versus 5 to 10 μm in diameter). P. weirii also resembles P. ferrugineofuscus (P. Karst.) Bourdot in macro-morphology, but the latter species is a saprophyte and has allantoid to almost lunate basidiospores. The studied specimens of P. weirii and P. sulphurascens are preserved at the herbaria of the Botanical Museum of the University of Helsinki, the Institute of Applied Ecology, and the Chinese Academy of Sciences as DAOM 8734, Lowe 6960, TAA 52744, 55644, and 103812, and Dai 988, 2053, 2061, 2527, and 5067. To my knowledge, this is the first report of P. weirii sensu stricto from China and the first report of laminated root rot on S. przewalskii. References: (1) Y. C. Dai and G. F. Qin. Fungal Sci. 13:101, 1998. (2) E. M. Hansen et al. Species limits for Phellinus weirii. Pages 119-127 in: Root and butt rots of forest tress. Int. Conf. Root and Butt Rots, INRA, France, 1998. (3) M. J. Larsen et al. Mycologia 86:121, 1994.


Fat mass and obesity gene and cognitive decline: the Atherosclerosis Risk in Communities Study.
To determine whether 4 genetic variants in the fat mass and obesity associated gene (FTO) identified in genome-wide association studies of diabetes and obesity are associated with cognitive change in midlife in the Atherosclerosis Risk in Communities (ARIC) Study. ARIC is a prospective cohort study of the development of atherosclerosis in 15,792 individuals aged 45 to 64 years at baseline from 1986 to 1989. FTO is highly expressed in human fetal and adult brain, and a single nucleotide polymorphism in FTO has previously been associated with reduced brain volume in cognitively normal subjects. Since a relationship between brain atrophy and diminished cognitive function has been demonstrated in ARIC participants, general linear models were used to evaluate the association between 6-year change in scores on 3 neuropsychological tests and FTO genotype. In a sample of 8,364 white and 2,083 African American men and women with no clinical history of stroke, significantly greater mean change in performance on the Delayed Word Recall Test was associated with 2 of 4 FTO single nucleotide polymorphisms examined (rs9939609, rs805136, rs17817449, and rs1421085) in whites but not in African Americans (p ≤ 0.002). The association of the FTO polymorphisms with cognitive change was independent of potential confounding clinical and demographic variables including age, gender, education, diabetes, hypertension, and body mass index. Further studies will be needed to clarify the biological mechanisms and genetic pathways through which variants in FTO can increase susceptibility to decline in verbal memory detectable in middle-aged, community-dwelling adults.


Interaction driver-bicyclist on rural roads: Effects of cross-sections and road geometric elements.
The interaction of motorists and bicyclists, particularly during passing maneuvers, is cited as one of the primary causes of bicyclist fatalities. This paper reports the results of a driving simulator study, which sought to analyze the effects that three cross-section configurations of a two-lane rural road and four geometric elements of the road have on driver behavior, during the interaction with a cyclist. A two-lane rural road, about 11km long, was designed and implemented in an advanced-interactive driving simulator. Three different cross-sections (all with same width, but with and without a bicycle lane and for different widths of bicycle lane) were tested. Forty participants carried out three driving sessions (one for each road alignment with different cross-section) and were exposed to the condition of bicycle traffic along four geometric elements of the alignment (2 tangents with different lengths, right curve and left curve). The driving simulator experiments were designed in such a way that, along the sections where the driver-cyclist interactions occurred, the oncoming traffic was absent. Overall, 468 speed profiles and 468 lateral position profiles were plotted to obtain the descriptive variables of the driver behavior during the interaction with the cyclist. The influences of cross-sections, geometric elements and bicycle traffic conditions on driver behavior were evaluated by a multivariate variance analysis. The presence of the cyclist determined different levels of influence on driver's trajectory for the three cross-sections. A wider bicycle lane ensured a higher later clearance distance between driver and cyclist, allowing safer overtaking maneuver. The interferences of the cyclist on driver's behavior depended on the geometric elements. On tangents, the lowest lateral clearances were recorded and no speed reduction was observed, compared to the cyclist absence condition. On the left curve, the higher lateral clearance was recorded, due to the concordant tendencies of the driver to move away from the cyclist and to cut the curve. This determined an excessive and risky displacement of the vehicle to the opposing lane, whose criticality was also emphasized by the high speed adopted by the driver. On the right curve, the lateral clearance was higher than that recorded on the tangents, probably due to the necessity of the driver to perform the demanding maneuver of entering in the right curve, which also determined a speed reduction compared to the cyclist absence condition. The obtained results provide suggestions for the most efficient cross-section reorganization of existing two-lane rural roads in order to improve the road safety.


Reproducing kernel potential energy surfaces in biomolecular simulations: Nitric oxide binding to myoglobin.
Multidimensional potential energy surfaces based on reproducing kernel-interpolation are employed to explore the energetics and dynamics of free and bound nitric oxide in myoglobin (Mb). Combining a force field description for the majority of degrees of freedom and the higher-accuracy representation for the NO ligand and the Fe out-of-plane motion allows for a simulation approach akin to a mixed quantum mechanics/molecular mechanics treatment. However, the kernel-representation can be evaluated at conventional force-field speed. With the explicit inclusion of the Fe-out-of-plane (Fe-oop) coordinate, the dynamics and structural equilibrium after photodissociation of the ligand are correctly described compared to experiment. Experimentally, the Fe-oop coordinate plays an important role for the ligand dynamics. This is also found here where the isomerization dynamics between the Fe-ON and Fe-NO state is significantly affected whether or not this co-ordinate is explicitly included. Although the Fe-ON conformation is metastable when considering only the bound (2)A state, it may disappear once the (4)A state is included. This explains the absence of the Fe-ON state in previous experimental investigations of MbNO.


TREM2 activation on microglia promotes myelin debris clearance and remyelination in a model of multiple sclerosis.
Multiple sclerosis (MS) is an inflammatory, demyelinating, and neurodegenerative disease of the central nervous system (CNS) triggered by autoimmune mechanisms. Microglia are critical for the clearance of myelin debris in areas of demyelination, a key step to allow remyelination. TREM2 is expressed by microglia and promotes microglial survival, proliferation, and phagocytic activity. Herein we demonstrate that TREM2 was highly expressed on myelin-laden phagocytes in active demyelinating lesions in the CNS of subjects with MS. In gene expression studies, macrophages from subjects with TREM2 genetic deficiency displayed a defect in phagocytic pathways. Treatment with a new TREM2 agonistic antibody promoted the clearance of myelin debris in the cuprizone model of CNS demyelination. Effects included enhancement of myelin uptake and degradation, resulting in accelerated myelin debris removal by microglia. Most importantly, antibody-dependent TREM2 activation on microglia increased density of oligodendrocyte precursors in areas of demyelination, as well as the formation of mature oligodendrocytes thus enhancing remyelination and axonal integrity. These results are relevant as they propose TREM2 on microglia as a potential new target to promote remyelination.


Counter-reporting sustainability from the bottom up: the case of the construction company WeBuild and dam-related conflicts.
Controversies around large-scale development projects offer many cases and insights which may be analyzed through the lenses of corporate social (ir)responsibility (CSIR) and business ethics studies. In this paper, we confront the CSR narratives and strategies of WeBuild (formerly known as Salini Impregilo), an Italian transnational construction company. Starting from the Global Atlas of Environmental Justice (EJAtlas), we collect evidence from NGOs, environmental justice organizations, journalists, scholars, and community leaders on socio-environmental injustices and controversies surrounding 38 large hydropower schemes built by the corporation throughout the last century. As a counter-reporting exercise, we code (un)sustainability discourses from a plurality of sources, looking at their discrepancy under the critical lenses of post-normal science and political ecology, with environmental justice as a normative framework. Our results show how the mismatch of narratives can be interpreted by considering the voluntary, self-reporting, non-binding nature of CSR accounting performed by a corporation wishing to grow in a global competitive market. Contributing to critical perspectives on political CS(I)R, we question the reliability of current CSR mechanisms and instruments, calling for the inclusion of complexity dimensions in and a re-politicization of CS(I)R accounting and ethics. We argue that the fields of post-normal science and political ecology can contribute to these goals. The online version contains supplementary material available at 10.1007/s10551-021-04946-6.


Impact of treatment and re-treatment with artemether-lumefantrine and artesunate-amodiaquine on selection of Plasmodium falciparum multidrug resistance gene-1 polymorphisms in the Democratic Republic of Congo and Uganda.
The emergence of resistance against artemisinin combination treatment is a major concern for malaria control. ACTs are recommended as the rescue treatment, however, there is limited evidence as to whether treatment and re-treatment with ACTs select for drug-resistant P. falciparum parasites. Thus, the purpose of the present study is to investigate the impact of (re-)treatment using artesunate-amodiaquine (ASAQ) and artemether-lumefantrine (AL) on the selection of P. falciparum multidrug resistance-1 (Pfmdr1) alleles in clinical settings. P. falciparum positive samples were collected from children aged 12-59 months in a clinical trial in DR Congo and Uganda. Pfmdr1 single nucleotide polymorphisms (SNPs) analysis at codons N86Y, Y184F, and D1246Y were performed at baseline and post-treatment with either AL or ASAQ as a rescue treatment using nested PCR followed by restriction fragment length polymorphism (RFLP) assays. The pre-treatment prevalence of Pfmdr1 N86 and D1246Y varied significantly between the sites, (p>0.001) and (p = 0.013), respectively. There was borderline significant directional selection for Pfmdr1 184F in recurrent malaria infections after treatment with AL in Uganda site (p = 0.05). Pfmdr1 NFD haplotype did not significantly change in post-treatment infections after re-treatment with either AL or ASAQ. Comparison between pre-treatment and post-treatment recurrences did not indicate directional selection of Pfmdr1 N86, D1246 alleles in the pre-RCT, RCT and post-RCT phases in both AL and ASAQ treatment arms. Pfmdr1 86Y was significantly associated with reduced risk of AL treatment failure (RR = 0.34, 95% CI:0.11-1.05, p = 0.04) while no evidence for D1246 allele (RR = 1.02; 95% CI: 0.42-2.47, p = 1.0). Survival estimates showed that the Pfmdr1 alleles had comparable mean-time to PCR-corrected recrudescence and new infections in both AL and ASAQ treatment arms. We found limited impact of (re-)treatment with AL or ASAQ on selection for Pfmdr1 variants and haplotypes associated with resistance to partner drugs. These findings further supplement the evidence use of same or alternative ACTs as a rescue therapy for recurrent P.falciparum infections. Continued monitoring of genetic signatures of resistance is warranted to timely inform malaria (re-)treatment policies and guidelines.


Accuracy of unloading with the anti-gravity treadmill.
Body weight (BW)-supported treadmill training has become increasingly popular in professional sports and rehabilitation. To date, little is known about the accuracy of the lower-body positive pressure treadmill. This study evaluated the accuracy of the BW support reported on the AlterG "Anti-Gravity" Treadmill across the spectrum of unloading, from full BW (100%) to 20% BW. Thirty-one adults (15 men and 16 women) with a mean age of 29.3 years (SD = 10.9), and a mean weight of 66.55 kg (SD = 12.68) were recruited. Participants were weighed outside the machine and then inside at 100-20% BW in 10% increments. Predicted BW, as presented by the AlterG equipment, was compared with measured BW. Significant differences between predicted and measured BW were found at all but 90% through 70% of BW. Differences were small (<5%), except at the extreme ends of the unloading spectrum. At 100% BW, the measured weight was lower than predicted (mean = 93.15%, SD = 1.21, p < 0.001 vs. predicted). At 30 and 20% BW, the measured weight was higher than predicted at 35.75% (SD = 2.89, p < 0.001), and 27.67% (SD = 3.76, p < 0.001), respectively. These findings suggest that there are significant differences between reported and measured BW support on the AlterG Anti-Gravity Treadmill®, with the largest differences (>5%) found at 100% BW and the greatest BW support (30 and 20% BW). These differences may be associated with changes in metabolic demand and maximum speed during walking or running and should be taken into consideration when using these devices for training and research purposes.


Improving the adoption of thyroid cancer clinical practice guidelines.
To present an overview of the barriers to the implementation of clinical practice guidelines (CPGs) in thyroid cancer management and to introduce a computer-based clinical support system. PubMed. A review of studies on adherence to CPGs was conducted. Awareness and adoption of CPGs is low in thyroid cancer management. Barriers to implementation include unfamiliarity with the CPGs and financial concerns. Effective interventions to improve adherence are possible, especially when they are readily accessible at the point of care delivery. Computerized clinical support systems show particular promise. The authors introduce the clinical decision making modules (CDMMs) of the Thyroid Cancer Care Collaborative, a thyroid cancer-specific electronic health record. These computer-based modules can assist clinicians with implementation of these recommendations in clinical practice. Computer-based support systems can help clinicians understand and adopt the thyroid cancer CPGs. By integrating patient characteristics and guidelines at the point of care delivery, the CDMMs can improve adherence to the guidelines and help clinicians provide high-quality, evidence-based, and individualized patient care in the management of differentiated thyroid cancer. Laryngoscope, 126:2640-2645, 2016.


Crystallographic comparison of manganese- and iron-dependent homoprotocatechuate 2,3-dioxygenases.
The X-ray crystal structures of homoprotocatechuate 2,3-dioxygenases isolated from Arthrobacter globiformis and Brevibacterium fuscum have been determined to high resolution. These enzymes exhibit 83% sequence identity, yet their activities depend on different transition metals, Mn2+ and Fe2+, respectively. The structures allow the origins of metal ion selectivity and aspects of the molecular mechanism to be examined in detail. The homotetrameric enzymes belong to the type I family of extradiol dioxygenases (vicinal oxygen chelate superfamily); each monomer has four betaalphabetabetabeta modules forming two structurally homologous N-terminal and C-terminal barrel-shaped domains. The active-site metal is located in the C-terminal barrel and is ligated by two equatorial ligands, H214NE1 and E267OE1; one axial ligand, H155NE1; and two to three water molecules. The first and second coordination spheres of these enzymes are virtually identical (root mean square difference over all atoms, 0.19 A), suggesting that the metal selectivity must be due to changes at a significant distance from the metal and/or changes that occur during folding. The substrate (2,3-dihydroxyphenylacetate [HPCA]) chelates the metal asymmetrically at sites trans to the two imidazole ligands and interacts with a unique, mobile C-terminal loop. The loop closes over the bound substrate, presumably to seal the active site as the oxygen activation process commences. An "open" coordination site trans to E267 is the likely binding site for O2. The geometry of the enzyme-substrate complexes suggests that if a transiently formed metal-superoxide complex attacks the substrate without dissociation from the metal, it must do so at the C-3 position. Second-sphere active-site residues that are positioned to interact with the HPCA and/or bound O2 during catalysis are identified and discussed in the context of current mechanistic hypotheses.


Development of a Footwear Sizing System in the National Football League.
Footwear performance and injury mitigation may be compromised if the footwear is not properly sized for an athlete. Additionally, poor fit may result in discomfort and foot injury such as fifth metatarsal stress fracture, foot deformities, turf toe, and blisters. Current footwear fitting methods consist of foot length and width measurements, which may not properly describe the shape of the individual foot, correlated with shoe size descriptors that are not standardized. Footwear manufacturers employ a range of sizing rubrics, which introduces shoe size and shape variability between and even within footwear companies. This article describes the synthesis of literature to inform the development and deployment of an objective footwear fitting system in the National Football League (NFL). The process may inform athletic footwear fitting at other levels of play and in other sports. Literature related to footwear fitting, sizing, and foot scanning from 1980 through 2017 was compiled using electronic databases. Reference lists of articles were examined for additional relevant studies. Sixty-five sources are included in this descriptive review. Descriptive review. Level 5. Current methods of footwear fitting and variability in the size and shape of athletic footwear complicate proper fitting of footwear to athletes. An objective measurement and recommendation system that can match the 3-dimensional shape of an athlete's foot to the internal shape of available shoe models can provide important guidance for footwear selection. One such system has been deployed in the NFL. An objective footwear fitting system based on 3-dimensional shape matching of feet and shoes can facilitate the selection of footwear that properly fits an athlete's foot.


[Burnout in psychiatric physicians. Results of an empirical study].
The study aimed at assessing burnout in psychiatrists and psychiatric residents, all of them working in the Canton of Zurich, Switzerland, and at identifying some of its correlates. Potentially burnout- related demographic, work, leisure activities, and personality (Munich Personality Test) variables were studied in three subgroups of 307 probands--psychiatric residents, psychiatrists working as staff members or in leading positions in psychiatric institutions, and psychiatrists working in private practice--the degree of burnout having been assessed with the Tedium Measure (TM). The mean TM score of all probands of 2.9 (SD = 0.7) was on a low level, high TM scores (> 3.5) were indicated by 57 (18%) participants. Significantly higher TM scores were indicated by psychiatric residents, who also scored higher with regard to neuroticism and lower with regard to frustration tolerance on MPT. Neuroticism alone explained a substantial proportion of the total TM variance in individual groups. Work-related variables turned out to be of a small importance only, whereas no influence could be demonstrated for different leisure activities.


Radiation doses to aquatic organisms from natural radionuclides.
A framework for protection of the environment is likely to require a methodology for assessing dose rates arising from naturally occurring radionuclides. This paper addresses this issue for European aquatic environments through a process of (a) data collation, mainly with respect to levels of radioactivity in water sediments and aquatic flora and fauna, (b) the use of suitable distribution coefficients, concentration factors and global data where data gaps are present and (c) the utilisation of a reference organism approach whereby a finite number of suitable geometries are selected to allow dose per unit concentration factors to be derived and subsequent absorbed dose calculations (weighted or unweighted) to be made. The majority of the calculated absorbed dose, for both marine and freshwater organisms, arises from internally incorporated alpha emitters, with 210Po and 226Ra being the major contributors. Calculated doses are somewhat higher for freshwater compared to marine organisms, and the range of doses is also much greater. This reflects both the much greater variability of radionuclide concentrations in freshwater as compared to seawater, and also variability or uncertainty in concentration factor values. This work has revealed a number of substantial gaps in published empirical data especially for European aquatic environments.


Comparison of real-time PCR and microscopy for malaria parasite detection in Malawian pregnant women.
New diagnostic tools for malaria are required owing to the changing epidemiology of malaria, particularly among pregnant women in sub-Saharan Africa. Real-time PCR assays targeting Plasmodium falciparum lactate dehydrogenase (pfldh) gene may facilitate the identification of a high proportion of pregnant women with a P. falciparum parasitaemia below the threshold of microscopy. These molecular methods will enable further studies on the effects of these submicroscopic infections on maternal health and birth outcomes. The pfldh real-time PCR assay and conventional microscopy were compared for the detection of P. falciparum from dried blood spots and blood smears collected from the peripheral blood of 475 Malawian women at delivery. A cycle threshold (Ct) of the real-time PCR was determined optimizing the sensitivity and specificity of the pfldh PCR assay compared to microscopy. A real-time PCR species-specific assay was applied to identify the contribution to malaria infections of three Plasmodium species (P. falciparum P. ovale and P. malariae) in 44 discordant smear and pfldh PCR assay results. Of the 475 women, P. falciparum was detected in 11 (2.3%) by microscopy and in 51 (10.7%) by real-time PCR; compared to microscopy, the sensitivity of real-time PCR was 90.9% and the specificity 91.2%. If a Ct value of 38 was used as a cut-off, specificity improved to 94.6% with no change in sensitivity. The real-time PCR species-specific assay detected P. falciparum alone in all but four samples: two samples were mixed infections with P. falciparum and P. malariae, one was a pure P. malariae infection and one was a pfldh PCR assay-positive/species-specific assay-negative sample. Of three P. malariae infections detected by microscopy, only one was confirmed by the species-specific assay. Although microscopy remains the most appropriate method for clinical malaria diagnosis in field settings, molecular diagnostics such as real-time PCR offer a more reliable means to detect malaria parasites, particularly at low levels. Determination of the possible contribution of these submicroscopic infections to poor birth outcomes and maternal health is critical. For future studies to investigate these effects, this pfldh real-time PCR assay offers a reliable detection method.


Cytosine methylation and hydroxymethylation mark DNA for elimination in Oxytricha trifallax.
Cytosine methylation of DNA is conserved across eukaryotes and plays important functional roles regulating gene expression during differentiation and development in animals, plants and fungi. Hydroxymethylation was recently identified as another epigenetic modification marking genes important for pluripotency in embryonic stem cells. Here we describe de novo cytosine methylation and hydroxymethylation in the ciliate Oxytricha trifallax. These DNA modifications occur only during nuclear development and programmed genome rearrangement. We detect methylcytosine and hydroxymethylcytosine directly by high-resolution nano-flow UPLC mass spectrometry, and indirectly by immunofluorescence, methyl-DNA immunoprecipitation and bisulfite sequencing. We describe these modifications in three classes of eliminated DNA: germline-limited transposons and satellite repeats, aberrant DNA rearrangements, and DNA from the parental genome undergoing degradation. Methylation and hydroxymethylation generally occur on the same sequence elements, modifying cytosines in all sequence contexts. We show that the DNA methyltransferase-inhibiting drugs azacitidine and decitabine induce demethylation of both somatic and germline sequence elements during genome rearrangements, with consequent elevated levels of germline-limited repetitive elements in exconjugant cells. These data strongly support a functional link between cytosine DNA methylation/hydroxymethylation and DNA elimination. We identify a motif strongly enriched in methylated/hydroxymethylated regions, and we propose that this motif recruits DNA modification machinery to specific chromosomes in the parental macronucleus. No recognizable methyltransferase enzyme has yet been described in O. trifallax, raising the possibility that it might employ a novel cytosine methylation machinery to mark DNA sequences for elimination during genome rearrangements.


Structural transitions in the RNA 7SK 5' hairpin and their effect on HEXIM binding.
7SK RNA, as part of the 7SK ribonucleoprotein complex, is crucial to the regulation of transcription by RNA-polymerase II, via its interaction with the positive transcription elongation factor P-TEFb. The interaction is induced by binding of the protein HEXIM to the 5' hairpin (HP1) of 7SK RNA. Four distinct structural models have been obtained experimentally for HP1. Here, we employ computational methods to investigate the relative stability of these structures, transitions between them, and the effects of mutations on the observed structural ensembles. We further analyse the results with respect to mutational binding assays, and hypothesize a mechanism for HEXIM binding. Our results indicate that the dominant structure in the wild type exhibits a triplet involving the unpaired nucleotide U40 and the base pair A43-U66 in the GAUC/GAUC repeat. This conformation leads to an open major groove with enough potential binding sites for peptide recognition. Sequence mutations of the RNA change the relative stability of the different structural ensembles. Binding affinity is consequently lost if these changes alter the dominant structure.


Central cancellation of self-produced tickle sensation.
A self-produced tactile stimulus is perceived as less ticklish than the same stimulus generated externally. We used fMRI to examine neural responses when subjects experienced a tactile stimulus that was either self-produced or externally produced. More activity was found in somatosensory cortex when the stimulus was externally produced. In the cerebellum, less activity was associated with a movement that generated a tactile stimulus than with a movement that did not. This difference suggests that the cerebellum is involved in predicting the specific sensory consequences of movements, providing the signal that is used to cancel the sensory response to self-generated stimulation.


The role of inflammation in the pathology of preeclampsia.
Preeclampsia (PE) affects 5-7% of all pregnancies in the United States and is the leading cause of maternal and prenatal morbidity. PE is associated with hypertension after week 20 of gestation, decreased renal function and small-for-gestational-age babies. Women with PE exhibit chronic inflammation and production of autoantibodies. It is hypothesized that during PE, placental ischaemia occurs as a result of shallow trophoblast invasion which is associated with an immune imbalance where pro-inflammatory CD4(+) T-cells are increased and T regulatory cells (Tregs) are decreased. This imbalance leads to chronic inflammation characterized by oxidative stress, pro-inflammatory cytokines and autoantibodies. Studies conducted in our laboratory have demonstrated the importance of this immune imbalance in causing hypertension in response to placental ischaemia in pregnant rats. These studies confirm that increased CD4(+) T-cells and decreased Tregs during pregnancy leads to elevated inflammatory cytokines, endothelin (ET-1), reactive oxygen species (ROS) and agonistic autoantibodies to the angiotensin II (Ang II), type 1 receptor (AT1-AA). All of these factors taken together play an important role in increasing the blood pressure during pregnancy. Specifically, this review focuses on the decrease in Tregs, and their associated regulatory cytokine interleukin (IL)-10, which is seen in response to placental ischaemia during pregnancy. This study will also examine the effect of regulatory immune cell repopulation on the pathophysiology of PE. These studies show that restoring the balance of the immune system through increasing Tregs, either by adoptive transfer or by infusing IL-10, reduces the blood pressure and pathophysiology associated with placental ischaemia in pregnant rats.


Cardioprotection with dexrazoxane for doxorubicin-containing therapy in advanced breast cancer.
To determine the cardioprotective effect of dexrazoxane (DZR) used in a doxorubicin-based combination therapy in advanced breast cancer. Between November 1988 and January 1991, 534 patients with advanced breast cancer were randomized to two multicenter, double-blind studies (088001 and 088006). Patients received fluorouracil, doxorubicin, and cyclophosphamide (FAC) with either DZR (DZR-to-doxorubicin ratio, 10:1) or placebo (PLA) every 3 weeks and were monitored with serial multiplegated acquisition (MUGA) scans. The hazards ratio (HR) of PLA to DZR for a cardiac event, which was predefined ejection fraction changes or congestive heart failure (CHF), was 2.63 (95% confidence interval [CI], 1.61 to 4.27; P < .001) for 088001 and 2.00 (95% CI, 1.01 to 3.96; P = .038) for 088006. The objective response rates for 088001 were 46.8% for DZR and 60.5% for PLA, a difference of 14% (95% CI, -25% to -2%; P = .019), and for 088006 were 53.7% for DZR and 49.3% for PLA, a difference of 4% (95% CI, -13% to 22%; P = .63). Time to progression and survival were not significantly different between treatment arms in either study. Toxicities on the DZR arms included lower granulocyte and platelet counts at nadir (P = .009 and P = .004, respectively) and more pain on injection (P = .001), with no difference in the rates of fever, infection, or hemorrhage. DZR had a significant cardioprotective effect as measured by noninvasive testing and clinical CHF. One of the two studies (088001) showed a lower response rate with DZR, but time to progression and survival were not significantly different. DZR is the first agent shown to reduce cardiotoxicity from doxorubicin.


Hospice Capacity to Provide General Inpatient Care: Emergency Department Utilization and Live Discharge Among Cancer Patients.
General inpatient (GIP) hospice care is used only minimally for hospice patients, and more than a quarter of Medicare hospice facilities do not provide GIP care. To determine the impact of hospices' capacity to provide on emergency department use during hospice enrollment and live discharge from hospice, we used Surveillance, Epidemiology, and End Results-Medicare linked data and CMS Provider of Services data from 2007 to 2013 from ten states and two metropolitan regions. Grouping hospices into three GIP care provision categories: 1) no-GIP; 2) GIP-contract; and 3) GIP-IHF where hospices directly provide GIP care in their own inpatient hospice facility (IHF), we built a multilevel logistic model that accounted for unobserved hospice characteristics. Nearly 9% of the study sample received GIP care, of which 82% received such care in the last week of discharge. GIP-IHF hospices had lower live discharge rates than no-GIP hospices (AOR: .61; 95% CI: .47-.79; P < .001) and GIP-contract hospices (AOR: .84; 95% CI: .70-1.00; P < .05). Similarly, GIP-contract hospices were also associated with a decreased risk of live discharge, compared to no-GIP hospices (AOR: .76; CI: .62-.92; P < .05). There was no difference in emergency department use between no-GIP hospices and hospices with such capacity. Our results suggest that hospices capable of providing GIP care have lower live discharge rates than their counterparts. However, the fact that GIP care tends to be provided too close to death limits its effectiveness in preventing avoidable emergency department use.


Role of Negative Orbit Vector in Orbital Blow-Out Fractures.
Negative orbit vector is defined as the most anterior globe portion protrudes past the malar eminence. The aim of the study was to evaluate the relationship between negative orbit vector and blow-out fracture location analyzing the distance between the anterior corneal surface and orbital bone with facial soft tissue in medial and orbital floor blow out fractures using orbital computed tomography (CT). Seventy-seven patients diagnosed with blow-out fractures involving the medial or orbital floor were included. Distances from the anterior cornea to lower lid fat, inferior orbital wall, inferior orbital rim, and anterior cheek mass were measured using orbital CT scans. The proportion of negative orbit vector and measured distanced were compared between medial wall fracture and orbital floor fracture. Medical records including age, sex, concomitant ophthalmic diagnosis, and nature of injury were retrospectively reviewed. Forty-three eyes from 43 patients diagnosed with medial wall fracture and 34 eyes from 34 patients diagnosed with orbital floor fracture were included. There was no significant difference in the distance from the anterior cornea to lower lid fat (P = 0.574), inferior orbital wall (P = 0.494), or orbital rim (P = 0.685). The distance from anterior cornea to anterior cheek mass was significantly different in medial wall fracture (-0.19 ± 3.49 mm) compared with orbital floor fracture (-1.69 ± 3.70 mm), P = 0.05. Negative orbit vector was significantly higher in orbital floor fracture patients (24 among 34 patients, 70.6%) compared with those with medial wall fractures (19 among 43 patients, 44.2%) (P = 0.04). Patients presenting with a negative orbit vector relationship when the most anterior portion of globe protruded past the anterior cheek mass and malar eminence were more likely to develop orbital floor fracture than medial wall fracture.


Engineering pyruvate decarboxylase-mediated ethanol production in the thermophilic host Geobacillus thermoglucosidasius.
This study reports the expression, purification, and kinetic characterization of a pyruvate decarboxylase (PDC) from Gluconobacter oxydans. Kinetic analyses showed the enzyme to have high affinity for pyruvate (120 μM at pH 5), high catalytic efficiency (4.75 × 10(5) M(-1) s(-1) at pH 5), a pHopt of approximately 4.5 and an in vitro temperature optimum at approximately 55 °C. Due to in vitro thermostablity (approximately 40 % enzyme activity retained after 30 min at 65 °C), this PDC was considered to be a suitable candidate for heterologous expression in the thermophile Geobacillus thermoglucosidasius for ethanol production. Initial studies using a variety of methods failed to detect activity at any growth temperature (45-55 °C). However, the application of codon harmonization (i.e., mimicry of the heterogeneous host's transcription and translational rhythm) yielded a protein that was fully functional in the thermophilic strain at 45 °C (as determined by enzyme activity, Western blot, mRNA detection, and ethanol productivity). Here, we describe the first successful expression of PDC in a true thermophile. Yields as high as 0.35 ± 0.04 g/g ethanol per gram of glucose consumed were detected, highly competitive to those reported in ethanologenic thermophilic mutants. Although activities could not be detected at temperatures approaching the growth optimum for the strain, this study highlights the possibility that previously unsuccessful expression of pdcs in Geobacillus spp. may be the result of ineffective transcription/translation coupling.


Circulating insulin-like growth factor (IGF)-I and IGF binding protein (IGFBP)-3 levels and postmenopausal breast cancer risk in the prostate, lung, colorectal, and ovarian cancer screening trial (PLCO) cohort.
Early prospective studies suggested circulating insulin-like growth factor (IGF)-I was positively associated with risk of premenopausal, but not postmenopausal, breast cancer; however, a recent, large analysis reported a statistically significant positive association with postmenopausal disease. Therefore, we conducted a large study nested within the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial cohort to assess the association between circulating IGF-I and IGF binding protein (IGFBP)-3 levels and subsequent postmenopausal breast cancer risk. We included 389 breast cancer cases and 470 controls, aged 55-74, not using exogenous hormones at blood draw, and matched by age at and date of serum collection. Mean follow-up was 8.5 years; mean time between serum collection and diagnosis was 4.0 years. We used Cox proportional hazards regression models to obtain hazard ratios (HRs) and 95% confidence intervals (95% CIs). Multivariate HRs for IGF-I, IGFBP-3, and the molar ratio IGF-I/IGFBP-3, comparing the highest quintile to the lowest, were 1.28 (95% CI, 0.67-2.44), 1.12 (95% 0.55-2.27), and 1.25 (95% 0.72-2.15), respectively. Multivariate HRs per one quintile increase were 1.07 (95% 0.92-1.25) for IGF-I, 1.01 (95% 0.86-1.18) for IGFBP-3, and 1.10 (95% 0.98-1.24) for the molar ratio. These models included accepted breast cancer risk factors and height, along with baseline BMI and serum estradiol, both of which increased the risk associated with IGF-I and the molar ratio. IGF-I and the IGF-I/IGFBP-3 molar ratio were positively, although not statistically significantly, associated with postmenopausal breast cancer risk. Further research should emphasize larger studies, including pooled analyses, analyses by cancer subtype, improved exposure assessment, and possible mechanisms.


Krüppel-like factor 9 promotes hepatic cytochrome P450 2D6 expression during pregnancy in CYP2D6-humanized mice.
Cytochrome P450 2D6 (CYP2D6), a major drug-metabolizing enzyme, is responsible for metabolism of approximately 25% of marketed drugs. Clinical evidence indicates that metabolism of CYP2D6 substrates is increased during pregnancy, but the underlying mechanisms remain unclear. To identify transcription factors potentially responsible for CYP2D6 induction during pregnancy, a panel of genes differentially expressed in the livers of pregnant versus nonpregnant CYP2D6-humanized (tg-CYP2D6) mice was compiled via microarray experiments followed by real-time quantitative reverse-transcription polymerase chain reaction(qRT-PCR) verification. As a result, seven transcription factors-activating transcription factor 5 (ATF5), early growth response 1 (EGR1), forkhead box protein A3 (FOXA3), JUNB, Krüppel-like factor 9 (KLF9), KLF10, and REV-ERBα-were found to be up-regulated in liver during pregnancy. Results from transient transfection and promoter reporter gene assays indicate that KLF9 itself is a weak transactivator of CYP2D6 promoter but significantly enhances CYP2D6 promoter transactivation by hepatocyte nuclear factor 4 (HNF4α), a known transcriptional activator of CYP2D6 expression. The results from deletion and mutation analysis of CYP2D6 promoter activity identified a KLF9 putative binding motif at -22/-14 region to be critical in the potentiation of HNF4α-induced transactivation of CYP2D6. Electrophoretic mobility shift assays revealed a direct binding of KLF9 to the putative KLF binding motif. Results from chromatin immunoprecipitation assay showed increased recruitment of KLF9 to CYP2D6 promoter in the livers of tg-CYP2D6 mice during pregnancy. Taken together, our data suggest that increased KLF9 expression is in part responsible for CYP2D6 induction during pregnancy via the potentiation of HNF4α transactivation of CYP2D6.


The selective reversible FAAH inhibitor, SSR411298, restores the development of maladaptive behaviors to acute and chronic stress in rodents.
Enhancing endogenous cannabinoid (eCB) signaling has been considered as a potential strategy for the treatment of stress-related conditions. Fatty acid amide hydrolase (FAAH) represents the primary degradation enzyme of the eCB anandamide (AEA), oleoylethanolamide (OEA) and palmitoylethanolamide (PEA). This study describes a potent reversible FAAH inhibitor, SSR411298. The drug acts as a selective inhibitor of FAAH, which potently increases hippocampal levels of AEA, OEA and PEA in mice. Despite elevating eCB levels, SSR411298 did not mimic the interoceptive state or produce the behavioral side-effects (memory deficit and motor impairment) evoked by direct-acting cannabinoids. When SSR411298 was tested in models of anxiety, it only exerted clear anxiolytic-like effects under highly aversive conditions following exposure to a traumatic event, such as in the mouse defense test battery and social defeat procedure. Results from experiments in models of depression showed that SSR411298 produced robust antidepressant-like activity in the rat forced-swimming test and in the mouse chronic mild stress model, restoring notably the development of inadequate coping responses to chronic stress. This preclinical profile positions SSR411298 as a promising drug candidate to treat diseases such as post-traumatic stress disorder, which involves the development of maladaptive behaviors.


Long-term Clinical Outcomes of Splanchnic Vein Thrombosis: Results of an International Registry.
Little information is available on the long-term clinical outcome of patients with splanchnic vein thrombosis (SVT). To assess the incidence rates of bleeding, thrombotic events, and mortality in a large international cohort of patients with SVT. A prospective cohort study was conducted beginning May 2, 2008, and completed January 30, 2014, at hospital-based centers specialized in the management of thromboembolic disorders; a 2-year follow-up period was completed January 30, 2014, and data analysis was conducted from July 1, 2014, to February 28, 2015. Participants included 604 consecutive patients with objectively diagnosed SVT; there were no exclusion critieria. Information was gathered on baseline characteristics, risk factors, and antithrombotic treatment. Clinical outcomes during the follow-up period were documented and reviewed by a central adjudication committee. Major bleeding, defined according to the International Society on Thrombosis and Hemostasis; bleeding requiring hospitalization; thrombotic events, including venous and arterial thrombosis; and all-cause mortality. Of the 604 patients (median age, 54 years; 62.6% males), 21 (3.5%) did not complete follow-up. The most common risk factors for SVT were liver cirrhosis (167 of 600 patients [27.8%]) and solid cancer (136 of 600 [22.7%]); the most common sites of thrombosis were the portal vein (465 of 604 [77.0%]) and the mesenteric veins (266 of 604 [44.0%]). Anticoagulation was administered to 465 patients in the entire cohort (77.0%) with a mean duration of 13.9 months; 175 of the anticoagulant group (37.6%) received parenteral treatment only, and 290 patients (62.4%) were receiving vitamin K antagonists. The incidence rates (reported with 95% CIs) were 3.8 per 100 patient-years (2.7-5.2) for major bleeding, 7.3 per 100 patient-years (5.8-9.3) for thrombotic events, and 10.3 per 100 patient-years (8.5-12.5) for all-cause mortality. During anticoagulant treatment, these rates were 3.9 per 100 patient-years (2.6-6.0) for major bleeding and 5.6 per 100 patient-years (3.9-8.0) for thrombotic events. After treatment discontinuation, rates were 1.0 per 100 patient-years (0.3-4.2) and 10.5 per 100 patient-years (6.8-16.3), respectively. The highest rates of major bleeding and thrombotic events during the whole study period were observed in patients with cirrhosis (10.0 per 100 patient-years [6.6-15.1] and 11.3 per 100 patient-years [7.7-16.8], respectively); the lowest rates were in patients with SVT secondary to transient risk factors (0.5 per 100 patient-years [0.1-3.7] and 3.2 per 100 patient-years [1.4-7.0], respectively). Most patients with SVT have a substantial long-term risk of thrombotic events. In patients with cirrhosis, this risk must be balanced against a similarly high risk of major bleeding. Anticoagulant treatment appears to be safe and effective in most patients with SVT.


Immediate loading of immediate mandibular implants with a full-arch fixed prosthesis: a preliminary study.
To determine the survival of immediate dental implants with immediate loading in the partially edentulous mandible, by use of a full-arch screw-retained provisional restoration. Patients who were partially edentulous in the mandible with indications for extraction of the remaining teeth and with a minimum follow-up of 12 months after implant placement were included in the study. They were treated in chronologic order by the insertion of 6 Defcon dental implants (Impladent, Sentmenat, Spain) subjected to immediate loading (4 interforaminal and 2 posterior placements). Implants with a minimum primary stability of 60 implant stability quotient were loaded. All resin screw-retained prostheses were inserted and loaded with fully functional occlusion within 24 hours of implant placement. Eleven patients were treated with immediate implants, although 2 patients were excluded from the study for having an implant stability quotient value below 60 in at least one of the implants after surgery and did not undergo restoration with immediate loading. Fifty-four implants were placed in 9 partially edentulous patients with immediate loading with a full-arch screw-fixed prosthesis. The patients wore this provisional prosthesis during the healing period (2 months) without complication and with a high level of comfort. The survival rate of the implants was 100% at 12 months of follow-up. Immediate mandibular loading with immediate full-arch implant-supported and screw-retained restorations is a viable treatment alternative, yielding a 100% success rate in this small series of patients.


First Report of Fusarium proliferatum Causing Rot on Garlic and Onion in Argentina.
In October 2001 and January 2002, in onion fields (Allium cepa L. cv Valencianita) in the Provinces of San Juan (SJ) and Mendoza (MZ), Argentina, plants were observed with chlorosis, dry leaf tips, and bulbs showing discoloration and rot. During the summer of 2002, a tan rot with white mycelium in rot cavities was also observed in stored garlic bulbs (Allium sativum) in MZ. Four monosporic cultures obtained with a micro punch adapted microscope (three from onion CSJ1, CMZ1, CMZ2 and one from garlic AMZ1) were characterized by morphology on PDA and carnation leaf agar (2). The isolates were deposited in the fungal collection of the Plant Mycosis Laboratory of the Integrated Unit Balcarce. The isolates produced abundant aerial white mycelium and a violet to vinaceous pigmentation. Club-shaped microconidia were abundant, in chains on both mono- and polyphialides. Slender, thin-walled and relatively straight macroconidia were produced only under black light and were mostly 3-septate. Chlamydospores were absent. The isolates were identified as Fusarium proliferatum. Crosses to confirm mating populations and to identify mating types were made in triplicate on carrot agar (3) with standard tester strains D-04853 (MATD-2) and D-04854 (MATD-1) as female parents and the field isolates as male parents. Crosses were examined weekly and were scored positive only if perithecia were seen oozing a cirrhus of ascospores. The identities of these isolates were confirmed as showing positive crosses with standard tester strains of Gibberella intermedia. Pathogenicity tests were conducted with healthy 45-day-old onion seedlings (cv. Valcatorce INTA). The roots of the onion seedlings were soaked in a conidial suspension (5 × 106 conidia/ml) of each isolate (CSJ1, CMZ1, CMZ2) for 2 h; the control was soaked in sterile water (SW). Seedlings were transplanted to pots in a sterile mixture of soil and sand (v/v). Five plants were used for each of 3 replications. The plants were placed in a greenhouse and irrigated with SW. After 3 weeks, symptoms were evaluated. All inoculated plants exhibited symptoms similar to those observed in the bulbs from which the pathogen was isolated and a brown rot appeared on the basal plate of the onion, later becoming dark brown. In garlic, the inoculation consisted of a wound 4.5 mm deep and 2 mm wide in superficially sterilized garlic cloves (cv. Nieve INTA). Inside the cavity, a drop (50 μl) was placed from a suspension of 5 × 106 conidia/ml (AMZ1), then covered with a drop of paraffin. Controls used SW. The garlic cloves were incubated in hermetically sealed trays at 22 ± 3°C in darkness for 3 weeks (1). Garlic showed tan rot and white mycelium in the wound. F. proliferatum was reisolated from inoculated onion seedlings and garlic cloves. The controls did not exhibit symptoms nor were any fungi recovered when tissue was excised from the inoculation points and plated on agar. F. proliferatum was previously reported in Argentina on asparagus (4) with symptoms similar to those of onion and garlic. To our knowledge, this is the first report of F. proliferatum attacking onion and garlic in Argentina. This pathogen has the potential risk of mycotoxin accumulation in contaminated bulbs. References: (1) F. M. Dugan et al. J. Phytopathol. 155:437, 2007. (2) W. Gerlach and H. Nirenberg. The genus Fusarium - A Pictorial Atlas. Mitt. Biol. Bundesanst. Land. Forstwirsch. Berl.-Dahlem, 1982. (3) C. J. R. Klittich and J. F. Leslie. Genetics 118:417, 1988. (4) G. Lori et al. Plant Dis. 82:1405, 1998.


Delayed response and biosonar perception explain movement coordination in trawling bats.
Animal coordinated movement interactions are commonly explained by assuming unspecified social forces of attraction, repulsion and alignment with parameters drawn from observed movement data. Here we propose and test a biologically realistic and quantifiable biosonar movement interaction mechanism for echolocating bats based on spatial perceptual bias, i.e. actual sound field, a reaction delay, and observed motor constraints in speed and acceleration. We found that foraging pairs of bats flying over a water surface swapped leader-follower roles and performed chases or coordinated manoeuvres by copying the heading a nearby individual has had up to 500 ms earlier. Our proposed mechanism based on the interplay between sensory-motor constraints and delayed alignment was able to recreate the observed spatial actor-reactor patterns. Remarkably, when we varied model parameters (response delay, hearing threshold and echolocation directionality) beyond those observed in nature, the spatio-temporal interaction patterns created by the model only recreated the observed interactions, i.e. chases, and best matched the observed spatial patterns for just those response delays, hearing thresholds and echolocation directionalities found to be used by bats. This supports the validity of our sensory ecology approach of movement coordination, where interacting bats localise each other by active echolocation rather than eavesdropping.


From multispectral imaging of autofluorescence to chemical and sensory images of lipid oxidation in cod caviar paste.
The potential of multispectral imaging of autofluorescence to map sensory flavour properties and fluorophore concentrations in cod caviar paste has been investigated. Cod caviar paste was used as a case product and it was stored over time, under different headspace gas composition and light exposure conditions, to obtain a relevant span in lipid oxidation and sensory properties. Samples were divided in two sets, calibration and test sets, with 16 and 7 samples, respectively. A third set of samples was prepared with induced gradients in lipid oxidation and sensory properties by light exposure of certain parts of the sample surface. Front-face fluorescence emission images were obtained for excitation wavelength 382 nm at 11 different channels ranging from 400 to 700 nm. The analysis of the obtained sets of images was divided in two parts: First, in an effort to compress and extract relevant information, multivariate curve resolution was applied on the calibration set and three spectral components and their relative concentrations in each sample were obtained. The obtained profiles were employed to estimate the concentrations of each component in the images of the heterogeneous samples, giving chemical images of the distribution of fluorescent oxidation products, protoporphyrin IX and photoprotoporphyrin. Second, regression models for sensory attributes related to lipid oxidation were constructed based on the spectra of homogeneous samples from the calibration set. These models were successfully validated with the test set. The models were then applied for pixel-wise estimation of sensory flavours in the heterogeneous images, giving rise to sensory images. As far as we know this is the first time that sensory images of odour and flavour are obtained based on multispectral imaging.


What is an 'elasmobranch'? The impact of palaeontology in understanding elasmobranch phylogeny and evolution.
The Subclass Elasmobranchii is widely considered nowadays to be the sister group of the Subclass Holocephali, although chimaeroid fishes were originally classified as elasmobranchs along with modern sharks and rays. While this modern systematic treatment provides an accurate reflection of the phylogenetic relationships among extant taxa, the classification of many extinct non-holocephalan shark-like chondrichthyans as elasmobranchs is challenged. A revised, apomorphy-based definition of elasmobranchs is presented in which they are considered the equivalent of neoselachians, i.e. a monophyletic group of modern sharks and rays which not only excludes all stem and crown holocephalans, but also many Palaeozoic shark-like chondrichthyans and even close extinct relatives of neoselachians such as hybodonts. The fossil record of elasmobranchs (i.e. neoselachians) is reviewed, focusing not only on their earliest records but also on their subsequent distribution patterns through time. The value and limitations of the fossil record in answering questions about elasmobranch phylogeny are discussed. Extinction is seen as a major factor in shaping early elasmobranch history, especially during the Triassic. Extinctions may also have helped shape modern lamniform diversity, despite uncertainties surrounding the phylogenetic affinities of supposedly extinct clades such as cretoxyrhinids, anacoracids and odontids. Apart from these examples, and the supposed Cretaceous extinction of 'sclerorhynchids', elasmobranch evolution since the Jurassic has mostly involved increased diversification (especially during the Cretaceous). The biogeographical distribution of early elasmobranchs may be obscured by sampling bias, but the earliest records of numerous groups are located within the Tethyan realm. The break-up of Gondwana, and particularly the opening of the South Atlantic Ocean (together with the development of epicontinental seaways across Brazil and Africa during the Cretaceous), provided repeated opportunities for dispersal from both eastern (European) and western (Caribbean) Tethys into newly formed ocean basins.


Prediction of postoperative pain after percutaneous nephrolithotomy: can preoperative experimental pain assessment identify patients at risk?
Postoperative pain remains a significant problem and the individual variance in postoperative pain is not fully understood. In recent years, there has been focus on identifying risk factors predicting patients with high postoperative pain intensity or consumption of analgesics, which may facilitate an improvement in rehabilitation. This study evaluates the relationship between preoperative experimental pain assessment and postoperative pain and opioid consumption. Forty-four patients with uni- or bilateral kidney stone disease scheduled for percutaneous nephrolithotomy were included. The preoperative pain thresholds were measured using electrical (single and 5 repeated) and pressure pain stimulation over the flank bilaterally (stone-side = operation side and control-side = non-operation side). Postoperative pain scores were recorded on a numerical rating scale and analgesic consumption was registered. The responses to repeated electrical stimuli (temporal summation) were preoperatively increased on the stone-side compared to the control-side (P = 0.016). Preoperative electrical pain thresholds from the control-side correlated inversely with postoperative opioid consumption (single stimuli: ρ = -0.43, P < 0.01; repeated stimuli: ρ = -0.45, P < 0.005). This correlation was more pronounced for the 22 patients with unilateral renal calculi (single stimuli: ρ = -0.54, P < 0.02; repeated stimuli: ρ = -0.58, P < 0.01). There were no other correlations between the preoperative sensory tests and postoperative pain or opioid consumption. This study showed a correlation between the preoperative electrical pain thresholds on the control-side and postoperative opioid consumption after percutaneous nephrolithotomy. Preoperative measurement of the electrical pain thresholds may, therefore, be useful as a screening tool to identify patients at high risk of postoperative pain.


Not all intravenous immunoglobulin preparations are equally well tolerated.
Intravenous immunoglobulin (IVIG) is used for many indications beyond the original substitution in primary antibody deficiency. Whereas many reports mention adverse reactions, no comparative data exist concerning the incidence of side-effects among the different brands of IVIG. We describe here our experience with the use of different IVIG formulations and their tolerability in a select cohort of 40 patients. The IVIG dose ranged from 0.4 to 3 g/kg/day and was given for 1-2742 days. Fourteen patients (35%) experienced mild to severe adverse reactions during or within 48 h of administration of standard IVIG preparation, which did not recur after switching to an alternative preparation. Adverse reactions included headache, fever, chills, nausea, emesis, hypotension and muscle cramps. One patient experienced a severe adverse reaction; he had a 3-day headache following IVIG infusion. Among the 16 patients who received alternative preparation initially, none experienced adverse reactions. In conclusion, this study shows that IVIG preparations are not all equally well tolerated in patients. The data suggest that, perhaps to a comparable extent to the preparation itself, the infusion rate has a major effect. If a reduction in the infusion rate does not minimize side-effects, one should consider switching the IVIG formulation.


Nanoleakage: leakage within the hybrid layer.
Most microleakage studies involve quantitating the magnitude of movement of a tracer molecule through a gap between restorative materials and the wall of cavity preparations. The present microscopic study examined the migration of silver nitrate into the interface between dentin and five different dentin bonding agents used to restore class 5 cavities, in the absence of gap formation. Several different leakage patterns were seen, but they all indicated leakage within the hybrid layer when viewed by SEM. The ranking of microleakage from most to least was: All-Bond 2 > Suberbond C&B > Scotchbond Multi-Purpose > Clearfil Liner Bond System > Kuraray Experimental System, KB-200. To distinguish this special type of microleakage within the basal, porous region of the hybrid layer in the absence of gap formation, we propose the term nanoleakage.


Why are metabolic scaling exponents so controversial? Quantifying variance and testing hypotheses.
The metabolic theory of ecology links physiology with ecology, and successfully predicts many allometric scaling relationships. In recent years, proponents and critics of metabolic theory have debated vigorously about the scaling of metabolic rate. We show that the controversy arose, in part, because researchers examined the mean exponent separately from the variance. We estimate both quantities simultaneously using linear mixed-effects models and data from 1242 animal species. Metabolic rate scaling converges on the predicted value of 3/4 but is highly heterogeneous: 50% of orders lie outside the range 0.68-0.82. These findings are robust to several forms of statistical uncertainty. We then test competing hypotheses about the variation. Metabolic theory is currently unable to explain differences in scaling among orders, but the patterns are not consistent with competing explanations either. We conclude that current theories are inadequate to explain the full range of metabolic scaling patterns observed in nature.


Symptomatic lower extremity vasculitis in giant cell arteritis: a case series.
To describe the clinical features and outcomes of 19 patients with giant cell arteritis (GCA) and symptomatic lower extremity (LE) vasculitis. We reviewed medical records of all patients diagnosed with GCA and symptomatic LE involvement between January 1, 1983, and June 30, 2007, for clinical features, laboratory and radiographic findings, and outcomes. From 6212 people evaluated for GCA at our institution between 1983 and 2007, we identified 19 cases of GCA with LE vasculitis, all women. Mean age at GCA diagnosis was 70 years (+/- standard deviation 7.99). Sixteen patients (84.2%) had LE symptoms preceding the diagnosis of GCA, median interval 3 months (range 1-48). Three patients (15.8%) had GCA prior to developing LE claudication, median interval 16 months (range 9-34). Cranial symptoms were absent in 42.1%. No patient had permanent visual loss. Erythrocyte sedimentation rate (ESR) was elevated in 16 patients (84.2%), with median ESR 42.5 mm/h (range 8-103). Imaging studies revealed stenotic, occlusive, or aneurysmal disease that was frequently bilateral and consistent with vasculitis. The superficial femoral arteries were most commonly affected. Five patients (26.3%) had upper extremity involvement. Hypertension was the most common cardiovascular risk factor. All patients received glucocorticoid therapy, with clinical improvement in 15 patients (79%). Median length of followup was 41 months (range 11-180 mo). Five patients (26.3%) underwent LE revascularization surgery. Two patients required LE amputation and 1 patient underwent toe amputation. Five patients received additional immunosuppressive therapy. Symptomatic LE vasculitis from GCA is rare. Patients typically present with rapidly progressive LE claudication and elevated inflammatory markers, while cranial symptoms may be absent. GCA with LE involvement is associated with significant morbidity; prompt diagnosis and treatment is essential.


Development and validation of an integrated cell culture-qRTPCR assay for simultaneous quantification of coxsackieviruses, echoviruses, and polioviruses in disinfection studies.
This study demonstrated the applicability of integrated cell culture-quantitative RTPCR (ICC-qRTPCR) for the simultaneous quantification of coxsackievirus, echovirus, and poliovirus in disinfection studies. Buffalo green monkey cells were inoculated with a 10-fold dilution series of mixed enteroviruses and incubated prior to qRTPCR quantification. Optimal assay conditions included three post infection washes and a 24-hour post infection incubation period based on successful differentiation between infectious and noninfectious viruses and significant and consistent viral replication rates. Ultraviolet disinfection studies were performed to validate the ICC-qRTPCR assay. Using the optimized assay, three-log microbial inactivation was achieved at UV doses of 30-44, 28-42, and 28-29 mJ/cm(2) for coxsackievirus B6, echovirus 12, and poliovirus 1, respectively. These results compare favorably to side-by-side assessments using conventional cultural techniques and values previously reported in the literature. This indicates that ICC-qRTPCR is a practical alternative for the simultaneous quantification of enteroviruses in disinfection studies.


Case studies for practical food effect assessments across BCS/BDDCS class compounds using in silico, in vitro, and preclinical in vivo data.
Practical food effect predictions and assessments were described using in silico, in vitro, and/or in vivo preclinical data to anticipate food effects and Biopharmaceutics Classification System (BCS)/Biopharmaceutics Drug Disposition Classification System (BDDCS) class across drug development stages depending on available data: (1) limited in silico and in vitro data in early discovery; (2) preclinical in vivo pharmacokinetic, absorption, and metabolism data at candidate selection; and (3) physiologically based absorption modeling using biorelevant solubility and precipitation data to quantitatively predict human food effects, oral absorption, and pharmacokinetic profiles for early clinical studies. Early food effect predictions used calculated or measured physicochemical properties to establish a preliminary BCS/BDDCS class. A rat-based preclinical BCS/BDDCS classification used rat in vivo fraction absorbed and metabolism data. Biorelevant solubility and precipitation kinetic data were generated via animal pharmacokinetic studies using advanced compartmental absorption and transit (ACAT) models or in vitro methods. Predicted human plasma concentration-time profiles and the magnitude of the food effects were compared with observed clinical data for assessment of simulation accuracy. Simulations and analyses successfully identified potential food effects across BCS/BDDCS classes 1-4 compounds with an average fold error less than 1.6 in most cases. ACAT physiological absorption models accurately predicted positive food effects in human for poorly soluble bases after oral dosage forms. Integration of solubility, precipitation time, and metabolism data allowed confident identification of a compound's BCS/BDDCS class, its likely food effects, along with prediction of human exposure profiles under fast and fed conditions.


Methylation in the p21WAF1/cip1 promoter of Apc+/-, p21+/- mice and lack of response to sulindac.
P21WAF1/cip1 plays a critical role in regulating intestinal cell proliferation, maturation and tumorigenesis. Our previous work demonstrated that inactivation of a single p21 allele in Apc1638+/- mice was sufficient to accelerate Apc-initiated tumor formation, and that inactivation of only one p21 allele was also sufficient to abrogate duodenal tumor inhibition by sulindac, a nonsteroidal anti-inflammatory drug. To dissect the role of p21 in sulindac inhibition of intestinal tumor development in Apc1638+/- mice, we quantified p21 expression from Apc+/-, p21+/+, +/- or -/- mice fed sulindac. In Apc+/-, p21 wild-type mice fed the sulindac supplemental diet, both p21 mRNA and protein were significantly increased in the flat mucosa and tumors of the duodenum. However, p21 was not induced by sulindac in the duodenal flat mucosa and tumors of Apc+/- mice in which one or both p21 alleles had been inactivated. Further investigation revealed that the cytosine residues in a CpG cluster in the promoter region of the mouse p21 gene displayed hypermethylation in the Apc+/-, p21+/- mice. This suggested that although the p21+/- mice retained a wild-type allele, this allele was functionally modulated by hypermethylation, and that the inability of sulindac to inhibit tumor formation in Apc+/-, p21+/- mice is likely due to the inability to induce expression of the wild-type, but differentially methylated, p21 allele.


CMV promotes recipient T-cell immunity following reduced-intensity T-cell-depleted HSCT, significantly modulating chimerism status.
Cytomegalovirus (CMV) remains a significant cause of morbidity after allogeneic hematopoietic stem cell transplantation (HSCT). Clinical risk varies according to a number of factors, including recipient/donor CMV serostatus. Current dogma suggests risk is greatest in seropositive recipient (R+)/seronegative donor (D-) transplants and is exacerbated by T-cell depletion. We hypothesized that in the setting of reduced-intensity T-cell-depleted conditioning, recipient-derived CMV-specific T cells escaping deletion may contribute significantly to CMV-specific immunity and might therefore also influence chimerism status. We evaluated 105 recipients of alemtuzumab-based reduced-intensity HSCT and collated details on CMV infection episodes and T-cell chimerism. We used CMV-specific HLA multimers to enumerate CMV-specific T-cell numbers and select cells to assess chimerism status in a subset of R+/D- and R+/seropositive donor patients. We show that in R+/D- patients, CMV-specific T cells are exclusively of recipient origin, can protect against recurrent CMV infections, and significantly influence the chimerism status toward recipients. The major findings were replicated in a separate validation cohort. T-cell depletion in the R+/D- setting may actually, therefore, foster more rapid reconstitution of protective antiviral immunity by reducing graft-vs-host directed alloreactivity and the associated elimination of the recipient T-cell compartment. Finally, conversion to donor chimerism after donor lymphocytes is associated with clinically occult transition to donor-derived immunity.


Stretch induction of cyclooxygenase-2 expression in human urothelial cells is calcium- and protein kinase C zeta-dependent.
Prostanoid synthesis via cyclooxygenase (COX)-2 induction during urothelial stretch is central to nociception, inflammation, contractility, and proliferation caused by urinary tract obstruction. We used our primary human urothelial cell stretch model published previously to evaluate the signaling mechanisms responsible for stretch-induced COX-2 expression in urothelial cells. To determine intracytosolic calcium concentrations ([Ca(2+)](i)), primary human urothelial cells were grown on flexible membranes and loaded with Fura-2 acetoxymethyl ester (AM). We determined [Ca(2+)](i) using a fluorescent scope during stretch. Additional cells were treated with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM, stretched, and COX-2 mRNA and protein were evaluated by real-time polymerase chain reaction and immunoblotting. To evaluate protein kinase C (PKC) in this system, cells were stretched and fractionated into membrane, cytosol, and nucleus. Fractions were immunoblotted for PKCalpha, beta1, and zeta, the predominant isoforms in urothelial cells. We treated additional cells with increasing concentrations of either bisindolylmaleimide-I or a peptide PKC pseudosubstrate inhibitor, and COX-2 mRNA and protein were evaluated after stretching. Furthermore, we transfected urothelial cells with siRNA against each of the inducible PKC isoforms in these cells and evaluated the stretch-induced COX-2 response. Stretch of urothelial cells activated calcium flux and PKC translocation to membrane and nucleus. Pharmacological inhibition indicated that stretch-induced COX-2 expression is dependent on calcium and PKC, and biochemical knockdown experiments indicated that PKCzeta is the predominant isoform mediating stretch-induced COX-2 expression. Elucidating the signaling mechanism of stretch-induced COX-2 expression may identify therapeutic targets.


A Pivotal Genetic Program Controlled by Thyroid Hormone during the Maturation of GABAergic Neurons.
Mammalian brain development critically depends on proper thyroid hormone signaling, via the TRα1 nuclear receptor. The downstream mechanisms by which TRα1 impacts brain development are currently unknown. In order to investigate these mechanisms, we used mouse genetics to induce the expression of a dominant-negative mutation of TRα1 specifically in GABAergic neurons, the main inhibitory neurons in the brain. This triggered post-natal epileptic seizures and a profound impairment of GABAergic neuron maturation in several brain regions. Analysis of the transcriptome and TRα1 cistrome in the striatum allowed us to identify a small set of genes, the transcription of which is upregulated by TRα1 in GABAergic neurons and which probably plays an important role during post-natal maturation of the brain. Thus, our results point to GABAergic neurons as direct targets of thyroid hormone during brain development and suggest that many defects seen in hypothyroid brains may be secondary to GABAergic neuron malfunction.


The Effect of Smoking Status on Successful Arthrodesis After Lumbar Instrumentation Supplemented with rhBMP-2.
The primary objective of this study is to examine the effects smoking status on rhBMP-2 supplementation in spinal fusion constructs. Patient records were reviewed retrospectively for a consecutive set of patients who underwent first-time posterolateral, instrumented fusion of the lumbar spine for degenerative spinal disease. All operations included arthrodesis supplementation with rhBMP-2. All patients were followed for at least 2 years. The primary endpoint of this study was reoperation for pseudarthrosis, instrumentation failure, or adjacent segment disease. After a rigorous sensitivity analysis, the measure of association was calculated with a multivariable logistic regression controlling for smoking, age, and number of spinal levels fused. Of the 110 patients in the study population, 82 (74.6%) were nonsmokers and 28 (25.5%) were smokers. Among perioperative predictors, smokers were younger in age (53.9 ± 9.6 vs. 61.1 ± 13.1 years; P = 0.008) and had shorter length of inpatient hospital stay (4.1 ± 1.8 vs. 5.3 ± 3.0; P = 0.039). After a mean follow-up of 59 months, the 32% incidence of reoperation for pseudarthrosis, instrumentation failure, or adjacent segment among smokers was statistically significantly higher than the 13.4% incidence in nonsmokers (P = 0.027). Following multivariable logistic regression, the odds of reoperation among smokers was 4.75-fold higher than for nonsmokers (P = 0.009; 95% confidence interval, 1.48-15.24). While rhBMP-2 supplements arthrodesis of instrumented lumbar fusion constructs, smoking status ascertains the strongest predictor of reoperation for pseudarthrosis, instrumentation failure, and adjacent segment.