Clinical value of real time 3D sonohysterography and 2D sonohysterography in comparison to hysteroscopy with subsequent histopathological examination in perimenopausal women with abnormal uterine bleeding.
In many publications the transvaginal ultrasound is regarded as the first step to diagnose the cause of uterine bleeding in perimenopausal women. In order to improve the sensitivity and specificity of the conventional ultrasound physiological saline solution was administered to the uterine cavity and after expansion of its walls the interior uterine cavity was examined. And this procedure is called 2D sonohysterography (SIS 2D). By the ultrasound scanners which enable to get 3D real time image a spatial evaluation of the uterine cavity is possible. Clinical value of the real time 3D sonohysterography and 2D sonohysterography compared to hysteroscopy with histopathological examination in perimenopausal women. The study concerned a group of 97 perimenopausal women with abnormal uterine bleeding. In all of them after a standard transvaginal ultrasonography a catheter was inserted into the uterine cavity. After expansion of the uterine walls by administering about 10 ml of 0,9% saline solution the uterine cavity was examined by conventional sonohysterography. Then a 3D imaging mode was activated and the uterine interior was examined by real time 3D ultrasonography. The ultrasound results were verified by hysteroscopy, the endometrial lesions were removed and underwent a histopathological examination. In two cases the SIS examination was impossible because of uterine cervix atresion. In the rest of examined group the SIS 2D sensitivity and specificity came up to 72 and 96% respectively. In the group of SIS 3D the sensitivity and specificity reached 83 and 99% respectively. Adding SIS 3D, a minimally invasive method, to conventional sonohysterography improves the precision of diagnosis of endometrial pathology, allows to get three-dimensional image of the uterine cavity and enables examination of endometrial lesions. The diagnostic precision of this procedure is similar to the results achieved by hysteroscopy.


High-resolution DNA melt-curve analysis for cost-effective mass screening of pairwise species interactions.
Ecological studies of pairwise interactions are constrained by the methods available for rapid species identification of the interacting organisms. The resolution of data required to characterize species interaction networks at multiple spatio-temporal scales can be intensive, and therefore laborious and costly to collect. We explore the utility of high-resolution DNA melt-curve analysis (HRM) as a rapid species identification method. An approach was developed to identify organisms at the pairwise interaction level, with particular application to cryptic species interactions that are traditionally difficult to study. Here, we selected a challenging application; to identify the presence/absence of pathogenic fungi (Sporothrix inflata, Ophiostoma nigrocarpum and Ophiostoma galeiforme) transported by bark beetle vectors (Hylastes ater and Hylurgus ligniperda). The technique was able to distinguish between different species of DNA within a single, pooled sample. In test applications, HRM was effective in the mass screening and identification of pathogenic fungal species carried by many individual bark beetle vectors (n = 455 beetles screened) across large geographic scales. For two of the fungal species, there was no difference in the frequency of association with either of their vectors, but for the third fungal species there was a shift in vector-pathogen associations across locations. This technique allows rapid, mass screening and characterization of species interactions at a fraction of the time and cost of traditional methods. It is anticipated that this method can be readily applied to explore other cryptic species interactions, or other studies requiring rapid generation of large data sets and/or high-throughput efficiency.


Crowding is size and eccentricity dependent.
Crowding is a form of lateral interaction in which flanking items interfere with the detection or discrimination of a target stimulus. It is believed that crowding is a property of peripheral vision only and that no crowding occurs at fixation. If these two claims are true, then there must be a change in the nature of crowding interactions across the visual field. In three different tasks, we determined target size and flanker separation at threshold for eccentricities of 0 to 16° in the lower visual field for 7 relative separations (1.25 to 8 times target size). In all three tasks, the magnitude of crowding increases with eccentricity; there was no crowding at fixation and extreme crowding at 16°. Using a novel double-scaling procedure, we show that the non-foveal data in all three tasks can be characterized as shifted versions of the same psychometric function such that different sections of the function characterize data at each eccentricity. This pattern of results can be understood in terms of size-dependent responses to the target and distance-dependent interference from the flankers. The data suggest that the distance-dependent interference increases with eccentricity.


Anti-ADAMTS5 monoclonal antibodies: implications for aggrecanase inhibition in osteoarthritis.
The extracellular matrix of articular cartilage is structurally specialized for efficient absorption of mechanical impact. In particular, giant aggregates of the large chondroitin sulfate proteoglycan, aggrecan, with the glycosaminoglycan, hyaluronan, allow cartilage to resist compressive load. Proteolysis of aggrecan by members of the proteinase family ADAMTS (A disintegrin-like and metalloproteinase domain with thrombospondin type 1 motif), was identified as an early step in the inexorable destruction of cartilage in osteoarthritis (OA). Of the investigated proteinases, ADAMTS5 has emerged as a principal mediator of aggrecan loss in OA, convincingly so in mouse models, and with high probability in humans. ADAMTS5 has a bipartite organization, comprising a proteinase domain and an ancillary domain containing exosites for interaction with aggrecan and other substrates. In a recent issue of this journal, Santamaria et al. characterized anti-ADAMTS5 monoclonal antibodies isolated from a phage display library. By blocking the catalytic site of the ADAMTS5 immunogen with a synthetic inhibitor, the authors of the paper biased selection of antibodies to the ancillary domain. This work, together with other antibodies targeting ADAMTS5, offers diverse, high-affinity and, as far as can be determined, selective aggrecanase inhibitors. Mapping of their epitopes provided novel insights into ADAMTS5 interactions with aggrecan. These monoclonal antibodies deserve continued investigation for potential arthritis therapy, although their successful use will require a comprehensive understanding of the physiological roles of ADAMTS5, and its regulation, intrinsic properties and intermolecular interactions.


Level of residual enzyme activity modulates the phenotype in phosphoglycerate kinase deficiency.
To study the variable clinical picture and exercise tolerance of patients with phosphoglycerate kinase (PGK) 1 deficiency and how it relates to residual PGK enzyme activity. In this case series study, we evaluated 7 boys and men from 5 families with PGK1 deficiency. Five had pure muscle symptoms, while 2 also had mild intellectual disability with or without anemia. Muscle glycolytic and oxidative capacities were evaluated by an ischemic forearm exercise test and by cycle ergometry. Enzyme levels of PGK were 4% to 9% of normal in red cells and 5% to10% in muscle in pure myopathy patients and 2.6% in both muscle and red cells in the 2 patients with multisystem involvement. Patients with pure myopathy had greater increases in lactate with ischemic exercise (2-3 mmol/L) vs the 2 multisystem-affected patients (<1 mmol/L). Myopathy patients had higher oxidative capacity in cycle exercise vs multisystem affected patients (≈30 vs ≈15 mL/kg per minute). One multisystem-affected patient developed frank myoglobinuria after the short exercise test. This case series study of PGK1 deficiency suggests that the level of impaired glycolysis in PGK deficiency is a major determinant of phenotype. Lower glycolytic capacity in PGK1 deficiency seems to result in multisystem involvement and increased susceptibility to exertional rhabdomyolysis.


Deciphering the role of IS6110 in a highly transmissible Mycobacterium tuberculosis Beijing strain, GC1237.
The capacity of infection and the ability of Mycobacterium tuberculosis strains belonging to the Beijing family to spread rapidly probably result from genetic advantages and unidentified mechanisms of virulence not yet thoroughly investigated. Among the mechanisms proposed to be responsible for the varying virulence phenotypes of M. tuberculosis strains we find IS6110 insertions, genetic reorganizations and deletions, which have strong influences on fitness. Beijing family is one of the lineages with the highest number of copies of IS6110. By studying genetic markers characteristic for this lineage, here we have characterized the clinical isolate M. tuberculosis GC1237 strain responsible for important epidemic outbreaks in the Gran Canary Island. We have identified and analyzed each point of insertion of IS6110 using a bacterial artificial chromosome (BAC) library of this strain, in addition to the use of other approximations. Nineteen copies of IS6110 have been localized in GC1237 genome of which, four copies of IS6110 can act as a promoter and we have focused in the characterization of one copy located 31 bp upstream of the essential gene Rv2179c and compared to the reference strain H37Rv.


Co-activation of synovial fibroblasts by laminin-111 and transforming growth factor-beta induces expression of matrix metalloproteinases 3 and 10 independently of nuclear factor-kappaB.
We showed previously that the attachment of synovial fibroblasts to laminin (LM)-111 in the presence of transforming growth factor-beta induces significant expression of the matrix metalloproteinase (MMP)-3. Here we go on to investigate the regulation of additional MMPs and their specific tissue inhibitors of matrix proteases (TIMPs). Changes in steady-state mRNA levels encoding TIMPs and MMPs were investigated by quantitative reverse transcription-polymerase chain reaction. Production of MMPs was monitored by a multiplexed immunoarray. Signal transduction pathways were studied by immunoblotting. Attachment of synovial fibroblasts to LM-111 in the presence of transforming growth factor-beta induced significant increases in MMP-3 mRNA (12.35-fold, p < 0.001) and protein (mean 62 ng/ml, sixfold, p < 0.008) and in expression of MMP-10 mRNA (11.68-fold, p < 0.05) and protein (54 ng/ml, 20-fold, p > or = 0.02). All other TIMPs and MMPs investigated failed to show this LM-111-facilitated transforming growth factor-beta response. No phosphorylation of nuclear factor-kappaB was observed. We conclude that co-stimulation of synovial fibroblasts by LM-111 together with transforming growth factor-beta suffices to induce significant expression of MMP-3 and MMP-10 by synovial fibroblasts and that this induction is independent of nuclear factor-kappaB phosphorylation.


Single crystalline and core-shell indium-catalyzed germanium nanowires-a systematic thermal CVD growth study.
Germanium nanowires were synthesized using thermal chemical vapor deposition (CVD) and indium as a catalyst. The process parameter space for successful growth was studied. By optimizing the growth temperature and gas pressure, high aspect ratio germanium nanowires have been obtained. Scanning electron microscopy investigations indicate that the final diameter of the nanowires is strongly influenced by the growth temperature and the germane partial pressure. High resolution transmission electron microscopy reveals that nanowires grow either as high quality single crystalline, or with a high quality single-crystalline core and a concentric amorphous shell. The occurrence of these two morphologies is found to only depend on the wire diameter. Chemical analysis of the nanowire tip indicates the presence of indium, validating its role as a catalyst. Raman spectroscopy measurements reveal a higher incidence of core-shell structures for nanowires synthesized at 30 Torr and indicate the presence of tensile strain. These results are important towards obtaining high quality germanium nanowires without the use of gold as a catalyst, which is known to degrade the wires' electrical and optical properties.


Rationale and design of the HIP fracture Accelerated surgical TreaTment And Care tracK (HIP ATTACK) Trial: a protocol for an international randomised controlled trial evaluating early surgery for hip fracture patients.
Annually, millions of adults suffer hip fractures. The mortality rate post a hip fracture is 7%-10% at 30 days and 10%-20% at 90 days. Observational data suggest that early surgery can improve these outcomes in hip fracture patients. We designed a clinical trial-HIP fracture Accelerated surgical TreaTment And Care tracK (HIP ATTACK) to determine the effect of accelerated surgery compared with standard care on the 90-day risk of all-cause mortality and major perioperative complications. HIP ATTACK is a multicentre, international, parallel group randomised controlled trial (RCT) that will include patients ≥45 years of age and diagnosed with a hip fracture from a low-energy mechanism requiring surgery. Patients are randomised to accelerated medical assessment and surgical repair (goal within 6 h) or standard care. The co-primary outcomes are (1) all-cause mortality and (2) a composite of major perioperative complications (ie, mortality and non-fatal myocardial infarction, pulmonary embolism, pneumonia, sepsis, stroke, and life-threatening and major bleeding) at 90 days after randomisation. All patients will be followed up for a period of 1 year. We will enrol 3000 patients. All centres had ethics approval before randomising patients. Written informed consent is required for all patients before randomisation. HIP ATTACK is the first large international trial designed to examine whether accelerated surgery can improve outcomes in patients with a hip fracture. The dissemination plan includes publishing the results in a policy-influencing journal, conference presentations, engagement of influential medical organisations, and providing public awareness through multimedia resources. NCT02027896; Pre-results.


The functions of the multi-tasking Pfh1Pif1 helicase.
Approximately, 1% of the genes in eukaryotic genomes encode for helicases, which make the number of helicases expressed in the cell considerably high. Helicases are motor proteins that participate in many central aspects of the nuclear and mitochondrial genomes, and based on their helicase motif conservation, they are divided into different helicase families. The Pif1 family of helicases is an evolutionarily conserved helicase family that is associated with familial breast cancer in humans. The Schizosaccharomyces pombe Pfh1 helicase belongs to the Pif1 helicase family and is a multi-tasking helicase that is important for replication fork progression through natural fork barriers, for G-quadruplex unwinding, and for Okazaki fragment maturation, and these activities are potentially shared by the human Pif1 helicase. This review discusses the known functions of the Pfh1 helicase, the study of which has led to a better understanding of nucleic acid metabolism in eukaryotes.


Intent to Refer: Exploring Bias Toward Specific Medication-Assisted Treatments by Community Corrections Employees.
As opioid use increases in the United States, especially in the correctional population, the most effective treatment approaches need to be utilized. Research has shown that medication-assisted treatment (MAT) provides better outcomes than traditional treatment approaches alone, but is underutilized among correctional-supervised populations. This article looks at how previously identified barriers to implementing MAT can create potential biases regarding the intent to refer individuals to either buprenorphine or methadone among treatment and correctional staff within community corrections. The varying advantages of each medication are discussed to highlight the importance of individualized treatment planning. Data were collected from 959 treatment specialists and community-corrections officers between 2010 and 2013. The participants were employed by one of 20 community corrections agencies that were selected and randomized within a cluster design. The data were part of a larger study that tested how the experimental condition of organizational linkages impacted the use of MAT in the criminal justice system. In order to analyze the data, multinomial logistic regression was used. Results showed that some community corrections employees were likely to refer clients to use methadone, but not buprenorphine (or vice versa) which was influenced by work setting, level of education, training, and negative perceptions of MAT as a substitute addiction. However, these biases could be minimized by proper training. These findings suggest that proper training should be mandatory for these employees and include information about various MATs while also addressing the negative perception that MAT is a substitute addiction.


Modulation of enhancer activity by the hormone responsive regulatory element from mouse mammary tumor virus.
Addition of the transcriptional enhancers present in the U3 region of the Harvey murine sarcoma virus (HaMuSV) long terminal repeat (LTR) to recombinant chimeras in which the HaMuSV transforming gene (Ha-v-ras) is expressed from the mouse mammary tumor virus (MMTV) promoter increases the ability of the MMTV v-ras chimeras to transform mouse fibroblasts in culture 50- to 100-fold. Significant stimulation of transfection efficiency occurs only when glucocorticoids are present in the culture medium. Glucocorticoids also elevate the steady-state concentration of MMTV-initiated v-ras mRNA in cell lines isolated from these transfections, and MMTV-v-ras fusion transcripts are initiated at the normal MMTV cap site; potential cryptic initiation events associated with the enhancer could not be detected. The ability of the enhancer to increase the transcriptional activity of the MMTV promoter was also studied in acute transfection assays where expression of the chloramphenical acetyl transferase (CAT) gene is driven by the MMTV promoter. In this system the strong positive effect on MMTV transcription is again obtained only when the cells are hormone treated. These experiments indicate that the hormone-regulatory region is capable of modulating the function of an exogenously introduced enhancer element.


Postoperative complications and functional results after total glossectomy with microvascular reconstruction.
Microsurgical reconstruction after total glossectomy can greatly improve quality of life; however, postoperative functional results are often unstable, and the effectiveness of total glossectomy remains questionable. To determine the problems of reconstruction after total glossectomy with laryngeal preservation and to examine the functional results of swallowing and speech, 30 patients who had undergone total glossectomy and reconstruction with free flaps were reviewed for this study. The patients ranged in age from 20 to 73 years, and 23 of the 30 had undergone reconstruction with a rectus abdominis musculocutaneous flap. Wider and thicker flaps were designed and transferred and were sutured to suspend the larynx. To maintain physiologic swallowing function after surgery, the extent of laryngeal suspension and cricopharyngeal myotomy was limited. Of the 30 patients, 21 (70 percent) could be decannulated with laryngeal preservation; 20 of these 21 could tolerate a normal/soft/pureed diet, and 1 was limited to a fluid diet. Speech was intelligible in 16 of the 19 patients evaluated. In 9 of the 30 patients, laryngeal function could not be preserved. In four of these nine patients, additional resection combined with total glossectomy caused severe aspiration and recurrent pneumonia. Two patients with preoperative cerebral dysfunction were also poor candidates for laryngeal preservation. Additionally, the transferred flap's lack of bulk in the oral cavity and the advanced age (73 years) of one patient and the poor motivation of another may have contributed to postoperative aspiration. Aspiration occurred in one patient because of local recurrence of a tumor. The presence of preoperative cerebral dysfunction (p = 0.025), resection of the epiglottis (p = 0.005), and postoperative orocutaneous fistulas (p = 0.04) were significantly associated with the failure of laryngeal preservation. However, because of the difficulty of enrolling a sufficient number of patients in the study and the inherent limitations of retrospective studies, multivariate analysis in this study showed that no factors, such as patient age, flap volume, and the type of neck dissection, were significant predictors of laryngeal preservation. Although prospective studies are necessary, the function of individual patients must be assessed so that the study experiences discussed here can be applied to subsequent patients.


The influence of acute starvation on the cardiovascular responses to lower body subatmospheric pressure or to standing in man.
The cardiovascular responses to lower body subatmospheric pressure or to standing were measured in the same nine normal male subjects after a 12 h and after a 48 h fast. After the 48 h fast there was a significant reduction in diastolic blood pressure and forearm vascular resistance (relative to the values after a 12 h fast) when subjects were supine. During exposure to lower body subatmospheric pressure, subjects who had fasted for 48 h showed an inability to maintain systolic blood pressure, accompanied by an impairment of forearm vasoconstriction and an exaggerated tachycardia (relative to their responses after a 12 h fast). Similar disorders of cardiovascular homoeostasis were seen on standing. The results are consistent with an inhibition of sympathetic nervous activity after a 48 h fast, but other possibilities are discussed.


Diffusion tensor imaging in a human PET/MR hybrid system.
The aim of this study was to test and demonstrate the feasibility of diffusion tensor imaging (DTI) with a hybrid positron emission tomography (PET)/magnetic resonance imaging system for simultaneous PET and magnetic resonance (MR) data acquisition. All measurements were performed with a prototype hybrid PET/MR scanner dedicated for brain and head imaging. The PET scanner, which is inserted into a conventional 3.0-Tesla high field MR imager equipped with a transmit/receive birdcage head coil, consists of 192 block detectors with a matrix of 12 x 12 lutetium oxyorthosilicate scintillation crystals combined with MR-compatible 3 x 3 avalanche photodiode arrays. In 7 volunteers and 4 patients with brain tumors, DTI was performed during simultaneous PET data readout applying a diffusion weighted echo planar sequence (12 noncollinear directions, echo time (TE)/repetition time (TR) 98 ms/5300 ms, b-value 800 s/mm). Image quality and accuracy of DTI were assessed in comparison with DTI images acquired after removal of the PET insert. The diffusion images showed good image quality in all volunteers regardless of simultaneous PET data readout or after removal of the PET scanner; however, significantly (P < 0.01) stronger rim artifacts were found in fractional anisotropy images computed from DTI images recorded during simultaneous PET acquisition, demonstrating higher eddy-current effects. In region of interest analysis, no notable differences were found in the computation of the direction of the principal eigenvector (P > 0.05) and fractional anisotropy values (P > 0.05). In the assessment of pathologies, in all 4 patients PET and DTI provided important clinical information in addition to conventional magnetic resonance imaging. Diffusion tensor imaging may be combined with simultaneous PET data acquisition, offering additional important morphologic and functional information for treatment planning in patients with brain tumors.


Blood lead screening practices among US pediatricians.
In 1991, the Centers for Disease Control and Prevention (CDC) decreased the blood lead level of concern to 10 micrograms/dL (0.48 mumol/L) and recommended universal screening. Because these guidelines continue to provoke controversy, we conducted a study to: 1) estimate the proportion of pediatricians who are members of the American Academy of Pediatrics (AAP) who report screening for elevated blood lead levels; 2) describe their clinical practices regarding screening for elevated blood lead levels; 3) compare attitudes of universal screeners, selective screeners, and nonscreeners; and 4) identify characteristics of pediatricians who universally screen. Confidential, cross-sectional survey of a nationally representative random sample of 1610 pediatricians conducted through the AAP Periodic Survey. The study included 1035 responders (64% response rate). Analysis was limited to the 734 pediatricians who provide well-child care (ie, primary-care pediatricians). Fifty-three percent of pediatricians reported screening all their patients aged 9 to 36 months, 39% reported screening some, and 8% reported screening none. Among those who screen, 96% use a blood lead assay. The primary risk factors for which selective screeners screen are: history of pica (94%); living in an older home with recent renovations (92%); living in an older home with peeling paint (93%); and having a sibling who had an elevated blood lead level (88%). Among primary-care pediatricians, 73% agree that blood lead levels > or = 10 micrograms/dL should be considered elevated, and 16% disagree. However, 89% of primary-care pediatricians believe that epidemiologic studies should be performed to determine which communities have high proportions of children with elevated blood lead levels, and 34% of primary-care pediatricians believe that the costs of screening exceed the benefits. Three years after the Centers for Disease Control and Prevention issued new guidelines for the management of elevated blood lead levels, a slight majority of primary-care pediatricians in the United States who are members of the AAP report that they universally screen their appropriately aged patients, while most of the remaining pediatricians report screening high-risk patients. Many pediatricians may want additional guidance about circumstances under which selective screening should be considered.


Knockdown of serine/threonine-protein kinase 24 promotes tumorigenesis and myeloid-derived suppressor cell expansion in an orthotopic immunocompetent gastric cancer animal model.
A higher incidence of gastric cancer has been found in East Asia compared to the incidence in other regions. Gastric cancer patients have a poor prognosis due to distant metastasis and advanced cancer stages. Tumor escape pathways include the expansion of the immunosuppressive myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment. We have successfully established an orthotopic immunocompetent gastric cancer model in C57BL/6 mice. The cell line is named M12 and was deposited at the Bioresource Collection and Research Center of Taiwan on Sep. 13, 2016 (Patent No. I604054). The orthotopic animal model of gastric cancer has similar biological characteristics as human gastric cancer. Serine/threonine-protein kinase 24 (STK24) is a member of the germinal center kinase (GCK)-III family. GCKs participate in cancer and immunological disorders. The effects of STK24 in gastric cancer are less well understood. CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 technology was used to induce a STK24 genetic knockout at the genomic DNA level in tumor cells. The knockdown of the STK24 gene increased the tumor growth in an orthotopic model of gastric cancer. The STK24 gene silencing in tumors induced the expansion of CD11b+Ly6C+ cells and F4/80+ macrophages in vivo. To our knowledge, we have developed the first orthotopic transplantable model of gastric cancer in syngeneic inbred mice. Our results further indicate that STK24 is important for immune regulation during the tumorigenesis of gastric cancer.


Magnetization transfer imaging and proton MR spectroscopy in the evaluation of axonal injury: correlation with clinical outcome after traumatic brain injury.
Current imaging does not permit quantification of neural injury after traumatic brain injury (TBI) and therefore limits both the development of new treatments and the appropriate counseling of patients concerning prognosis. We evaluated the utility of magnetization transfer ratio (MTR) and proton MR spectroscopy in identifying patients with neuronal injury after TBI. Thirty patients with TBI (21-77 years old; mean age, 42 years; admission Glasgow Coma Scale (GOS) scores 3-15; mean score, 11) were studied on a 1.5-T system with magnetization transfer imaging and MR spectroscopy of the splenium. Magnetization transfer imaging was also performed in the brain stem in all patients, and other areas of the brain were sampled in one patient. The splenium of the corpus callosum and brain stem were studied because these are often affected by diffuse axonal injury. Scans were obtained 2 to 1129 days after injury (median, 41 days). MTR was considered abnormal if it was more than 2 SD below normal. Proton MR spectroscopy was used to calculate the N-acetylaspartate (NAA)/creatine (Cr) ratio. GOS was determined at least 3 months after injury. In 10 patients with a GOS of 1 to 4, the mean NAA/Cr was 1.24 +/- 0.28; two of these patients had abnormal MTR in normal-appearing white matter (NAWM). In 20 patients with a GOS of 5, the mean NAA/Cr was 1.53 +/- 0.37 (P < .05); four of these patients had abnormal MTR in NAWM. MTR abnormalities in NAWM were identified in six patients, but these changes did not correlate with GOS or MR spectroscopy changes. MTR and MR spectroscopy can quantify damage after TBI, and NAA levels may be a sensitive indicator of the neuronal damage that results in a worse clinical outcome.


Pharmacological and molecular studies on the interaction of varenicline with different nicotinic acetylcholine receptor subtypes. Potential mechanism underlying partial agonism at human α4β2 and α3β4 subtypes.
To determine the structural components underlying differences in affinity, potency, and selectivity of varenicline for several human (h) nicotinic acetylcholine receptors (nAChRs), functional and structural experiments were performed. The Ca2+ influx results established that: (a) varenicline activates (μM range) nAChR subtypes with the following rank sequence: hα7>hα4β4>hα4β2>hα3β4>>>hα1β1γδ; (b) varenicline binds to nAChR subtypes with the following affinity order (nM range): hα4β2~hα4β4>hα3β4>hα7>>>Torpedo α1β1γδ. The molecular docking results indicating that more hydrogen bond interactions are apparent for α4-containing nAChRs in comparison to other nAChRs may explain the observed higher affinity; and that (c) varenicline is a full agonist at hα7 (101%) and hα4β4 (93%), and a partial agonist at hα4β2 (20%) and hα3β4 (45%), relative to (±)-epibatidine. The allosteric sites found at the extracellular domain (EXD) of hα3β4 and hα4β2 nAChRs could explain the partial agonistic activity of varenicline on these nAChR subtypes. Molecular dynamics simulations show that the interaction of varenicline to each allosteric site decreases the capping of Loop C at the hα4β2 nAChR, suggesting that these allosteric interactions limit the initial step in the gating process. In conclusion, we propose that in addition to hα4β2 nAChRs, hα4β4 nAChRs can be considered as potential targets for the clinical activity of varenicline, and that the allosteric interactions at the hα3β4- and hα4β2-EXDs are alternative mechanisms underlying partial agonism at these nAChRs.


Eukaryotic viruses in wastewater samples from the United States.
Human fecal matter contains a large number of viruses, and current bacterial indicators used for monitoring water quality do not correlate with the presence of pathogenic viruses. Adenoviruses and enteroviruses have often been used to identify fecal pollution in the environment; however, other viruses shed in fecal matter may more accurately detect fecal pollution. The purpose of this study was to develop a baseline understanding of the types of viruses found in raw sewage. PCR was used to detect adenoviruses, enteroviruses, hepatitis B viruses, herpesviruses, morbilliviruses, noroviruses, papillomaviruses, picobirnaviruses, reoviruses, and rotaviruses in raw sewage collected throughout the United States. Adenoviruses and picobirnaviruses were detected in 100% of raw sewage samples and 25% and 33% of final effluent samples, respectively. Enteroviruses and noroviruses were detected in 75% and 58% of raw sewage samples, respectively, and both viral groups were found in 8% of final effluent samples. This study showed that adenoviruses, enteroviruses, noroviruses, and picobirnaviruses are widespread in raw sewage. Since adenoviruses and picobirnaviruses were detected in 100% of raw sewage samples, they are potential markers of fecal contamination. Additionally, this research uncovered previously unknown sequence diversity in human picobirnaviruses. This baseline understanding of viruses in raw sewage will enable educated decisions to be made regarding the use of different viruses in water quality assessments.


Time-resolved observation and control of superexchange interactions with ultracold atoms in optical lattices.
Quantum mechanical superexchange interactions form the basis of quantum magnetism in strongly correlated electronic media. We report on the direct measurement of superexchange interactions with ultracold atoms in optical lattices. After preparing a spin-mixture of ultracold atoms in an antiferromagnetically ordered state, we measured coherent superexchange-mediated spin dynamics with coupling energies from 5 hertz up to 1 kilohertz. By dynamically modifying the potential bias between neighboring lattice sites, the magnitude and sign of the superexchange interaction can be controlled, thus allowing the system to be switched between antiferromagnetic and ferromagnetic spin interactions. We compare our findings to predictions of a two-site Bose-Hubbard model and find very good agreement, but are also able to identify corrections that can be explained by the inclusion of direct nearest-neighbor interactions.


Celecoxib induces heme-oxygenase expression in glomerular mesangial cells.
Nonsteroidal anti-inflammatory drugs (NSAIDs) are frequently used as analgesics. They inhibit cyclooxygenases (COX), preventing the formation of prostaglandins, including prostacyclin and thromboxane. A serious side effect of COX-1 and COX-2 inhibitors is renal damage. To investigate the molecular basis of the renal injury, we evaluated the expression of the stress marker, heme oxygenase-1 (HO-1), in celecoxib-stimulated mesangial cells. We report here that a COX-2 selective NSAID, celecoxib, induced a concentration- and time-dependent increase of HO-1 expression in glomerular mesangial cells. Celecoxib-induced HO-1 protein expression was inhibited by actinomycin D and cycloheximide, suggesting that de novo transcription and translation are required in this process. N-acetylcysteine, a free radical scavenger, strongly decreased HO-1 expression, suggesting the involvement of reactive oxygen species (ROS). Celecoxib-induced HO-1 expression was attenuated by pretreatment of the cells with SP 600125 (a specific JNK inhibitor), but not SB 203580 (a specific p38 MAPK inhibitor), or PD 98059 (a specific MEK inhibitor). Consistently, celecoxib activated c-Jun N-terminal kinase (JNK) as demonstrated by kinase assays and by increasing phosphorylation of this kinase. N-acetylcysteine reduced the stimulatory effect of celecoxib on stress kinase activities, suggesting an involvement of JNK in HO-1 expression. On the other hand, LY 294002, a phosphatidylinositol 3-kinase (PI-3K)-specific inhibitor, prevented the enhancement of HO-1 expression. This effect was correlated with inhibition of the phosphorylation of the PDK-1 downstream substrate Akt/protein kinase B (PKB). In conclusion, our data suggest that celecoxib-induced HO-1 expression in glomerular mesangial cells may be mediated by ROS via the JNK-PI-3K cascade.


Psychometric evaluation of the Working Alliance Inventory-Therapist version: Current and new short forms.
Background: The Working Alliance Inventory (WAI) and its short forms are widely used, although the properties of the therapists' versions have been little studied. Method: We examined the psychometric properties of two short forms (WAI-S-T, WAI-SR-T), and explored the creation of a psychometrically stronger short form using contemporary measure development techniques. Well-fitting items from the full 36-item WAI were identified in a development sample (131 therapists, 688 patients) using multi-level Bayesian Structural Equation Modeling, accounting for therapist rated effects. Multi-level Item Response Theory (IRT) methods aided creation of a revised short form (WAI-S-T-IRT). Factor structures of the three forms were assessed using multi-level ML estimation with robust standard errors. Results: Collinearity problems for the Goal and Task dimensions led to testing a two-factor model (Goal-Task, Bond). All three measures showed satisfactory fit; the WAI-S-T-IRT fit slightly better but differences were minor. Testing the structures in an independent sample (N = 1117) yielded essentially the same results. No version showed strong measurement invariance. Discussion: Continued use of current therapist forms is supported; differentiation of theoretical dimensions is difficult with current measures, and may not be possible with self-report forms.


Distinctive Effect of Donepezil Treatment on P300 and N200 Subcomponents of Auditory Event-Related Evoked Potentials in Alzheimer Disease Patients.
Latency of P300 subcomponent of event-related potentials (ERPs) increases in Alzheimer disease (AD) patients, which correlate well with cognitive impairment. Cholinesterase inhibitors (ChEIs) reduce P300 latency in AD patients with parallel improvement in cognition. It is not known whether N200 response to ChEIs is similar to that of P300. The aim of this study was to evaluate and compare characteristics of P300 and N200 in AD patients, treatment-naïve and on stable donepezil treatment, matched by age, education, sex, and cognitive function. We recruited 22 consecutive treatment-naïve AD patients (AD-N group), 22 AD patients treated with a stable donepezil dose of 10 mg/day for at least 3 months (AD-T group), and 50 healthy controls were recruited. Neuropsychological testing (MMSE, ADAS-Cog, and additional tests) and ERP recording was performed and analyzed. All groups did not differ according to age, duration of education, or sex (p>0.05). AD-N and AD-T groups did not differ according to cognitive function. The AD-T group had longer duration of disease than the AD-N group (p<0.001). The AD-T and AD-N groups did not differ in P300 latencies (p=0.49). N200 latency was longer in the AD-T group (p<0.001). The general linear model showed that significant predictors of P300 latency were age (p=0.019) and AD treatment status (p<0.001). Duration of AD was a significant predictor of N200 latency (p=0.004). The response of N200 latency to donepezil treatment differs from the response of P300. P300 is a better marker of ChEI treatment-dependent cognitive functions. N200 is more dependent on the duration of AD.


Cranial growth in infants─A longitudinal three-dimensional analysis of the first months of life.
In the first months of life, any deviation from a physiological growth pattern can cause skull deformity. As there has not been any longitudinal three-dimensional (3D) study investigating the physiological growth of the infant skull, the aim of the present study was to acquire such data. We performed 3D stereophotogrammetric scans of 40 infants without cranial asymmetry at four regular 2-month intervals from the 4th to the 10th month of age. Six growth-related parameters (circumference, length, width, height, cephalic index [CI; width-length ratio] and total head volume) were used to analyse skull growth longitudinally. With exception of the CI, all parameters showed significant increases, with maximum percentage growth from the 4th to the 6th month. The CI initially remained unchanged until the 6th month, before showing a significant reduction that continued throughout the study period. Male infants had larger heads than female infants, but a similar width-length ratio at all measurement times. This prospective study is the first longitudinal 3D analysis to examine the physiological growth dynamics of infants' heads within the first months of life. Understanding patterns of skull growth in all three dimensions is important for gaining further insights into physiological and pathophysiological skull development.


[Health and its determinants in the immigrant population of the region of Madrid].
To describe health status and its determinants in immigrants living in the region of Madrid and to compare these factors with those in the native-born population. We performed a descriptive, cross-sectional study based on data from the Madrid Regional Health Survey 2007 (n=12,190). Subjects were classified as native-born or immigrants born in medium-to-low-income countries and with less or more than 5 years of residence in Spain. The results were adjusted for age. Proportions and means were compared through the ji square test and linear regression. Compared with native-born residents, immigrants were younger, carried out mainly precarious manual jobs that did not match their educational level and had a lower income and less space in their dwellings. Both immigrant men and women smoked and drank less, but women showed a higher prevalence of overweight (10% more) and physical inactivity. Adherence to smear test and blood pressure screening was up to a 16% lower. Attendance at primary care and emergency facilities was less frequent in men and women with less residence time than in those with longer residence in Spain. Self-perceived health and mental health were worse in women with 5 or more years of residence. Chronic diseases were similar in both populations but were less prevalent in immigrants. Immigrants' health status is not as unfavorable as could be expected from the higher prevalence of harmful determinants of health.


Modification of the behavioral effects of (+/-)BAY k 8644, cocaine and d-amphetamine by L-type calcium channel blockers in squirrel monkeys.
Behavioral effects of the 1,4-dihydropyridine L-type calcium channel activator (+/-)BAY k 8644 [(+/-)methyl-1,4-dihydro-2,6-dimethyl-3- nitro-4-(2-trifluoromethylphenyl)-pyridine-5-carboxylate] the monoamine reuptake inhibitor cocaine and the monoamine releaser d-amphetamine were determined alone and after pretreatment with the structurally distinct L-type calcium channel blockers nimodipine (1,4-dihydropyridine), verapamil (phenylakylamine), diltiazem (benzothiazepine) and flunarizine (diphenylalkylamine) in squirrel monkeys responding under a 10-response fixed-ratio schedule of stimulus-shock termination. When administered alone, (+/-)BAY k 8644 (0.1-0.56 mg/kg) produced dose-dependent decreases in rates of responding. Pretreatment with nimodipine (3.0-10 mg/kg) or verapamil (1.0-3.0 mg/kg) produced dose-dependent rightward shifts of the (+/-)BAY k 8644 dose-response curve. In contrast, pretreatment with flunarizine (3.0 mg/kg) produced a leftward and downward shift of the (+/-)BAY k 8644 dose-response curve. Pretreatment with diltiazem (10-17.8 mg/kg) did not modify the (+/-)BAY k 8644 dose-effect curve. In addition, stereoselectivity was evident in the behavioral effects of (+/-)BAY k 8644 with the S(-)-enantiomer being approximately 3-fold more potent than the racemate. When administered alone, cocaine (0.1-5.6 mg/kg) and d-amphetamine (0.1-3.0 mg/kg) produced dose-dependent decreases in rates of responding. Pretreatment with flunarizine (3.0 mg/kg) produced rightward shifts of the cocaine and d-amphetamine dose-response curves. Pretreatment with nimodipine (10 mg/kg), verapamil (3 mg/kg) or diltiazem (17.8 mg/kg) did not modify the effects of cocaine or d-amphetamine. The results of the present study suggest that the behavioral effects of (+/-)BAY k 8644 are differentially modified by L-type calcium channel blockers interacting with different sites on the channel and also suggest that the calcium channel blockers can be distinguished based on their differing interactions with (+/-)BAY k 8644, cocaine and d-amphetamine.


Bone resorption by cells isolated from rheumatoid synovium.
Cellular mechanisms accounting for the osteolysis of rheumatoid erosions are poorly understood. Cells were isolated and characterised from the synovium of 16 patients with rheumatoid arthritis (RA) and four patients with osteoarthritis and their ability to resorb bone was assessed using a scanning electron microscope bone resorption assay. Macrophages were the major cell type isolated from the synovium of patients with RA. These produced extensive roughening of the bone surface without resorption pit formation. This low grade type of bone resorption was not affected by systemic (calcitonin, parathyroid hormone, 1,25-dihydroxyvitamin D3) or local (interleukin 1, prostaglandin E2) factors influencing bone resorption. Macrophage mediated bone resorption differs qualitatively and quantitatively from that of osteoclasts but is likely to play an important part in the development of marginal erosions in RA.


Physical contests for females in the Japanese beetle, Popillia japonica.
Abstract We conducted field observations of physical competition for mates, in which a single male attempts to usurp a female from another male, in male Japanese beetles, Popillia japonica Newman (Coleoptera; Scarabaeidae). Physical contests for mates were relatively rare, but when they occurred the challenger male was able to successfully takeover females by dislodging the previously paired resident male in only 18% of contests, suggesting that a substantial prior residency advantage exists in this species. Challenger males that were successful in takeover attempts were significantly larger than the resident male. In contrast, no size pattern was found between intruding males and residents in unsuccessful takeover attempts. The frequency of contests for existing pairs was examined throughout the day. Pair frequency was greatest in early morning and in the evening but contest frequency was highest during the middle of the day. Contest frequency was negatively related to beetle density but not related to temperature. Overall, physical contests for mates appear to be an important part of the mating behavior in Japanese beetles. The frequency of the contests relates to the time of day and social conditions and contest outcome is related to prior residency and the size of the intruding male relative to the paired male.


Ephrin B2/EphB4 pathway in hepatic stellate cells stimulates Erk-dependent VEGF production and sinusoidal endothelial cell recruitment.
Chemotaxis signals between hepatic stellate cells (HSC) and sinusoidal endothelial cells (SEC) maintain hepatic vascular homeostasis and integrity and also regulate changes in sinusoidal structure in response to liver injury. Our prior studies have demonstrated that the bidirectional chemotactic signaling molecules EphrinB2 and EphB4 are expressed in HSC. The aim of our present study was to explore whether and how the EphrinB2/EphB4 system in HSC could promote SEC recruitment, which is essential for sinusoidal structure and remodeling. Stimulation of human HSC (hHSC) with chimeric agonists (2 microg/ml) of either EphrinB2 or EphB4 (EphrinB2 Fc or EphB4 Fc, respectively) significantly increased VEGF mRNA levels in hHSC as assessed by quantitative PCR, with respective small interfering RNAs for EphrinB2 and EphB4 inhibiting this increase (P < 0.05, n = 3). EphrinB2 agonist-induced increase in VEGF mRNA levels in hHSC was associated with increased phosphorylation of Erk and was significantly blocked by U0126 (20 microM), an inhibitor of MEK, which is a kinase upstream from Erk (P < 0.05, n = 3). The EphB4 agonist also significantly increased human VEGF promoter activity (P < 0.05, n = 3) as assessed by promoter reporter luciferase assay in transfected LX2-HSC. This was associated with upregulation of the vasculoprotective transcription factor, Kruppel-like factor 2 (KLF2). In Boyden chamber assays, conditioned media from hHSC stimulated with agonists of EphrinB2 or EphB4 increased SEC chemotaxis in a VEGF-dependent manner, compared with control groups that included basal media with agonists of EphrinB2, EphB4, or HSC-conditioned media from HSC in absence of agonist stimulation (P < 0.05, n = 3). EphB4 expression was detected in situ within liver sinusoidal vessels of rats after carbon tetrachloride-induced liver injury. In summary, activation of the EphrinB2/EphB4 signaling pathway in HSC promotes chemotaxis of SEC through a pathway that involves Erk, KLF2, and VEGF. These studies identify EphrinB2 or EphB4 as a key intermediary that links HSC signal transduction pathways with angiogenesis and sinusoidal remodeling.


MCB-613 exploits a collateral sensitivity in drug resistant EGFR-mutant non-small cell lung cancer through covalent inhibition of KEAP1.
Targeted therapies have revolutionized cancer chemotherapy. Unfortunately, most patients develop multifocal resistance to these drugs within a matter of months. Here, we used a high-throughput phenotypic small molecule screen to identify MCB-613 as a compound that selectively targets EGFR-mutant, EGFR inhibitor-resistant non-small cell lung cancer (NSCLC) cells harboring diverse resistance mechanisms. Subsequent proteomic and functional genomic screens involving MCB-613 identified its target in this context to be KEAP1, revealing that this gene is selectively essential in the setting of EGFR inhibitor resistance. In-depth molecular characterization demonstrated that (1) MCB-613 binds KEAP1 covalently; (2) a single molecule of MCB-613 is capable of bridging two KEAP1 monomers together; and, (3) this modification interferes with the degradation of canonical KEAP1 substrates such as NRF2. Surprisingly, NRF2 knockout sensitizes cells to MCB-613, suggesting that the drug functions through modulation of an alternative KEAP1 substrate. Together, these findings advance MCB-613 as a new tool for exploiting the selective essentiality of KEAP1 in drug-resistant, EGFR-mutant NSCLC cells.


Foot segment kinematics during normal walking using a multisegment model of the foot and ankle complex.
Gait analysis using optical tracking equipment has been demonstrated to be a clinically useful tool for measuring three-dimensional kinematics and kinetics of the human body. However, in current practice, the foot is treated as a single rigid segment that articulates with the lower leg, meaning the motions of the joints of the foot cannot be measured. A multisegment kinematic model of the foot was developed for use in a gait analysis laboratory. The foot was divided into hindfoot, talus, midfoot, and medial and lateral forefoot segments. Six functional joints were defined: Ankle and subtalar joints, frontal and transverse plane motions of the hindfoot relative to midfoot, supination-pronation twist of the forefoot relative to midfoot, and medial longitudinal arch height-to-length ratio. Twelve asymptomatic subjects were tested during barefoot walking with a six-camera optical stereometric system and passive markers organized in triads. Repeatability of reported motions was tested using coefficients of multiple correlation. Ankle and subtalar joint motions and twisting of the forefoot were most repeatable. Hindfoot motions were least repeatable both within subjects and between subjects. Hindfoot and forefoot pronations in the frontal place were found to coincide with dropping of the medial longitudinal arch between early to midstance, followed by supination and rising of the arch in late stance and swing phase. This multisegment foot model overcomes a major shortcoming in current gait analysis practice-the inability to measure motion within the foot. Such measurements are crucial if gait analysis is to remain relevant in orthopaedic and rehabilitative treatment of the foot and ankle.


Spectroscopic analysis of myoglobin and cytochrome c dynamics in isolated cardiomyocytes during hypoxia and reoxygenation.
Raman microspectroscopy was applied to monitor the intracellular redox state of myoglobin and cytochrome c from isolated adult rat cardiomyocytes during hypoxia and reoxygenation. The nitrite reductase activity of myoglobin leads to the production of nitric oxide in cells under hypoxic conditions, which is linked to the inhibition of mitochondrial respiration. In this work, the subsequent reoxygenation of cells after hypoxia is shown to lead to increased levels of oxygen-bound myoglobin relative to the initial levels observed under normoxic conditions. Increased levels of reduced cytochrome c in ex vivo cells are also observed during hypoxia and reoxygenation by Raman microspectroscopy. The cellular response to reoxygenation differed dramatically depending on the method used in the preceding step to create hypoxic conditions in the cell suspension, where a chemical agent, sodium dithionite, leads to reduction of cytochromes in addition to removal of dissolved oxygen, and bubbling-N2 gas leads to displacement of dissolved oxygen only. These results have an impact on the assessment of experimental simulations of hypoxia in cells. The spectroscopic technique employed in this work will be used in the future as an analytical method to monitor the effects of varying levels of oxygen and nutrients supplied to cardiomyocytes during either the preconditioning of cells or the reperfusion of ischaemic tissue.


Assessing the educational needs of Canadian gastroenterologists and gastroenterology nurses: challenges to optimal care in Crohn's Disease.
A national needs assessment of Canadian gastroenterologists and gastroenterology nurses was undertaken to determine the perceived and unperceived educational and performance barriers to caring for patients with Crohn's disease (CD). A triangulated, mixed-method approach (qualitative and quantitative) was used to determine the nature and extent of knowledge gaps and barriers in the care of patients with CD. Qualitative interviews were conducted with nine gastroenterologists, four gastroenterology nurses and nine patients with CD. Based on this exploratory research, a survey was designed and launched nationally (37 gastroenterologists, 36 gastroenterology nurses). Findings indicated that Canadian gastroenterologists and gastroenterology nurses lacked clarity regarding their roles and responsibilities across the continuum of CD care, and face communication gaps within the health care team, undermining their effectiveness. Gastroenterologists identified challenges in optimal diagnosis due to unclear testing and diagnostic criteria. They recognized knowledge gaps when treating patient subgroups and in prescribing biological therapies. Furthermore, gastroenterologists self-identified gaps in skill, knowledge, and confidence in monitoring disease progression and effectively assessing response to therapy. When managing patients with CD, gastroenterologists expressed challenges with patient issues outside their domain of medical expertise, particularly with the skills needed to facilitate effective patient communication and education that would enhance adherence to recommended treatments. Educational initiatives should address diagnostic and treatment guidelines, as well as enhancement of clinical performance gaps in health care team processes and the patient-professional therapeutic relationship. To impact care and patient outcomes, these initiatives must be relevant to clinical practice settings and applicable to the practice context.


Molecular paleontology and complexity in the last eukaryotic common ancestor.
Eukaryogenesis, the origin of the eukaryotic cell, represents one of the fundamental evolutionary transitions in the history of life on earth. This event, which is estimated to have occurred over one billion years ago, remains rather poorly understood. While some well-validated examples of fossil microbial eukaryotes for this time frame have been described, these can provide only basic morphology and the molecular machinery present in these organisms has remained unknown. Complete and partial genomic information has begun to fill this gap, and is being used to trace proteins and cellular traits to their roots and to provide unprecedented levels of resolution of structures, metabolic pathways and capabilities of organisms at these earliest points within the eukaryotic lineage. This is essentially allowing a molecular paleontology. What has emerged from these studies is spectacular cellular complexity prior to expansion of the eukaryotic lineages. Multiple reconstructed cellular systems indicate a very sophisticated biology, which by implication arose following the initial eukaryogenesis event but prior to eukaryotic radiation and provides a challenge in terms of explaining how these early eukaryotes arose and in understanding how they lived. Here, we provide brief overviews of several cellular systems and the major emerging conclusions, together with predictions for subsequent directions in evolution leading to extant taxa. We also consider what these reconstructions suggest about the life styles and capabilities of these earliest eukaryotes and the period of evolution between the radiation of eukaryotes and the eukaryogenesis event itself.


Academia's chilly climate for primary care.
To describe the attitudes toward and perceptions of primary care education and practice among academic health center constituents. Descriptive study using confidential telephone interviews (October 1993 to March 1994) of national stratified probability samples of first- and fourth-year medical students, residents, clinical faculty, internal medicine and pediatrics residency training directors and chairs, and deans (N=2293). Five areas were examined: respondents' specialty orientation, attitudes toward the competence of primary care physicians, encouragement and positive regard for primary care, exposure to primary care-related educational experiences, and socioemotional orientation. The response rate was 84%. Respondents generally perceive primary care tasks as not requiring high levels of expertise; nearly half believe that generalists are not the best physicians to manage patients with serious illness and that the quality of primary care research is inferior to that in other fields. Attitudes are more positive toward the quality of primary care teaching. Learners perceive little encouragement for generalist careers and negative attitudes toward generalists among faculty, and view the quality of their primary care training as inferior to that for specialty practice. Those who have progressed further in the academic medicine hierarchy generally report lower levels of socioemotional orientation than individuals at earlier phases of career development. Despite changes in the health care system and in education, students and residents encounter an atmosphere that is chilly toward primary care. If medical educators seek to optimize enthusiasm and preparation for primary care careers, they must develop approaches to changing the attitudes, values and composition of their faculties.


Emx2 regulates the proliferation of stem cells of the adult mammalian central nervous system.
The appropriate control of proliferation of neural precursors has fundamental implications for the development of the central nervous system and for cell homeostasis/replacement within specific brain regions throughout adulthood. The role of genetic determinants in this process is largely unknown. We report the expression of the homeobox transcription factor Emx2 within the periventricular region of the adult telencephalon. This neurogenetic area displays a large number of multipotent stem cells. Adult neural stem cells isolated from this region do express Emx2 and down-regulate it significantly upon differentiation into neurons and glia. Abolishing or, increasing Emx2 expression in adult neural stem cells greatly enhances or reduces their rate of proliferation, respectively. We determined that altering the expression of Emx2 affects neither the cell cycle length of adult neural stem cells nor their ability to generate neurons and glia. Rather, when Emx2 expression is abolished, the frequency of symmetric divisions that generate two stem cells increases, whereas it decreases when Emx2 expression is enhanced.


Japanese founder duplications/triplications involving BHLHA9 are associated with split-hand/foot malformation with or without long bone deficiency and Gollop-Wolfgang complex.
Limb malformations are rare disorders with high genetic heterogeneity. Although multiple genes/loci have been identified in limb malformations, underlying genetic factors still remain to be determined in most patients. This study consisted of 51 Japanese families with split-hand/foot malformation (SHFM), SHFM with long bone deficiency (SHFLD) usually affecting the tibia, or Gollop-Wolfgang complex (GWC) characterized by SHFM and femoral bifurcation. Genetic studies included genomewide array comparative genomic hybridization and exome sequencing, together with standard molecular analyses. We identified duplications/triplications of a 210,050 bp segment containing BHLHA9 in 29 SHFM patients, 11 SHFLD patients, two GWC patients, and 22 clinically normal relatives from 27 of the 51 families examined, as well as in 2 of 1,000 Japanese controls. Families with SHFLD- and/or GWC-positive patients were more frequent in triplications than in duplications. The fusion point was identical in all the duplications/triplications and was associated with a 4 bp microhomology. There was no sequence homology around the two breakpoints, whereas rearrangement-associated motifs were abundant around one breakpoint. The rs3951819-D17S1174 haplotype patterns were variable on the duplicated/triplicated segments. No discernible genetic alteration specific to patients was detected within or around BHLHA9, in the known causative SHFM genes, or in the exome. These results indicate that BHLHA9 overdosage constitutes the most frequent susceptibility factor, with a dosage effect, for a range of limb malformations at least in Japan. Notably, this is the first study revealing the underlying genetic factor for the development of GWC, and demonstrating the presence of triplications involving BHLHA9. It is inferred that a Japanese founder duplication was generated through a replication-based mechanism and underwent subsequent triplication and haplotype modification through recombination-based mechanisms, and that the duplications/triplications with various haplotypes were widely spread in Japan primarily via clinically normal carriers and identified via manifesting patients. Furthermore, genotype-phenotype analyses of patients reported in this study and the previous studies imply that clinical variability is ascribed to multiple factors including the size of duplications/triplications as a critical factor.


Relations between plasma oxytocin and cortisol: The stress buffering role of social support.
Stress responses in humans can be attenuated by exogenous oxytocin administration, and these stress-buffering properties may be moderated by social factors. Yet, the influence of acute stressors on circulating endogenous oxytocin levels have been inconsistent, and limited information is available concerning the influence of social support in moderating this relationship. In the current investigation, undergraduate women (N = 67) were assessed in the Trier Social Stress Test (TSST) with either social support available from a close female friend or no social support being available. An additional set of women served as controls. The TSST elicited marked elevations of state anxiety and negative emotions, which were largely attenuated among women who received social support. Furthermore, baseline oxytocin levels were inversely related to women's general feelings of distrust, as well as basal plasma cortisol levels. Despite these associations, oxytocin levels were unaffected by the TSST, and this was the case irrespective of oral contraceptive use or estrogen levels. In contrast, plasma cortisol elevations were elicited by the psychosocial stressor, but only in women using oral contraceptives, an effect that was prevented when social support was available. Taken together, these data provisionally suggest that changes in plasma oxytocin might not accompany the stress attenuating effects of social support on cortisol levels. Moreover, as plasma oxytocin might not reliably reflect brain oxytocin levels, the linkage between oxytocin and prosocial behaviors remains tenuous.


BMI status in Swedish children and young adults in relation to caries prevalence.
Overweight and obesity are increasing as health problems at global level. Dental caries and obesity are both multifactorial diseases and are associated with dietary habits. The aim of the present study was to investigate the relationship between body weight status and caries prevalence in an unselected population followed from pre-school years to young adulthood. The present investigation was designed as a longitudinal analysis of the association between overweight/obesity and dental caries in one population at 3, 6, 15 and 20 years of age. The result shows that adolescents (15 years) and young adults (20 years) who are overweight/obese had a statistically significantly higher caries prevalence than normal-weight young people. At 6 years of age, the odds (OR) of having caries among obese children are 2.5 times higher than the odds for caries among six-year-old children of normal weight (p = 0.04). At 3 years of age, no association between overweight/obesity and caries was found. To conclude, overweight and obese adolescents and young adults had more caries than normal-weight individuals. The present study emphasises the need for multidisciplinary approaches to change the lifestyle factors causing both overweight/obesity and dental caries.


Catheter ablation of cardiac arrhythmias guided by electroanatomic imaging (CARTO): a single-center experience.
Catheter ablation is assuming a larger role in the management of patients with cardiac arrhythmias. Conventional fluoroscopic catheter mapping has limited spatial resolution and involves prolonged fluoroscopy. The non-fluoroscopic electroanatomic mapping technique (CARTO) has been developed to overcome these drawbacks. To report the early and late outcome in patients with different arrhythmias treated with radiofrequency ablation combined with the CARTO mapping and navigation system. The study cohort comprised 125 consecutive patients with different cardiac arrhythmias referred to our center from January 1999 to July 2005 for mapping and/or ablation procedures using the CARTO system. Forty patients (32%) had previous failed conventional ablation or mapping procedures and were referred by other centers. The arrhythmia included atrial fibrillation (n = 13), atrial flutter (n = 38), atrial tachycardia (n = 25), ventricular tachycardia (n = 24), arrhythmogenic right ventricular dysplasia (n = 9), and supraventricular tachycardia (n = 16). During the study period, a total of 125 patients (mean age 49 +/- 19 years, 59% males) underwent electrophysiological study and electroanatomic mapping of the heart chambers. Supraventricular arrhythmias were identified in 92 patients (73%) and ventricular arrhythmias in 33 (27%). Acute and late success rates, defined as termination of the arrhythmia without anti-arrhythmic drugs, were 87% and 76% respectively. One patient (0.8%) developed a clinically significant complication. The CARTO system advances our understanding of arrhythmias, and increases the safety, efficacy and efficiency of radiofrequency ablation.


The Development and Implementation of an Electronic Health Record Tool for Monitoring Metabolic Syndrome Indices in Patients with Serious Mental Illness.
Objectives1. A quality performance improvement (QI) project to implement an electronic screening and monitoring tool to record components of the metabolic syndrome (e-MSD) during clinic visits by persons with serious mental illness (SMI). 2. To encourage psychiatrists to use this tool in their documentation.MethodsWorking with the information technology staff; five psychiatrists developed, tested, revised and embedded the e-MSD tool into the medication management document within the electronic health record. A continuing medical education program on metabolic syndrome was developed, and released to psychiatrists and mental health clinicians. Psychiatrist offices at one clinic were equipped with weighing scales, sphygmomanometers, waist circumference tapes and a QI project was initiated.ResultsAt one month, 9 to 12% of the anthropometric measures (height, weight, body mass index, waist circumference, and blood pressure) were recorded in 974 unique patient encounters, and 1 year later the numbers moved upwards - 15 to 41%. Towards the end of Year 1, a patient care associate was hired to measure the anthropometric measures, and one year later, the documented rates increased to 75-80%. Laboratory recordings (glucose and lipids) remained ≤ 8% throughout the first year, but moved upwards to 25% in Year 2.DiscussionNotwithstanding significant administrative and technical support for this QI project, changing clinician practice to screen, monitor and document metabolic indices in persons with SMI in the ambulatory setting changed significantly after the hiring of a patient care associate. Efforts to obtain laboratory measures in real-time remain a challenge. Next steps include interventions to promote weight loss and smoking cessation in SMI patients, and effective communication with their primary care doctors.


Characteristics of coagulase-negative staphylococci that help differentiate these species and other members of the family Micrococcaceae.
One hundred reference strains and 1,240 clinical isolates representing 26 species of the family Micrococcaceae were used to evaluate the potential of tests for synergistic hemolysis, adherence to glass, pyroglutamyl-beta-naphthylamide hydrolysis, and susceptibility to a set of five antimicrobial agents for differentiating these species and strains within the species. Sixty-eight percent of the clinical isolates exhibited synergistic hemolysis; 69% of the clinical staphylococci but none of the micrococci or stomatococci were adherence positive, and 92% of the strong positive adherence reactions were produced by strains of Staphylococcus epidermidis. Strains from 15 of the species were pyroglutamyl-beta-naphthylamide positive, but this test separated Staphylococcus xylosus from other novobiocin-resistant staphylococci and Staphylococcus intermedius from other coagulase-positive species. A polymyxin B disk helped differentiate S. epidermidis from most other coagulase-negative staphylococci, and a bacitracin disk (10 U) helped differentiate Staphylococcus haemolyticus from most other novobiocin-susceptible staphylococci. All strains that were susceptible to furazolidone and resistant to Taxo A disks (bacitracin, 0.04 U; BBL Microbiology Systems, Cockeysville, Md.) were staphylococci. We observed a 91% correlation between species identification obtained with the Staph-Ident system (Analytab Products, Plainview, N.Y.) and conventional methods; but the micrococci and stomatococci were incorrectly identified as staphylococci with Staph-Ident, and several isolates of S. epidermidis were misidentified as Staphylococcus hominis because they were alkaline phosphatase negative. Both these problems can be prevented by adding the simple tests we describe to those already recommended when the Staph-Ident system is used to identify isolates of gram-positive, catalase-positive cocci.


Response kinetics of tethered Rhodobacter sphaeroides to changes in light intensity.
Rhodobacter sphaeroides can swim toward a wide range of attractants (a process known as taxis), propelled by a single rotating flagellum. The reversals of motor direction that cause tumbles in Eschericia coli taxis are replaced by brief motor stops, and taxis is controlled by a complex sensory system with multiple homologues of the E. coli sensory proteins. We tethered photosynthetically grown cells of R. sphaeroides by their flagella and measured the response of the flagellar motor to changes in light intensity. The unstimulated bias (probability of not being stopped) was significantly larger than the bias of tethered E. coli but similar to the probability of not tumbling in swimming E. coli. Otherwise, the step and impulse responses were the same as those of tethered E. coli to chemical attractants. This indicates that the single motor and multiple sensory signaling pathways in R. sphaeroides generate the same swimming response as several motors and a single pathway in E. coli, and that the response of the single motor is directly observable in the swimming pattern. Photo-responses were larger in the presence of cyanide or the uncoupler carbonyl cyanide 4-trifluoromethoxyphenylhydrazone (FCCP), consistent with the photo-response being detected via changes in the rate of electron transport.


Experimental ocular onchocerciasis in cynomolgus monkeys.
Infection of cynomolgus monkeys with microfilariae (Mf) of Onchocerca lienalis was studied as a model for human ocular onchocerciasis. Normal monkeys and immunized monkeys were given intracorneal/subconjunctival, intracameral, or intravitreal injections of Mf or bovine serum albumin (control). Selected animals were given diethylcarbamazine citrate (DEC) orally (15 mg/kg) daily after the ocular infection. Following intravitreal challenge, living Mf and fibrinous exudates were visible by slit-lamp in both the anterior chamber and vitreous. Eosinophils and macrophages surrounded the Mf in the vitreous, with degranulated eosinophils adherent to the Mf. Eosinophils infiltrated the uvea and surrounded the retinal vessels. After intracorneal injection of Mf, living Mf were visible by slit-lamp biomicroscopy in the cornea for 3 days, with minimal inflammation of the corneas occurring over the 7 days after injection. Intracamerally injected Mf induced anterior uveitis. The extent of the inflammatory reactions was not substantially altered by DEC treatment following intraocular injection of Mf. In vitro proliferative responses of peripheral blood leukocytes to a crude Mf antigen were not observed in infected monkeys and proliferative responses to mitogen declined in these animals. Responses to mitogen were inhibited by addition of Mf antigen in vitro in normal monkeys. Circulating IgG antibodies were present in the sensitized, intracorneally, and intravitreally challenged animals. No obvious correlations were present between IgG antibody level and ocular inflammation.


Helicobacter pylori infection: An overview of bacterial virulence factors and pathogenesis.
Helicobacter pylori pathogenesis and disease outcomes are mediated by a complex interplay between bacterial virulence factors, host, and environmental factors. After H. pylori enters the host stomach, four steps are critical for bacteria to establish successful colonization, persistent infection, and disease pathogenesis: (1) Survival in the acidic stomach; (2) movement toward epithelium cells by flagella-mediated motility; (3) attachment to host cells by adhesins/receptors interaction; (4) causing tissue damage by toxin release. Over the past 20 years, the understanding of H. pylori pathogenesis has been improved by studies focusing on the host and bacterial factors through epidemiology researches and molecular mechanism investigations. These include studies identifying the roles of novel virulence factors and their association with different disease outcomes, especially the bacterial adhesins, cag pathogenicity island, and vacuolating cytotoxin. Recently, the development of large-scale screening methods, including proteomic, and transcriptomic tools, has been used to determine the complex gene regulatory networks in H. pylori. In addition, a more available complete genomic database of H. pylori strains isolated from patients with different gastrointestinal diseases worldwide is helpful to characterize this bacterium. This review highlights the key findings of H. pylori virulence factors reported over the past 20 years.


Electromyography of the fin musculature of the cuttlefish Sepia officinalis.
The musculature of the fins of the cuttlefish Sepia officinalis (Mollusca, Cephalopoda) was studied with electromyography to test predictions of the functional role of the various muscle masses. Previous research had shown the fins to consist of a tightly packed, three-dimensional array of muscle with distinct zones of anaerobic glycolytic and oxidative muscle fibres. In addition, a network of crossed oblique connective tissue fibres was observed within the musculature. In a previous paper a model of the function of the muscle and connective tissue was presented. In the present paper, we present recordings of electrical activity from the various muscle bundles in the fin, in conjunction with the output from an electronic movement-monitoring device, and correlate muscle activity with both the phase and the intensity of the fin-beat cycle. The results obtained here support the hypothesis that the oxidative muscle fibres produce gentle fin movements and are consistent with the hypothesis that the network of crossed oblique connective tissue fibres provides skeletal support. The results also support predictions that the anaerobic glycolytic muscle fibres both produce vigorous fin movements and provide support for that movement. This study provides a critical test of models of the role of the tightly packed, three-dimensional array of muscle found in muscular hydrostats such as the arms and tentacles of cephalopods and tongues of mammals and lizards.


Endocranial anatomy of the charadriiformes: sensory system variation and the evolution of wing-propelled diving.
Just as skeletal characteristics provide clues regarding behavior of extinct vertebrates, phylogenetically-informed evaluation of endocranial morphology facilitates comparisons among extinct taxa and extant taxa with known behavioral characteristics. Previous research has established that endocranial morphology varies across Aves; however, variation of those systems among closely related species remains largely unexplored. The Charadriiformes (shorebirds and allies) are an ecologically diverse clade with a comparatively rich fossil record, and therefore, are well suited for investigating interspecies variation, and potential links between endocranial morphology, phylogeny, ecology and other life history attributes. Endocranial endocasts were rendered from high resolution X-ray computed tomography data for 17 charadriiforms (15 extant and two flightless extinct species). Evaluation of endocranial character state changes on a phylogeny for Charadriiformes resulted in identification of characters that vary in taxa with distinct feeding and locomotor ecologies. In comparison with all other charadriiforms, stem and crown clade wing-propelled diving Pan-Alcidae displayed compressed semicircular canals, and indistinct occipital sinuses and cerebellar fissures. Flightless wing-propelled divers have relatively smaller brains for their body mass and smaller optic lobes than volant pan-alcids. Observed differences between volant and flightless wing-propelled sister taxa are striking given that flightless pan-alcids continue to rely on the flight stroke for underwater propulsion. Additionally, the brain of the Black Skimmer Rynchops niger, a taxon with a unique feeding ecology that involves continuous forward aerial motion and touch-based prey detection used both at day and night, is discovered to be unlike that of any other sampled charadriiform in having an extremely large wulst as well as a small optic lobe and distinct occipital sinus. Notably, the differences between the Black Skimmer and other charadriiforms are more pronounced than between wing-propelled divers and other charadriiforms. Finally, aspects of endosseous labyrinth morphology are remarkably similar between divers and non-divers, and may deserve further evaluation.


The role of the interferon gamma release assay in assessing recent tuberculosis transmission in a hospital incident.
In 2007, an extensive contact screening investigation into onward transmission of tuberculosis was instigated at a hospital in Northern Ireland following diagnosis of pulmonary multi-drug resistant TB in a healthcare worker. Interferon gamma release assays (IGRAs) were used to test 333 patients and 98 staff. We investigated for evidence of onward transmission and recent infection based on analysis of clinical, demographic and IGRA data. We also described within-patient variability of IGRA results. Among patients and staff, increasing age of patients was the only factor associated with IGRA positivity. Greatest within-subject variability of IU/mL in serially-tested patients/staff was seen in those with a positive IGRA test and this did not correlate with increased exposure to the index case. IGRA positivity being largely explained by increasing age in patients and previous TB contact in staff lends weight to the conclusion that IGRA positivity reflected previous infection rather than recent transmission.


Trematode parasites exceed aquatic insect biomass in Oregon stream food webs.
Although parasites are increasingly recognized for their ecosystem roles, it is often assumed that free-living organisms dominate animal biomass in most ecosystems and therefore provide the primary pathways for energy transfer. To examine the contributions of parasites to ecosystem energetics in freshwater streams, we quantified the standing biomass of trematodes and free-living organisms at nine sites in three streams in western Oregon, USA. We then compared the rates of biomass flow from snails Juga plicifera into trematode parasites relative to aquatic vertebrate predators (sculpin, cutthroat trout and Pacific giant salamanders). The trematode parasite community had the fifth highest dry biomass density among stream organisms (0.40 g/m2 ) and exceeded the combined biomass of aquatic insects. Only host snails (3.88 g/m2 ), sculpin (1.11 g/m2 ), trout (0.73 g/m2 ) and crayfish (0.43 g/m2 ) had a greater biomass. The parasite 'extended phenotype', consisting of trematode plus castrated host biomass, exceeded the individual biomass of every taxonomic group other than snails. The substantial parasite biomass stemmed from the high snail density and infection prevalence, and the large proportional mass of infected hosts that consisted of trematode tissue (M = 31% per snail). Estimates of yearly biomass transfer from snails into trematodes were slightly higher than the combined estimate of snail biomass transfer into the three vertebrate predators. Pacific giant salamanders accounted for 90% of the snail biomass consumed by predators. These results demonstrate that trematode parasites play underappreciated roles in the ecosystem energetics of some freshwater streams.


Pheromone biosynthetic pathways in the moths Helicoverpa zea and Helicoverpa assulta.
Sex pheromones of many Lepidopteran species have relatively simple structures consisting of a hydrocarbon chain with a functional group and usually one to several double bonds. The sex pheromones are usually derived from fatty acids through a specific biosynthetic pathway. We investigated the incorporation of deuterium-labeled palmitic and stearic acid precursors into pheromone components of Helicoverpa zea and Helicoverpa assulta. The major pheromone component for H. zea is (Z)11-hexadecenal (Z11-16:Ald) while H. assulta utilizes (Z)9-hexadecenal (Z9-16:Ald). We found that H. zea uses palmitic acid to form Z11-16:Ald via delta 11 desaturation and reduction, but also requires stearic acid to biosynthesize the minor pheromone components Z9-16:Ald and Z7-16:Ald. The Z9-16:Ald is produced by delta 11 desaturation of stearic acid followed by one round of chain-shortening and reduction to the aldehyde. The Z7-16:Ald is produced by delta 9 desaturation of stearic acid followed by one round of chain-shortening and reduction to the aldehyde. H. assulta uses palmitic acid as a substrate to form Z9-16:Ald, Z11-16:Ald and 16:Ald. The amount of labeling indicated that the delta 9 desaturase is the major desaturase present in the pheromone gland cells of H. assulta; whereas, the delta 11 desaturase is the major desaturase in pheromone glands of H. zea. It also appears that H. assulta lacks chain-shortening enzymes since stearic acid did not label any of the 16-carbon aldehydes.


Multivoxel patterns in face-sensitive temporal regions reveal an encoding schema based on detecting life in a face.
More than a decade of research has demonstrated that faces evoke prioritized processing in a 'core face network' of three brain regions. However, whether these regions prioritize the detection of global facial form (shared by humans and mannequins) or the detection of life in a face has remained unclear. Here, we dissociate form-based and animacy-based encoding of faces by using animate and inanimate faces with human form (humans, mannequins) and dog form (real dogs, toy dogs). We used multivariate pattern analysis of BOLD responses to uncover the representational similarity space for each area in the core face network. Here, we show that only responses in the inferior occipital gyrus are organized by global facial form alone (human vs dog) while animacy becomes an additional organizational priority in later face-processing regions: the lateral fusiform gyri (latFG) and right superior temporal sulcus. Additionally, patterns evoked by human faces were maximally distinct from all other face categories in the latFG and parts of the extended face perception system. These results suggest that once a face configuration is perceived, faces are further scrutinized for whether the face is alive and worthy of social cognitive resources.


Pesticide Residues on Three Cut Flower Species and Potential Exposure of Florists in Belgium.
In order to assess the prevalence of pesticide contamination and the risk of florists' exposure when handling cut flowers, sampling and analysis of 90 bouquets of the most commonly sold cut flowers in Belgium (50 bouquets of roses; 20 of gerberas, and 20 of chrysanthemums) were carried out. The bouquets were collected from 50 florists located in the seven largest cities of Belgium (Antwerp, Brussels, Charleroi, Ghent, Leuven, Liege, and Namur) and from five supermarkets located in the different regions. To have a better understanding of the route of exposure and professional practices a questionnaire was also addressed to a group of 25 florists who volunteered to take part in the survey. All florists were interviewed individually when collecting the questionnaire. The residual pesticide deposit values on cut flowers were determined in an accredited laboratory using a multi-residue (QuEChERS Quick Easy Cheap Effective Rugged Safe) method and a combination of gas chromatography (GC) and liquid chormatograhphy (LC) analysis. A total of 107 active substances were detected from all samples; i.e., an average of about 10 active substances per bouquet. The most severely contaminated bouquet accumulated a total concentration of residues up to 97 mg/kg. Results show that roses are the most contaminated cut flowers; with an average of 14 substances detected per sample and a total concentration per rose sample of 26 mg/kg. Some active substances present an acute toxicity (acephate, methiocarb, monocrotophos, methomyl, deltamethrin, etc.) and exposure can generate a direct effect on the nervous system of florists. Nevertheless, fungicides (dodemorph, propamocarb, and procymidone) were the most frequently detected in samples and had the highest maximum concentrations out of all the active substances analysed. Dodemorph was the most frequently detected substance with the highest maximum concentration (41.9 mg/kg) measured in the rose samples. It appears from the survey that, despite being exposed to high deposits of residues, florists usually do not protect themselves from contact with residues even if they spend several hours handling cut flowers and preparing bouquets (from 2 to 6 h/day, depending on the time of year and/or selling periods) daily. Bad habits (eating, drinking, or smoking at work) and absence of personal protective equipment of most florists also increase the risk of contact with pesticide residues.


Neural Network Approach for Characterizing Structural Transformations by X-Ray Absorption Fine Structure Spectroscopy.
The knowledge of the coordination environment around various atomic species in many functional materials provides a key for explaining their properties and working mechanisms. Many structural motifs and their transformations are difficult to detect and quantify in the process of work (operando conditions), due to their local nature, small changes, low dimensionality of the material, and/or extreme conditions. Here we use an artificial neural network approach to extract the information on the local structure and its in situ changes directly from the x-ray absorption fine structure spectra. We illustrate this capability by extracting the radial distribution function (RDF) of atoms in ferritic and austenitic phases of bulk iron across the temperature-induced transition. Integration of RDFs allows us to quantify the changes in the iron coordination and material density, and to observe the transition from a body-centered to a face-centered cubic arrangement of iron atoms. This method is attractive for a broad range of materials and experimental conditions.


Tracing the De Novo Origin of Protein-Coding Genes in Yeast.
De novo genes are very important for evolutionary innovation. However, how these genes originate and spread remains largely unknown. To better understand this, we rigorously searched for de novo genes in Saccharomyces cerevisiae S288C and examined their spread and fixation in the population. Here, we identified 84 de novo genes in S. cerevisiae S288C since the divergence with their sister groups. Transcriptome and ribosome profiling data revealed at least 8 (10%) and 28 (33%) de novo genes being expressed and translated only under specific conditions, respectively. DNA microarray data, based on 2-fold change, showed that 87% of the de novo genes are regulated during various biological processes, such as nutrient utilization and sporulation. Our comparative and evolutionary analyses further revealed that some factors, including single nucleotide polymorphism (SNP)/indel mutation, high GC content, and DNA shuffling, contribute to the birth of de novo genes, while domestication and natural selection drive the spread and fixation of these genes. Finally, we also provide evidence suggesting the possible parallel origin of a de novo gene between S. cerevisiae and Saccharomyces paradoxus Together, our study provides several new insights into the origin and spread of de novo genes.IMPORTANCE Emergence of de novo genes has occurred in many lineages during evolution, but the birth, spread, and function of these genes remain unresolved. Here we have searched for de novo genes from Saccharomyces cerevisiae S288C using rigorous methods, which reduced the effects of bad annotation and genomic gaps on the identification of de novo genes. Through this analysis, we have found 84 new genes originating de novo from previously noncoding regions, 87% of which are very likely involved in various biological processes. We noticed that 10% and 33% of de novo genes were only expressed and translated under specific conditions, therefore, verification of de novo genes through transcriptome and ribosome profiling, especially from limited expression data, may underestimate the number of bona fide new genes. We further show that SNP/indel mutation, high GC content, and DNA shuffling could be involved in the birth of de novo genes, while domestication and natural selection drive the spread and fixation of these genes. Finally, we provide evidence suggesting the possible parallel origin of a new gene.


Computational prediction and experimental validation of evolutionarily conserved microRNA target genes in bilaterian animals.
In many eukaryotes, microRNAs (miRNAs) bind to complementary sites in the 3'-untranslated regions (3'-UTRs) of target messenger RNAs (mRNAs) and regulate their expression at the stage of translation. Recent studies have revealed that many miRNAs are evolutionarily conserved; however, the evolution of their target genes has yet to be systematically characterized. We sought to elucidate a set of conserved miRNA/target-gene pairs and to analyse the mechanism underlying miRNA-mediated gene regulation in the early stage of bilaterian evolution. Initially, we extracted five evolutionarily conserved miRNAs (let-7, miR-1, miR-124, miR-125/lin-4, and miR-34) among five diverse bilaterian animals. Subsequently, we designed a procedure to predict evolutionarily conserved miRNA/target-gene pairs by introducing orthologous gene information. As a result, we extracted 31 orthologous miRNA/target-gene pairs that were conserved among at least four diverse bilaterian animals; the prediction set showed prominent enrichment of orthologous miRNA/target-gene pairs that were verified experimentally. Approximately 84% of the target genes were regulated by three miRNAs (let-7, miR-1, and miR-124) and their function was classified mainly into the following categories: development, muscle formation, cell adhesion, and gene regulation. We used a reporter gene assay to experimentally verify the downregulation of six candidate pairs (out of six tested pairs) in HeLa cells. The application of our new method enables the identification of 31 miRNA/target-gene pairs that were expected to have been regulated from the era of the common bilaterian ancestor. The downregulation of all six candidate pairs suggests that orthologous information contributed to the elucidation of the primordial set of genes that has been regulated by miRNAs; it was also an efficient tool for the elimination of false positives from the predicted candidates. In conclusion, our study identified potentially important miRNA-target pairs that were evolutionarily conserved throughout diverse bilaterian animals and that may provide new insights into early-stage miRNA functions.


Application of GRADE: making evidence-based recommendations about diagnostic tests in clinical practice guidelines.
Accurate diagnosis is a fundamental aspect of appropriate healthcare. However, clinicians need guidance when implementing diagnostic tests given the number of tests available and resource constraints in healthcare. Practitioners of health often feel compelled to implement recommendations in guidelines, including recommendations about the use of diagnostic tests. However, the understanding about diagnostic tests by guideline panels and the methodology for developing recommendations is far from completely explored. Therefore, we evaluated the factors that guideline developers and users need to consider for the development of implementable recommendations about diagnostic tests. Using a critical analysis of the process, we present the results of a case study using the Grading of Recommendations Applicability, Development and Evaluation (GRADE) approach to develop a clinical practice guideline for the diagnosis of Cow Milk Allergy with the World Allergy Organization. To ensure that guideline panels can develop informed recommendations about diagnostic tests, it appears that more emphasis needs to be placed on group processes, including question formulation, defining patient-important outcomes for diagnostic tests, and summarizing evidence. Explicit consideration of concepts of diagnosis from evidence-based medicine, such as pre-test probability and treatment threshold, is required to facilitate the work of a guideline panel and to formulate implementable recommendations. This case study provides useful guidance for guideline developers and clinicians about what they ought to demand from clinical practice guidelines to facilitate implementation and strengthen confidence in recommendations about diagnostic tests. Applying a structured framework like the GRADE approach with its requirement for transparency in the description of the evidence and factors that influence recommendations facilitates laying out the process and decision factors that are required for the development, interpretation, and implementation of recommendations about diagnostic tests.


Genome-wide SNP analyses reveal population structure of Portunus pelagicus along Vietnam coastline.
The blue swimming crab (Portunus pelagicus Linnaeus, 1758) is one of the commercially exploited crab fishery resources in Vietnam. This is the first study to provide a broad survey of genetic diversity, population structure and migration patterns of P. pelagicus along the Vietnamese coastline. The crab samples were collected from northern, central and southern Vietnam. Here, we used a panel of single nucleotide polymorphisms (SNPs) generated from restriction site-associated DNA sequencing (RADseq). After removing 32 outlier loci, 306 putatively neutral SNPs from 96 individuals were used to assess fine-scale population structure of blue swimming crab. The mean observed heterozygosity (Ho) and expected heterozygosity (He) per locus was 0.196 and 0.223, respectively. Pairwise Fst and hierarchical AMOVA supported significant differentiation of central and northern from southern populations (P<0.01). Population structure analyses revealed that P. pelagicus in the south is a separate fisheries unit from the north and center. Contemporary migration patterns supported high migration between northern and central populations and restricted genetic exchange within the southern population. In contrast, historic gene flow provides strong evidence for single panmictic population. The results are useful for understanding current status of P. pelagicus in the wild under an environment changing due to natural and anthropogenic stresses, with implications for fisheries management.