Non-suicidal self-injury and suicidal behaviour in children and adolescents accessing residential or intensive home-based mental health services.
There is a dearth of Canadian research with clinical samples of youth who self-harm, and no studies could be located on self-harm in children and youth accessing residential or intensive home-based treatment. The purposes of this report were to explore the proportion and characteristics of children and youth identified as self-harming at admission by clinicians compared to youth not identified as self-harming, compare self-harming children to adolescents, and to compare caregiver ratings of self-harm at intake to clinician ratings at admission. This report was developed from a larger longitudinal, observational study involving 210 children and youth accessing residential and home-based treatment and their caregivers in partnership with five mental health treatment centres in southwestern Ontario. Agency data were gleaned from files, and caregivers reported on symptom severity at 12 to 18 months and 36 to 40 months post-discharge. Fifty-seven (34%) children and youth were identified as self-harming at admission. The mean age was 11.57 (SD 2.75). There were statistically significant differences on symptom severity at intake between those identified as self-harming and those not so identified; most of these differences were no longer present at follow up. Children were reported to have higher severity of conduct disorder symptoms than adolescents at intake, and there was some consistency between caregiver-rated and clinician-rated self-harm. Children were reported to engage in a wide range of self-harming behaviours. These findings suggest that youth who were identified as self-harming at admission have elevated scores of symptom severity, self-harm can occur in young children and while many improve, there remains a concern for several children and youth who did not improve by the end of service. Children engage in some of the same types of self-harm behaviours as adolescents, and they also engage in behaviours unique to children.


Lymphocytic colitis: a clue to bacterial etiology.
To find out the role of bacteria as a possible etiological factor in lymphocytic colitis. Twenty patients with histopathological diagnosis of lymphocytic colitis and 10 normal controls were included in this study. Colonoscopic biopsies were obtained from three sites (hepatic and splenic flexures and rectosigmoid region). Each biopsy was divided into two parts. A fresh part was incubated on special cultures for bacterial growth. The other part was used for the preparation of histologic tissue sections that were examined for the presence of bacteria with the help of Giemsa stain. Culture of tissue biopsies revealed bacterial growth in 18 out of 20 patients with lymphocytic colitis mostly Escherichia coli (14/18), which was found in all rectosigmoid specimens (14/14), but only in 8/14 and 6/14 of splenic and hepatic flexure specimens respectively. In two of these cases, E coli was associated with proteus. Proteus was found only in one case, Klebsiella in two cases, and Staphylococcus aureus in one case. In the control group, only 2 out of 10 controls showed the growth of E coli in their biopsy cultures. Histopathology showed rod-shaped bacilli in the tissue sections of 12 out of 14 cases with positive E coli in their specimen's culture. None of the controls showed these bacteria in histopathological sections. This preliminary study reports an association between E coli and lymphocytic colitis, based on histological and culture observations. Serotyping and molecular studies are in process to assess the role of E coli in the pathogenesis of lymphocytic colitis.


Repetitive reversible labeling of proteins at polyhistidine sequences for single-molecule imaging in live cells.
Sensitive live-cell fluorescence microscopy and single-molecule imaging are severely limited by rapid photobleaching of fluorescent probes. Herein, we show how to circumvent this problem using a novel, generic labeling strategy. Small nickel-nitrilotriacetate fluorescent probes are reversibly bound to oligohistidine sequences of exposed proteins on cell surfaces, permitting selective observation of the proteins by fluorescence microscopy. Photobleached probes are removed by washing and replaced by new fluorophores, thus enabling repetitive acquisition of single-molecule trajectories on the same cell and allowing variation of experimental conditions between acquisitions. This method offers free choice of fluorophores while being minimally perturbing. The strength of the method is demonstrated by labeling engineered polyhistidine sequences of the serotonin-gated 5-HT(3) receptor on the surface of live mammalian cells. Single-molecule microscopy reveals pronounced heterogeneous mobility patterns of the 5-HT(3) receptor. After activating the receptor with serotonin, the number of immobile receptors increases substantially, which might be important for receptor regulation at synapses.


Management of allergy to penicillins and other beta-lactams.
The Standards of Care Committee of the British Society for Allergy and Clinical Immunology (BSACI) and an expert panel have prepared this guidance for the management of immediate and non-immediate allergic reactions to penicillins and other beta-lactams. The guideline is intended for UK specialists in both adult and paediatric allergy and for other clinicians practising allergy in secondary and tertiary care. The recommendations are evidence based, but where evidence is lacking, the panel reached consensus. During the development of the guideline, all BSACI members were consulted using a Web-based process and all comments carefully considered. Included in the guideline are epidemiology of allergic reactions to beta-lactams, molecular structure, formulations available in the UK and a description of known beta-lactam antigenic determinants. Sections on the value and limitations of clinical history, skin testing and laboratory investigations for both penicillins and cephalosporins are included. Cross-reactivity between penicillins and cephalosporins is discussed in detail. Recommendations on oral provocation and desensitization procedures have been made. Guidance for beta-lactam allergy in children is given in a separate section. An algorithm to help the clinician in the diagnosis of patients with a history of penicillin allergy has also been included.


Direct evidence that apoptosis enhances tumor responses to fractionated radiotherapy.
Currently, the contribution of cellular apoptotic sensitivity to tumor response after radiation therapy remains controversial. To address this issue, the survival of Rat-1 fibroblasts containing a 4-hydroxytamoxifen-regulated c-Myc allele, c-MycER (T. D. Littlewood et al., Nucleic Acids Res., 23: 1686-1690, 1995), after single and fractionated doses of radiation was investigated. This model system allows pharmacological regulation of apoptosis sensitivity in the same cells in vitro and as xenograft tumors derived from these cells in vivo (G. I. Evan et al., Cell, 69: 119-128, 1992; R. M. Alarcon et al., Cancer Res., 56: 4315-4319, 1996). Activating c-MycER in vitro resulted in marked sensitization of Rat-1 fibroblasts to the effects of both single-dose and fractionated irradiation as measured by the induction of apoptosis and clonogenic survival. Overexpression of the antiapoptosis protein Bcl-2 suppressed the induction of apoptosis and increased clonogenic survival in cells with activated c-Myc after single-dose and fractionated radiation. Systemic time-release implant delivery of 4-hydroxytamoxifen to severe combined immunodeficient mice bearing Rat-1-MycER tumors over the course of either single-dose (10 Gy) or fractionated (five fractions of 2 Gy) radiotherapy resulted in prolonged tumor growth delay relative to identical tumors from mice that received placebo implants. Furthermore, tumors derived from Rat-1-MycER cells that overexpressed Bcl-2 exhibited shorter tumor growth delays relative to similarly treated Rat-1-MycER tumors. The length of tumor growth delay after single-dose or fractionated radiotherapy strongly correlated with the extent of radiation-induced apoptosis in the xenograft tumors as measured by terminal deoxynucleotidyl transferase-mediated nick end labeling. These in vivo results provide direct evidence that increasing the sensitivity of tumor cells to die by apoptosis increases the efficacy of fractionated radiotherapy by reducing tumor cell clonogenic survival.


From molecule to market: steroid hormones and financial risk-taking.
Little is known about the role of the endocrine system in financial decision-making. Here, we survey research on steroid hormones and their cognitive effects, and examine potential links to trader performance in the financial markets. Preliminary findings suggest that cortisol codes for risk and testosterone for reward. A key finding of this endocrine research is the different cognitive effects of acute versus chronic exposure to hormones: acutely elevated steroids may optimize performance on a range of tasks; but chronically elevated steroids may promote irrational risk-reward choices. We present a hypothesis suggesting that the irrational exuberance and pessimism observed during market bubbles and crashes may be mediated by steroid hormones. If hormones can exaggerate market moves, then perhaps the age and sex composition among traders and asset managers may affect the level of instability witnessed in the financial markets.


Perioperative and delayed complications associated with the surgical treatment of cervical spondylotic myelopathy based on 302 patients from the AOSpine North America Cervical Spondylotic Myelopathy Study.
Rates of complications associated with the surgical treatment of cervical spondylotic myelopathy (CSM) are not clear. Appreciating these risks is important for patient counseling and quality improvement. The authors sought to assess the rates of and risk factors associated with perioperative and delayed complications associated with the surgical treatment of CSM. Data from the AOSpine North America Cervical Spondylotic Myelopathy Study, a prospective, multicenter study, were analyzed. Outcomes data, including adverse events, were collected in a standardized manner and externally monitored. Rates of perioperative complications (within 30 days of surgery) and delayed complications (31 days to 2 years following surgery) were tabulated and stratified based on clinical factors. The study enrolled 302 patients (mean age 57 years, range 29-86) years. Of 332 reported adverse events, 73 were classified as perioperative complications (25 major and 48 minor) in 47 patients (overall perioperative complication rate of 15.6%). The most common perioperative complications included minor cardiopulmonary events (3.0%), dysphagia (3.0%), and superficial wound infection (2.3%). Perioperative worsening of myelopathy was reported in 4 patients (1.3%). Based on 275 patients who completed 2 years of follow-up, there were 14 delayed complications (8 minor, 6 major) in 12 patients, for an overall delayed complication rate of 4.4%. Of patients treated with anterior-only (n = 176), posterior-only (n = 107), and combined anterior-posterior (n = 19) procedures, 11%, 19%, and 37%, respectively, had 1 or more perioperative complications. Compared with anterior-only approaches, posterior-only approaches had a higher rate of wound infection (0.6% vs 4.7%, p = 0.030). Dysphagia was more common with combined anterior-posterior procedures (21.1%) compared with anterior-only procedures (2.3%) or posterior-only procedures (0.9%) (p < 0.001). The incidence of C-5 radiculopathy was not associated with the surgical approach (p = 0.8). The occurrence of perioperative complications was associated with increased age (p = 0.006), combined anterior-posterior procedures (p = 0.016), increased operative time (p = 0.009), and increased operative blood loss (p = 0.005), but it was not associated with comorbidity score, body mass index, modified Japanese Orthopaedic Association score, smoking status, anterior-only versus posterior-only approach, or specific procedures. Multivariate analysis of factors associated with minor or major complications identified age (OR 1.029, 95% CI 1.002-1.057, p = 0.035) and operative time (OR 1.005, 95% CI 1.002-1.008, p = 0.001). Multivariate analysis of factors associated with major complications identified age (OR 1.054, 95% CI 1.015-1.094, p = 0.006) and combined anterior-posterior procedures (OR 5.297, 95% CI 1.626-17.256, p = 0.006). For the surgical treatment of CSM, the vast majority of complications were treatable and without long-term impact. Multivariate factors associated with an increased risk of complications include greater age, increased operative time, and use of combined anterior-posterior procedures.


Vascular imaging of solid tumors in rats with a radioactive arsenic-labeled antibody that binds exposed phosphatidylserine.
We recently reported that anionic phospholipids, principally phosphatidylserine, become exposed on the external surface of vascular endothelial cells in tumors, probably in response to oxidative stresses present in the tumor microenvironment. In the present study, we tested the hypothesis that a chimeric monoclonal antibody that binds phosphatidylserine could be labeled with radioactive arsenic isotopes and used for molecular imaging of solid tumors in rats. Bavituximab was labeled with (74)As (beta(+), T(1/2) 17.8 days) or (77)As (beta(-), T(1/2) 1.6 days) using a novel procedure. The radionuclides of arsenic were selected because their long half-lives are consistent with the long biological half lives of antibodies in vivo and because their chemistry permits stable attachment to antibodies. The radiolabeled antibodies were tested for the ability to image subcutaneous Dunning prostate R3227-AT1 tumors in rats. Clear images of the tumors were obtained using planar gamma-scintigraphy and positron emission tomography. Biodistribution studies confirmed the specific localization of bavituximab to the tumors. The tumor-to-liver ratio 72 h after injection was 22 for bavituximab compared with 1.5 for an isotype-matched control chimeric antibody of irrelevant specificity. Immunohistochemical studies showed that the bavituximab was labeling the tumor vascular endothelium. These results show that radioarsenic-labeled bavituximab has potential as a new tool for imaging the vasculature of solid tumors.


Long-term progression and therapeutic response of visceral metastatic disease non-invasively monitored in mouse urine using beta-human choriogonadotropin secreting tumor cell lines.
Historically, the use of mouse models of metastatic disease to evaluate anticancer therapies has been hampered because of difficulties in detection and quantification of such lesions without sacrificing the mice, which in turn may also be dictated by institutional or ethical guidelines. Advancements in imaging technologies have begun to change this situation. A new method to non-invasively measure tumor burden, as yet untested to monitor spontaneous metastases, is the use of transplanted tumors expressing secretable human beta-chorionic gonadotropin (beta-hCG) that can be measured in urine. We describe examples of beta-hCG-transfected tumor cell lines for evaluating the effect of different therapies on metastatic disease, which in some cases involved monitoring tumor growth for >100 days. We used beta-hCG-tagged mouse B16 melanoma and erbB-2/Her-2-expressing human breast cancer MDA-MB-231 models, and drug treatments included metronomic low-dose cyclophosphamide chemotherapy with or without a vascular endothelial growth factor receptor 2-targeting antibody (DC101) or trastuzumab, the erbB-2/Her-2-targeting antibody. Both experimental and spontaneous metastasis models were studied; in the latter case, an increase in urine beta-hCG always foreshadowed the development of lung, liver, brain, and kidney metastases. Metastatic disease was unresponsive to DC101 or trastuzumab monotherapy treatment, as assessed by beta-hCG levels. Our results also suggest that beta-hCG levels may be set as an end point for metastasis studies, circumventing guidelines, which have often hampered the use of advanced disease models. Collectively, our data indicates that beta-hCG is an effective noninvasive preclinical marker for the long term monitoring of untreated or treated metastatic disease.


The quest for epigenetic regulation underlying unisexual flower development in Cucumis melo.
Melon (Cucumis melo) is an important vegetable crop from the Cucurbitaceae family and a reference model specie for sex determination, fruit ripening and vascular fluxes studies. Nevertheless, the nature and role of its epigenome in gene expression regulation and more specifically in sex determination remains largely unknown. We have investigated genome wide H3K27me3 and H3K9ac histone modifications and gene expression dynamics, in five melon organs. H3K9ac and H3K27me3 were mainly distributed along gene-rich regions and constrained to gene bodies. H3K9ac was preferentially located at the TSS, whereas H3K27me3 distributed uniformly from TSS to TES. As observed in other species, H3K9ac and H3K27me3 correlated with high and low gene expression levels, respectively. Comparative analyses of unisexual flowers pointed out sex-specific epigenetic states of TFs involved in ethylene response and flower development. Chip-qPCR analysis of laser dissected carpel and stamina primordia, revealed sex-specific histone modification of MADS-box genes. Using sex transition mutants, we demonstrated that the female promoting gene, CmACS11, represses the expression of the male promoting gene CmWIP1 via deposition of H3K27me3. Our findings reveal the organ-specific landscapes of H3K9ac and H3K27me3 in melon. Our results also provide evidence that the sex determination genes recruit histone modifiers to orchestrate unisexual flower development in monoecious species.


Right hemisphere language functions and schizophrenia: the forgotten hemisphere?
This review highlights the importance of right hemisphere language functions for successful social communication and advances the hypothesis that the core deficit in psychosis is a failure of segregation of right from left hemisphere functions. Lesion studies of stroke patients and dichotic listening and functional imaging studies of healthy people have shown that some language functions are mediated by the right hemisphere rather than the left. These functions include discourse planning/comprehension, understanding humour, sarcasm, metaphors and indirect requests, and the generation/comprehension of emotional prosody. Behavioural evidence indicates that patients with typical schizophrenic illnesses perform poorly on tests of these functions, and aspects of these functions are disturbed in schizo-affective and affective psychoses. The higher order language functions mediated by the right hemisphere are essential to an accurate understanding of someone's communicative intent, and the deficits displayed by patients with schizophrenia may make a significant contribution to their social interaction deficits. We outline a bi-hemispheric theory of the neural basis of language that emphasizes the role of the sapiens-specific cerebral torque in determining the four-chambered nature of the human brain in relation to the origins of language and the symptoms of schizophrenia. Future studies of abnormal lateralization of left hemisphere language functions need to take account of the consequences of a failure of lateralization of language functions to the right as well as the left hemisphere.


Generation of 12-fs pulses from a diode-pumped Kerr-lens mode-locked Cr:LiSAF laser.
We demonstrate 12-fs-pulse generation from a diode-pumped Kerr-lens mode-locked Cr:LiSAF laser. This is to our knowledge the shortest pulse ever produced from a mode-locked Cr:LiSAF laser. Based on an improved estimation of the intracavity dispersions, we utilized BK7 glass for the Brewster-prism-pair material.


Haloarchaeal diversity in 23, 121 and 419 MYA salts.
DNA was extracted from surface-sterilized salt of different geological ages (23, 121, 419 million years of age, MYA) to investigate haloarchaeal diversity. Only Haloarcula and Halorubrum DNA was found in 23 MYA salt. Older crystals contained unclassified groups and Halobacterium. The older crystals yielded a unique 55-bp insert within the 16S rRNA V2 region. The secondary structure of the V2 region completely differed from that in haloarchaea of modern environments. The DNA demonstrates that unknown haloarchaea and the Halobacterium were key components in ancient hypersaline environments. Halorubrum and Haloarcula appear to be a dominant group in relatively modern hypersaline habitats.


Inferential optimization for simultaneous fitting of multiple components into a CryoEM map of their assembly.
Models of macromolecular assemblies are essential for a mechanistic description of cellular processes. Such models are increasingly obtained by fitting atomic-resolution structures of components into a density map of the whole assembly. Yet, current density-fitting techniques are frequently insufficient for an unambiguous determination of the positions and orientations of all components. Here, we describe MultiFit, a method used for simultaneously fitting atomic structures of components into their assembly density map at resolutions as low as 25 A. The component positions and orientations are optimized with respect to a scoring function that includes the quality-of-fit of components in the map, the protrusion of components from the map envelope, and the shape complementarity between pairs of components. The scoring function is optimized by our exact inference optimizer DOMINO (Discrete Optimization of Multiple INteracting Objects) that efficiently finds the global minimum in a discrete sampling space. MultiFit was benchmarked on seven assemblies of known structure, consisting of up to seven proteins each. The input atomic structures of the components were obtained from the Protein Data Bank, as well as by comparative modeling based on a 16-99% sequence identity to a template structure. A near-native configuration was usually found as the top-scoring model. Therefore, MultiFit can provide initial configurations for further refinement of many multicomponent assembly structures described by electron microscopy.


Scotopic vision deficits in young monkeys exposed to lead.
Rhesus monkeys were reared on diets designed to produce blood lead concentrations of 14 (untreated), 55, or 85 micrograms per 100 milliliters for the first year of life. Eighteen months later, blood lead levels were normal in all animals. At this time, however, visual discrimination performance in the 85-microgram group was impaired under dim light relative both to their own performance under bright light and to the performance of the other groups under all light levels used. We interpret these results to reflect a deleterious, enduring impairment of scotopic visual function (night blindness) as a result of early lead intoxication.


Genome sequencing and analysis of Yersina pestis KIM D27, an avirulent strain exempt from select agent regulation.
Yersinia pestis is the causative agent of the plague. Y. pestis KIM 10+ strain was passaged and selected for loss of the 102 kb pgm locus, resulting in an attenuated strain, KIM D27. In this study, whole genome sequencing was performed on KIM D27 in order to identify any additional differences. Initial assemblies of 454 data were highly fragmented, and various bioinformatic tools detected between 15 and 465 SNPs and INDELs when comparing both strains, the vast majority associated with A or T homopolymer sequences. Consequently, Illumina sequencing was performed to improve the quality of the assembly. Hybrid sequence assemblies were performed and a total of 56 validated SNP/INDELs and 5 repeat differences were identified in the D27 strain relative to published KIM 10+ sequence. However, further analysis showed that 55 of these SNP/INDELs and 3 repeats were errors in the KIM 10+ reference sequence. We conclude that both 454 and Illumina sequencing were required to obtain the most accurate and rapid sequence results for Y. pestis KIMD27. SNP and INDELS calls were most accurate when both Newbler and CLC Genomics Workbench were employed. For purposes of obtaining high quality genome sequence differences between strains, any identified differences should be verified in both the new and reference genomes.


Empirically derived major dietary patterns and their associations with overweight in Korean preschool children.
Prevailing dietary patterns and their association with nutritional outcomes are poorly understood, particularly for children in Korea. Our purposes were to identify major dietary patterns and to examine their associations with overweight among young children in Korea. For 1441 preschool children, usual diet was assessed by a FFQ, from which thirty-three food groups were created and entered into a factor analysis. We identified three dietary patterns by relative intake frequency of (1) vegetables, seaweeds, beans, fruits, milk and dairy products (Korean healthy pattern); (2) beef, pork, poultry, fish and fast foods (animal foods pattern); and (3) ice cream, soda, chocolate, cookies and candies (sweets pattern). The Korean healthy pattern was associated with better health status. As compared with the lowest quintile, the multivariate-adjusted OR of the highest quintile for health status inferior or similar to their peers was 0.59 (95 % CI 0.42, 0.84). Likelihood of being overweight was higher among those in the highest quintile (OR 1.77 (95 % CI 1.06, 2.94)) v. the lowest quintile regarding the animal foods pattern. These findings suggest that major dietary patterns are predictors of overweight and health status in Korean preschool children.


Correlation of serum cholylglycine level with hepatic dysfunction in children with sickle cell anemia.
Hepatic dysfunction occurs commonly in children with sickle cell disease (SCD). Although the etiology is multifactorial, cholestasis is a prominent feature. Serum cholylglycine (CG) has been found to be a very sensitive indicator of cholestasis. Our objective was to determine whether CG levels are elevated in children with SCD and whether they are predictive of hepatic dysfunction. Blood samples were obtained from 97 children with SCD. Liver function tests were done and serum CG concentrations were measured. Patients were followed up for 2 years. Thirty-eight percent of the patients had an elevated CG level. During the 2 years of follow-up, 16% of the children with a previously elevated CG level developed abnormal liver function test results or required a cholecystectomy as compared with 13% with a previously normal CG level (p = 0.92). We conclude that although CG level was elevated in 38% of the patients with SCD, it did not appear to predict liver dysfunction during the ensuring 2 years.


Marked Involvement of the Striatal Efferent System in TAR DNA-Binding Protein 43 kDa-Related Frontotemporal Lobar Degeneration and Amyotrophic Lateral Sclerosis.
Recent pathological studies indicate that neuronal loss and/or TAR DNA-binding protein-43 kDa (TDP-43) inclusions are frequent in the striatum of patients with TDP-43-related frontotemporal lobar degeneration (FTLD-TDP) and amyotrophic lateral sclerosis (ALS-TDP). However, no investigations have clarified the impact of such pathological changes on striatal neuronal outputs in these diseases. We analyzed pathological changes in the striatal efferent system of 59 consecutively autopsied patients with sporadic FTLD-TDP or ALS-TDP. The axon terminals of striatal efferent neurons were immunohistochemically assessed in the substantia nigra pars reticulata (SNr) and globus pallidus (GP). All of the FTLD-TDP patients exhibited a marked depletion of axon terminals, irrespective of disease duration. In particular, losses of substance-P-positive projections to the SNr and internal segment of GP were consistently severe. Similar findings were also observed in 69.0% of the ALS-TDP patients, although the severity was much less than that in the FTLD-TDP patients (p < 0.001). The accumulation of phosphorylated TDP-43 was observed in the striatal efferent neurons, efferent tracts, or their axon terminals in the SNr and GP in both groups. Thus, striatal efferent projections are essentially and commonly involved in the TDP-43-related FTLD/ALS disease spectrum.


In vitro characterization of genetically expressed absorbing proteins using photoacoustic spectroscopy.
Genetically expressed fluorescent proteins have been shown to provide photoacoustic contrast. However, they can be limited by low photoacoustic generation efficiency and low optical absorption at red and near infrared wavelengths, thus limiting their usefulness in mammalian small animal models. In addition, many fluorescent proteins exhibit low photostability due to photobleaching and transient absorption effects. In this study, we explore these issues by synthesizing and characterizing a range of commonly used fluorescent proteins (dsRed, mCherry, mNeptune, mRaspberry, AQ143, E2 Crimson) and novel non-fluorescent chromoproteins (aeCP597 and cjBlue and a non-fluorescent mutant of E2 Crimson). The photoacoustic spectra, photoacoustic generation efficiency and photostability of each fluorescent protein and chromoprotein were measured. Compared to the fluorescent proteins, the chromoproteins were found to exhibit higher photoacoustic generation efficiency due to the absence of radiative relaxation and ground state depopulation, and significantly higher photostability. The feasibility of converting an existing fluorescent protein into a non-fluorescent chromoprotein via mutagenesis was also demonstrated. The chromoprotein mutant exhibited greater photoacoustic signal generation efficiency and better agreement between the photoacoustic and the specific extinction coefficient spectra than the original fluorescent protein. Lastly, the genetic expression of a chromoprotein in mammalian cells was demonstrated. This study suggests that chromoproteins may have potential for providing genetically encoded photoacoustic contrast.


Changes in HbA1c and weight, and treatment persistence, over the 18 months following initiation of second-line therapy in patients with type 2 diabetes: results from the United Kingdom Clinical Practice Research Datalink.
Intensification of metformin monotherapy with additional glucose-lowering drugs is often required in patients with type 2 diabetes (T2D). This study evaluated changes in HbA1c and weight, as well as treatment persistence, associated with different second-line therapies used in UK clinical practice. The UK Clinical Practice Research Datalink was used to identify patients with T2D who initiated second-line therapy after metformin monotherapy between 1 August 2013 and 14 June 2016. Treatment persistence and changes in HbA1c and weight were assessed at 6-month intervals up to 18 months. In total, 9097 patients (mean age 61.2 years, 57.2% men, mean [standard deviation] HbA1c 9.0% [1.8]/ 75 mmol/mol [19.7]) were included in the analysis, with a median 2.3 years between initiating metformin monotherapy and initiating second-line therapy. Patients were stratified according to second-line therapy: metformin in combination with sulfonylurea (SU; n = 4655 [51.2%]), a dipeptidyl peptidase-4 inhibitor (DPP-4 inhibitor; n = 2899 [31.9%]), or a sodium-glucose cotransporter-2 inhibitor (SGLT-2 inhibitor; n = 441 [4.9%]) or other therapies (all other second-line treatments; n = 1102 [12.1%]). At 18 months, the cumulative proportion of patients changing treatment was lowest for those who received metformin plus an SGLT-2 inhibitor (42.3%), followed by patients on metformin plus SU or metformin plus a DPP-4 inhibitor (46.8%). HbA1c reductions were seen with all second-line therapies, with an overall mean (standard error) reduction of -1.23% (0.05)/-13.4 mmol/mol (0.5). Changes were directly, but not linearly, related to baseline HbA1c and were greater in those with higher HbA1c at baseline. Weight loss from baseline was greatest in patients treated with metformin plus either an SGLT-2 inhibitor (-4.2 kg) or a DPP-4 inhibitor (-1.5 kg). The highest proportion of patients who achieved the composite outcome of HbA1c reduction ≥ 0.5%, body weight loss ≥ 2.0 kg and treatment persistence for 18 months was observed in those receiving metformin plus an SGLT-2 inhibitor (36.5%). In this population-based cohort, all second-line therapies added to metformin monotherapy improved glycaemic control, but the lowest treatment change/discontinuation rate and most sustained weight loss was seen with patients receiving metformin plus an SGLT-2 inhibitor.


Integrated primary care could play a starring role in the Amazon-JP Morgan-Buffet health care enterprise.
Earlier this year, Amazon, J. P. Morgan, and Berkshire Hathaway announced plans to launch a joint health care company with more than a million covered lives. Dr. Atul Gawande, the prominent Boston-based surgeon and health care thought leader, will become the first chief executive officer of this health care venture, with a mission to get the best results at the lowest costs and remain free from profit-making incentives and constraints. In this presidential column, the author explores what this has to do with the mission in the Collaborative Family Health care Association (CFHA) and the promulgation of integrated health care. (PsycINFO Database Record


Intermittent preventive sulfadoxine-pyrimethamine treatment of primigravidae reduces levels of plasma immunoglobulin G, which protects against pregnancy-associated Plasmodium falciparum malaria.
Pregnancy-associated malaria (PAM) is an important cause of maternal and neonatal suffering. It is caused by Plasmodium falciparum capable of inhabiting the placenta through expression of particular variant surface antigens (VSA) with affinity for proteoglycans such as chondroitin sulfate A. Protective immunity to PAM develops following exposure to parasites inhabiting the placenta, and primigravidae are therefore particularly susceptible to PAM. The adverse consequences of PAM in primigravidae are preventable by intermittent preventive treatment (IPTp), where women are given antimalarials at specified intervals during pregnancy, but this may interfere with acquisition of protective PAM immunity. We found that Kenyan primigravidae receiving sulfadoxine-pyrimethamine IPTp had significantly lower levels of immunoglobulin G (IgG) with specificity for the type of parasite-encoded VSA-called VSA(PAM)-that specifically mediate protection against PAM than did women receiving a placebo. VSA(PAM)-specific IgG levels depended on the number of IPTp doses received and were sufficiently low to be of clinical concern among multidose recipients. Our data suggest that IPTp should be extended to women of all parities, in line with current World Health Organization recommendations.


Long-term efficacy of perioperative chemoradiotherapy on esophageal squamous cell carcinoma.
To investigate the role of perioperative chemoradiotherapy (CRT) in the treatment of locally advanced thoracic esophageal squamous cell carcinoma (ESCC). Using preoperative computed tomography (CT)-based staging criteria, 238 patients with ESCC (stage II-III) were enrolled in this prospective study between January 1997 and June 2004. With informed consent, patients were randomized into 3 groups: preoperative CRT (80 cases), postoperative CRT (78 cases) and surgery alone (S) (80 cases). The 1-, 3-, 5- and 10-year survival were followed up. Progression-free survival (PFS) was chosen as the primary endpoint by treatment arm measured from study entry until documented progression of disease or death from any cause. The secondary endpoint was overall survival (OS) determined as the time (in months) between the date of therapy and the date of death. Other objectives were surgical and adjuvant therapy complications. With median follow-up of 45 mo for all the enrolled patients, significant differences in the 1-, 3-, 5-, 10-year OS (91.3%, 63.5%, 43.5%, 24.5% vs 91%, 62.8%, 42.3%, 24.4% vs 87.5%, 51.3%, 33.8%, 12.5%, P = 0.0176) and PFS (89.3%, 61.3%, 37.5%, 18.1% vs 89.1%, 61.1%, 37.2%, 17.8% vs 84.5%, 49.3%, 25.9%, 6.2%, P = 0.0151) were detected among the 3 arms. There were no significant differences in OS and PFS between the preoperative CRT and postoperative CRT arm (P > 0.05). For the patients who had radical resection, significant differences in median PFS (48 mo vs 61 mo vs 39.5 mo, P = 0.0331) and median OS (56.5 mo vs 72 mo vs 41.5 mo, P = 0.0153) were detected among the 3 arms, but there were no significant differences in OS and PFS between the preoperative CRT and postoperative CRT arm (P > 0.05). The local recurrence rates in the preoperative CRT, postoperative CRT group and S group were 11.3%, 14.1% and 35%, respectively (P < 0.05). No significant differences were detected among the 3 groups when comparing complications but tended to be in favor of the postoperative CRT and S groups (P > 0.05). Toxicities of CRT in the preoperative or postoperative CRT arms were mostly moderate, and could be quickly alleviated by adequate therapy. Rational application of preoperative or postoperative CRT can provide a benefit in PFS and OS in patients with locally advanced ESCC.


Hyperinsulinemia-induced vascular smooth muscle cell (VSMC) migration and proliferation is mediated by converging mechanisms of mitochondrial dysfunction and oxidative stress.
Atherosclerosis is one of the major complications of diabetes and involves endothelial dysfunction, matrix alteration, and most importantly migration and proliferation of vascular smooth muscle cells (VSMCs). Although hyperglycemia and hyperinsulinemia are known to contribute to atherosclerosis, little is known about the specific cellular signaling pathways that mediate the detrimental hyperinsulinemic effects in VSMCs. Therefore, we investigated the cellular mechanisms of hyperinsulinemia-induced migration and proliferation of VSMCs. VSMCs were treated with insulin (100 nM) for 6 days and subjected to various physiological and molecular investigations. VSMCs subjected to hyperinsulinemia exhibited increased migration and proliferation, and this is paralleled by oxidative stress [increased NADPH oxidase activity, NADPH oxidase 1 mRNA expression, and reactive oxygen species (ROS) generation], alterations in mitochondrial physiology (membrane depolarization, decreased mitochondrial mass, and increased mitochondrial ROS), changes in mitochondrial biogenesis-related genes (mitofusin 1, mitofusin 2, dynamin-related protein 1, peroxisome proliferator-activated receptor gamma coactivator 1-alpha, peroxisome proliferator-activated receptor gamma coactivator 1-beta, nuclear respiratory factor 1, and uncoupling protein 2), and increased Akt phosphorylation. Diphenyleneiodonium, a known NADPH oxidase inhibitor significantly inhibited migration and proliferation of VSMCs and normalized all the above physiological and molecular perturbations. This study suggests a plausible crosstalk between mitochondrial dysfunction and oxidative stress under hyperinsulinemia and emphasizes counteracting mitochondrial dysfunction and oxidative stress as a novel therapeutic strategy for atherosclerosis.


pH-dependent internalization of muramyl peptides from early endosomes enables Nod1 and Nod2 signaling.
Nod1 and Nod2 are members of the Nod-like receptor family that detect intracellular bacterial peptidoglycan-derived muramyl peptides. The biological effects of muramyl peptides have been described for over three decades, but the mechanism underlying their internalization to the cytosol remains unclear. Using the human epithelial cell line HEK293T as a model system, we demonstrate here that Nod1-activating ligands entered cells through endocytosis, most likely by the clathrin-coated pit pathway, as internalization was dynamin-dependent but not inhibited by methyl-beta-cyclodextrin. In the endocytic pathway, the cytosolic internalization of Nod1 ligands was pH-dependent, occurred prior to the acidification mediated by the vacuolar ATPase, and was optimal at pH ranging from 5.5 to 6. Similarly, the Nod2 ligand MDP was internalized into host cytosol through a similar pathway with optimal pH for internalization ranging from 5.5 to 6.5. Moreover, Nod1-activating muramyl peptides likely required processing by endosomal enzymes, prior to transport into the cytosol, suggesting the existence of a sterically gated endosomal transporter for Nod1 ligands. In support for this, we identified a role for SLC15A4, an oligopeptide transporter expressed in early endosomes, in Nod1-dependent NF-kappaB signaling. Interestingly, SLC15A4 expression was also up-regulated in colonic biopsies from patients with inflammatory bowel disease, a disorder associated with mutations in Nod1 and Nod2. Together, our results shed light on the mechanisms by which muramyl peptides get access to the host cytosol, where they are detected by Nod1 and Nod2, and might have implications for the understanding of human diseases, such as inflammatory bowel disease.


Increased population sampling confirms low genetic divergence among Pteropus (Chiroptera: Pteropodidae) fruit bats of Madagascar and other western Indian Ocean islands.
Fruit bats of the genus Pteropus occur throughout the Austral-Asian region west to islands off the eastern coast of Africa. Recent phylogenetic analyses of Pteropus from the western Indian Ocean found low sequence divergence and poor phylogenetic resolution among several morphologically defined species. We reexamine the phylogenetic relationships of these taxa by using multiple individuals per species. In addition, we estimate population genetic structure in two well-sampled taxa occurring on Madagascar and the Comoro Islands (P. rufus and P. seychellensis comorensis). Despite finding a similar pattern of low sequence divergence among species, increased sampling provides insight into the phylogeographic history of western Indian Ocean Pteropus, uncovering high levels of gene flow within species.


Does increased standardisation in health care mean less responsiveness towards individual patients' expectations? A register-based study in Swedish primary care.
We explore whether standardisation in health care based on evidence on group level and a public health perspective is in conflict with responsiveness towards individual patient's expectations in Swedish primary care. Using regression analysis, we study the association between patient views about providers' responsiveness and indicators reflecting provider's adherence to evidence-based guidelines, controlled for characteristics related to providers, including patient mix and degree of competition facing providers. Data were taken from two Swedish regions in years 2012 and 2013. Patients' views about responsiveness are positively correlated with variables reflecting provider's adherence to evidence-based guidelines regarding treatment of elderly and risk groups, drug reviews and prescription of antibiotics. A high overall illness, private ownership and a high proportion of all visits being with a doctor are positively associated with patient views about responsiveness. The opposite relation was found for a high social deprivation among enrolled individuals and size of practice. There was no systematic variation with respect to the degree of competition facing providers. Results suggest that responsiveness towards individual patient expectations is compatible with increased standardisation in health care. This is encouraging for health care providers as they are challenged to balance increased demands from both patients and payers.


Natural selection and molecular evolution in primate PAX9 gene, a major determinant of tooth development.
Large differences in relation to dental size, number, and morphology among and within modern human populations and between modern humans and other primate species have been observed. Molecular studies have demonstrated that tooth development is under strict genetic control, but, the genetic basis of primate tooth variation remains unknown. The PAX9 gene, which codes for a paired domain-containing transcription factor that plays an essential role in the development of mammal dentition, has been associated with selective tooth agenesis in humans and mice, which mainly involves the posterior teeth. To determine whether this gene is polymorphic in humans, we sequenced approximately 2.1 kb of the entire four-exon region (exons 1, 2, 3 and 4; 1,026 bp) and exon-intron (1.1 kb) boundaries of 86 individuals sampled from Asian, European, and Native American populations. We provided evidence that human PAX9 polymorphisms are limited to exon 3 only and furnished details about the distribution of a mutation there in 350 Polish subjects. To investigate the pattern of selective pressure on exon 3, we sequenced ortholog regions of this exon in four species of New World monkeys and one gorilla. In addition, orthologous sequences of PAX9 available in public databases were also analyzed. Although several differences were identified between humans and other species, our findings support the view that strong purifying selection is acting on PAX9. New World and Old World primate lineages may, however, have different degrees of restriction for changes in this DNA region.


Effects of Cryopreservation on Cell Metabolic Activity and Function of Biofabricated Structures Laden with Osteoblasts.
Biofabrication and maturation of bone constructs is a long-term task that requires a high degree of specialization. This specialization falls onto the hierarchy complexity of the bone tissue that limits the transfer of this technology to the clinic. This work studied the effects of the short-term cryopreservation on biofabricated osteoblast-containing structures, with the final aim to make them steadily available in biobanks. The biological responses studied include the osteoblast post-thawing metabolic activity and the recovery of the osteoblastic function of 3D-bioprinted osteoblastic structures and beta tricalcium phosphate (β-TCP) scaffolds infiltrated with osteoblasts encapsulated in a hydrogel. The obtained structures were cryopreserved at -80 °C for 7 days using dimethyl sulfoxide (DMSO) as cryoprotectant additive. After thawing the structures were cultured up to 14 days. The results revealed fundamental biological aspects for the successful cryopreservation of osteoblast constructs. In summary, immature osteoblasts take longer to recover than mature osteoblasts. The pre-cryopreservation culture period had an important effect on the metabolic activity and function maintain, faster recovering normal values when cryopreserved after longer-term culture (7 days). The use of β-TCP scaffolds further improved the osteoblast survival after cryopreservation, resulting in similar levels of alkaline phosphatase activity in comparison with the non-preserved structures. These results contribute to the understanding of the biology of cryopreserved osteoblast constructs, approaching biofabrication to the clinical practice.


Environmental Predictors of US County Mortality Patterns on a National Basis.
A growing body of evidence has found that mortality rates are positively correlated with social inequalities, air pollution, elevated ambient temperature, availability of medical care and other factors. This study develops a model to predict the mortality rates for different diseases by county across the US. The model is applied to predict changes in mortality caused by changing environmental factors. A total of 3,110 counties in the US, excluding Alaska and Hawaii, were studied. A subset of 519 counties from the 3,110 counties was chosen by using systematic random sampling and these samples were used to validate the model. Step-wise and linear regression analyses were used to estimate the ability of environmental pollutants, socio-economic factors and other factors to explain variations in county-specific mortality rates for cardiovascular diseases, cancers, chronic obstructive pulmonary disease (COPD), all causes combined and lifespan across five population density groups. The estimated models fit adequately for all mortality outcomes for all population density groups and, adequately predicted risks for the 519 validation counties. This study suggests that, at local county levels, average ozone (0.07 ppm) is the most important environmental predictor of mortality. The analysis also illustrates the complex inter-relationships of multiple factors that influence mortality and lifespan, and suggests the need for a better understanding of the pathways through which these factors, mortality, and lifespan are related at the community level.


Somatic rearrangements across cancer reveal classes of samples with distinct patterns of DNA breakage and rearrangement-induced hypermutability.
Whole-genome sequencing using massively parallel sequencing technologies enables accurate detection of somatic rearrangements in cancer. Pinpointing large numbers of rearrangement breakpoints to base-pair resolution allows analysis of rearrangement microhomology and genomic location for every sample. Here we analyze 95 tumor genome sequences from breast, head and neck, colorectal, and prostate carcinomas, and from melanoma, multiple myeloma, and chronic lymphocytic leukemia. We discover three genomic factors that are significantly correlated with the distribution of rearrangements: replication time, transcription rate, and GC content. The correlation is complex, and different patterns are observed between tumor types, within tumor types, and even between different types of rearrangements. Mutations in the APC gene correlate with and, hence, potentially contribute to DNA breakage in late-replicating, low %GC, untranscribed regions of the genome. We show that somatic rearrangements display less microhomology than germline rearrangements, and that breakpoint loci are correlated with local hypermutability with a particular enrichment for transversions.


Comparison of dietary intake of overweight postpartum mothers practicing breastfeeding or formula feeding.
Weight gain in the postpartum period is a risk factor for long-term obesity. Investigations of dietary intake among lactating and nonlactating overweight women might identify nutritional concerns specific to this population. To compare nutrient, meal, and snack intakes, food-group servings and prevalence of dieting among fully breastfeeding (BF), mixed breast and formula feeding (MF), and formula feeding (FF) overweight and obese women. The second aim was to compare nutrient intakes and food-group servings to the Dietary Reference Intake and MyPyramid recommendations, respectively. Data were collected from September 2004 through April 2006 in Durham, NC. Infant feeding practices and dietary information were collected on 450 women between 6 and 9 weeks postpartum. Two 24-hour dietary recalls were completed by phone, using Nutrition Data Systems for Research. Analysis of covariance was used to compare infant feeding groups in dietary quality (nutrient intake per 1,000 kcal) and food-group servings, controlling for prepregnancy body mass index, race, age, education, income, and marital status. χ² analysis was performed to determine differences in meal and snack intake and dieting among infant feeding groups. BF women consumed more energy (2,107 ± 50 kcal) than MF (1,866 ± 56 kcal) or FF (1,657 ± 50 kcal) women (P < 0.001). Adjusted nutrient intake did not differ between groups. All groups were at risk for inadequate intakes of vitamins A, E, C, and folate and did not meet recommended servings of all food groups. BF women consumed lunch and snacks more frequently, were less likely to diet, and reported higher intakes of grains and desserts than MF and FF women. To help increase intakes of nutrients lacking in the diet and prevent postpartum weight gain, overweight women should be encouraged to increase fruits, vegetables, low-fat dairy, whole grains, legumes, and healthy types of fat, while decreasing refined grains, regular soda, sweetened beverages, and desserts.


Bioactive self-assembled peptide nanofibers for corneal stroma regeneration.
Defects in the corneal stroma caused by trauma or diseases such as macular corneal dystrophy and keratoconus can be detrimental for vision. Development of therapeutic methods to enhance corneal regeneration is essential for treatment of these defects. This paper describes a bioactive peptide nanofiber scaffold system for corneal tissue regeneration. These nanofibers are formed by self-assembling peptide amphiphile molecules containing laminin and fibronectin inspired sequences. Human corneal keratocyte cells cultured on laminin-mimetic peptide nanofibers retained their characteristic morphology, and their proliferation was enhanced compared with cells cultured on fibronectin-mimetic nanofibers. When these nanofibers were used for damaged rabbit corneas, laminin-mimetic peptide nanofibers increased keratocyte migration and supported stroma regeneration. These results suggest that laminin-mimetic peptide nanofibers provide a promising injectable, synthetic scaffold system for cornea stroma regeneration.


The evolution of misbelief.
From an evolutionary standpoint, a default presumption is that true beliefs are adaptive and misbeliefs maladaptive. But if humans are biologically engineered to appraise the world accurately and to form true beliefs, how are we to explain the routine exceptions to this rule? How can we account for mistaken beliefs, bizarre delusions, and instances of self-deception? We explore this question in some detail. We begin by articulating a distinction between two general types of misbelief: those resulting from a breakdown in the normal functioning of the belief formation system (e.g., delusions) and those arising in the normal course of that system's operations (e.g., beliefs based on incomplete or inaccurate information). The former are instances of biological dysfunction or pathology, reflecting "culpable" limitations of evolutionary design. Although the latter category includes undesirable (but tolerable) by-products of "forgivably" limited design, our quarry is a contentious subclass of this category: misbeliefs best conceived as design features. Such misbeliefs, unlike occasional lucky falsehoods, would have been systematically adaptive in the evolutionary past. Such misbeliefs, furthermore, would not be reducible to judicious - but doxastically noncommittal - action policies. Finally, such misbeliefs would have been adaptive in themselves, constituting more than mere by-products of adaptively biased misbelief-producing systems. We explore a range of potential candidates for evolved misbelief, and conclude that, of those surveyed, only positive illusions meet our criteria.


Sleep Hygiene and Recovery Strategies in Elite Soccer Players.
In elite soccer, players are frequently exposed to various situations and conditions that can interfere with sleep (e.g., playing night matches interspersed with 3 days; performing activities demanding high levels of concentration close to bedtime; use of products containing caffeine or alcohol in the period preceding bedtime; regular daytime napping throughout the week; variable wake-up times or bedtime), potentially leading to sleep deprivation. We outline simple, practical, and pharmaceutical-free sleep strategies that are coordinated to the constraints of elite soccer in order to promote sleep. Sleep deprivation is best alleviated by sleep extension; however, sleep hygiene strategies (i.e., consistent sleep pattern, appropriate napping, and active daytime behaviors) can be utilized to promote restorative sleep. Light has a profound impact on sleep, and sleep hygiene strategies that support the natural environmental light-dark cycle (i.e., red-light treatment prior to sleep, dawn-simulation therapy prior to waking) and prevent cycle disruption (i.e., filtering short wavelengths prior to sleep) may be beneficial to elite soccer players. Under conditions of inordinate stress, techniques such as brainwave entrainment and meditation are promising sleep-promoting strategies, but future studies are required to ascertain the applicability of these techniques to elite soccer players. Consuming high-electrolyte fluids such as milk, high-glycemic index carbohydrates, some forms of protein immediately prior to sleep, as well as tart cherry juice concentrate and tryptophan may promote rehydration, substrate stores replenishment, muscle-damage repair and/or restorative sleep. The influence of cold water immersion performed close to bedtime on subsequent sleep is still debated. Conversely, the potential detrimental effects of sleeping medication must be recognized. Sleep initiation is influenced by numerous factors, reinforcing the need for future research to identify such factors. Efficient and individualized sleep hygiene strategies may consequently be proposed.


Effects of cytosine modifications on DNA flexibility and nucleosome mechanical stability.
Cytosine can undergo modifications, forming 5-methylcytosine (5-mC) and its oxidized products 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC). Despite their importance as epigenetic markers and as central players in cellular processes, it is not well understood how these modifications influence physical properties of DNA and chromatin. Here we report a comprehensive survey of the effect of cytosine modifications on DNA flexibility. We find that even a single copy of 5-fC increases DNA flexibility markedly. 5-mC reduces and 5-hmC enhances flexibility, and 5-caC does not have a measurable effect. Molecular dynamics simulations show that these modifications promote or dampen structural fluctuations, likely through competing effects of base polarity and steric hindrance, without changing the average structure. The increase in DNA flexibility increases the mechanical stability of the nucleosome and vice versa, suggesting a gene regulation mechanism where cytosine modifications change the accessibility of nucleosomal DNA through their effects on DNA flexibility.


Why is it difficult to wash aphids off from superhydrophobic kale?
Many varieties of the cabbage family have leaves covered with superhydrophobic epicuticular wax, which provides them with self-cleaning characteristics. Since the wax also lowers insect adhesion, rinsing of the leaves with water should be an effective way of removing the insects. Conversely, we report that superhydrophobicity of tuscan kale increases resistance of aphids to hydrodynamic removal. The exterior surface of the insects is also superhydrophobic and acts as an extension of the leaf's surface. As a result even at moderate impact velocities impinging water drops cannot penetrate under the pests. Consequently, liquid impact aids the insect's adhesion by increasing the normal compressive forces they experience. We show that on a hydrophilic arugula leaf this mechanism is absent, and aphids can be easily washed off with water, as it is able to penetrate underneath them. As for removal of aphids from Tuscan kale, we show that lower surface tension liquids, such as oils and soapy water are more effective, because they are able to wet both the plant and insect surfaces. We also show that aerodynamic removal of aphids consisting of simply exposing the invaded leaf to an air flow is most effective.


Influence of practice patterns on outcome among countries enrolled in the SYNTAX trial: 5-year results between percutaneous coronary intervention and coronary artery bypass grafting.
To examine differences among participating countries in baseline characteristics, clinical practice, medication strategies and outcomes of patients randomized to coronary artery bypass grafting and percutaneous coronary intervention in the SYNTAX trial. In SYNTAX, centres in 18 different countries enrolled 1800 patients, of which 8 countries enrolled ≥80 patients, what was projected to be a large enough sample size to be included in the analysis. Baseline characteristics, practice patterns and clinical outcomes were compared between the USA (n = 245), the UK (n = 267), Italy (n = 197), France (n = 208), Germany (n = 179), Netherlands (n = 148), Belgium (n = 91) and Hungary (n = 83). The remaining patients from other participating countries were pooled together (n = 382). Five-year results demonstrated significantly different outcomes between countries. After adjustment, percutaneous coronary intervention patients in France had lower rates of major adverse cardiac and cerebrovascular events [hazard ratio (HR) = 0.60, 95% confidence interval (CI) 0.37-0.98], while the incidence of repeat revascularization was higher in Hungary (HR = 1.89, 95% CI 1.14-3.42). Coronary artery bypass grafting showed the lowest rate of repeat revascularization in the UK (HR = 0.32, 95% CI 0.12-0.85). There were numerous differences in the risk profile of patients between participating countries, as well as marked differences in surgical practice across countries in the use of blood cardioplegia (range 3.1-89.0%; P < 0.001), bilateral internal mammary artery usage (range 7.8-68.2%; P < 0.001) and off-pump procedures (range 3.9-44.4%; P < 0.001). Variation was also found for percutaneous coronary intervention in the number of implanted stents (range 4.0 ± 2.3 to 6.1 ± 2.6; P < 0.001) as well as for the entire stents length (range 69.0 ± 45.1 to 124.1 ± 60.9; P < 0.001). Remarkable differences were observed in the prescription of post-coronary artery bypass grafting medication in terms of acetylsalicylic acid (range 79.6-95.0%; P = 0.004), thienopyridine (6.8-31.1%; P < 0.001) and statins (41.3-89.1%; P < 0.001). Patient characteristics and clinical patterns are significantly different between countries, resulting in significantly different 5-year outcomes. This article presents specific data that can further improve outcomes in each country. NCT00114972.


Adaptive smoothing as inference strategy: more specificity for unequally sized or neighbouring regions.
Although spatial smoothing of fMRI data can serve multiple purposes, increasing the sensitivity of activation detection is probably its greatest benefit. However, this increased detection power comes with a loss of specificity when non-adaptive smoothing (i.e. the standard in most software packages) is used. Simulation studies and analysis of experimental data was performed using the R packages neuRosim and fmri. In these studies, we systematically investigated the effect of spatial smoothing on the power and number of false positives in two particular cases that are often encountered in fMRI research: (1) Single condition activation detection for regions that differ in size, and (2) multiple condition activation detection for neighbouring regions. Our results demonstrate that adaptive smoothing is superior in both cases because less false positives are introduced by the spatial smoothing process compared to standard Gaussian smoothing or FDR inference of unsmoothed data.


Inherited mitochondrial disorders.
Though inherited mitochondrial disorders (MIDs) are most well known for their syndromic forms, for which widely known acronyms (MELAS, MERRF, NARP, LHON etc.) have been coined, the vast majority of inherited MIDs presents in a non-syndromic form. Since MIDs are most frequently multisystem disorders already at onset or during the disease course, a MID should be suspected if there is a combination of neurological and non-neurological abnormalities. Neurological abnormalities occurring as a part of a MID include stroke-like episodes, epilepsy, migraine-like headache, movement disorders, cerebellar ataxia, visual impairment, encephalopathy, cognitive impairment, dementia, psychosis, hypopituitarism, aneurysms, or peripheral nervous system disease, such as myopathy, neuropathy, or neuronopathy. Non-neurological manifestations concern the ears, the endocrine organs, the heart, the gastrointestinal tract, the kidneys, the bone marrow, and the skin. Whenever there is an unexplained combination of neurological and non-neurological disease in a patient or kindred, a MID should be suspected and appropriate diagnostic measures initiated. Genetic testing should be guided by the phenotype, the biopsy findings, and the biochemical results.


Audio computer assisted self interview and face to face interview modes in assessing response bias among STD clinic patients.
Audio computer assisted self interview (ACASI) may minimise social desirability bias in the ascertainment of sensitive behaviours. The aim of this study was to describe the difference in reporting risk behaviour in ACASI compared to a face to face interview (FFI) among public sexually transmitted diseases (STD) clinic attendees. Randomly selected patients attending a public STD clinic in Baltimore, Maryland, sequentially took an ACASI formatted risk behaviour assessment followed by an FFI conducted by a single clinician, with both interview modalities surveying sexual and drug use behaviours. Binary responses were compared using the sign test, and categorical responses were compared using the Wilcoxon signed rank test to account for repeated measures. 671 (52% men, mean age 30 years, 95% African American) of 795 clinic attendees screened consented to participate. Subjects affirmed sensitive sexual behaviours such as same sex contact (p = 0.012), receptive rectal sexual exposure (p < 0.001), orogenital contact (p < 0.001), and a greater number of sex partners in the past month (p < 0.001) more frequently with ACASI than with an FFI. However, there were no differences in participant responses to questions on use of illicit drugs or needle sharing. Among STD clinic patients, reporting of sensitive sexual risk behaviours to clinicians was much more susceptible to social desirability bias than was reporting of illegal drug use behaviours. In STD clinics where screening of sexual risk is an essential component of STD prevention, the use of ACASI may be a more reliable assessment method than traditional FFI.


Chromosome 15q11-13 duplication syndrome brain reveals epigenetic alterations in gene expression not predicted from copy number.
Chromosome 15q11-13 contains a cluster of imprinted genes essential for normal mammalian neurodevelopment. Deficiencies in paternal or maternal 15q11-13 alleles result in Prader-Willi or Angelman syndromes, respectively, and maternal duplications lead to a distinct condition that often includes autism. Overexpression of maternally expressed imprinted genes is predicted to cause 15q11-13-associated autism, but a link between gene dosage and expression has not been experimentally determined in brain. Postmortem brain tissue was obtained from a male with 15q11-13 hexasomy and a female with 15q11-13 tetrasomy. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was used to measure 10 15q11-13 transcripts in maternal 15q11-13 duplication, Prader-Willi syndrome, and control brain samples. Southern blot, bisulfite sequencing and fluorescence in situ hybridisation were used to investigate epigenetic mechanisms of gene regulation. Gene expression and DNA methylation correlated with parental gene dosage in the male 15q11-13 duplication sample with severe cognitive impairment and seizures. Strikingly, the female with autism and milder Prader-Willi-like characteristics demonstrated unexpected deficiencies in the paternally expressed transcripts SNRPN, NDN, HBII85, and HBII52 and unchanged levels of maternally expressed UBE3A compared to controls. Paternal expression abnormalities in the female duplication sample were consistent with elevated DNA methylation of the 15q11-13 imprinting control region (ICR). Expression of non-imprinted 15q11-13 GABA receptor subunit genes was significantly reduced specifically in the female 15q11-13 duplication brain without detectable GABRB3 methylation differences. Our findings suggest that genetic copy number changes combined with additional genetic or environmental influences on epigenetic mechanisms impact outcome and clinical heterogeneity of 15q11-13 duplication syndromes.


Phosphorylation-state-dependent regulation of NMDA receptor short-term plasticity modifies hippocampal dendritic Ca2+ transients.
N-methyl-D-aspartate (NMDA) receptor-mediated currents are enhanced by phosphorylation. We have investigated effects of phosphorylation-dependent short-term plasticity of NMDA receptor-mediated excitatory postsynaptic currents (EPSCs) on the induction of long-term depression (LTD). We confirmed in whole cell clamped CA1 pyramidal neurons that LTD is induced by pairing stimulus protocols. However, after serine-threonine phosphorylation was modified by postsynaptic introduction of a protein phosphatase-1 (PP1) inhibitor, the same pairing protocol evoked long-term potentiation (LTP). We determined effects of modification of phosphatase activity on evoked NMDA EPSCs during LTD induction protocols. During LTD induction, using a protocol pairing depolarization to -40 mV and 0.5 Hz stimulation, NMDA receptor-mediated EPSCs undergo a short-term enhancement at the start of the protocol. In neurons in which PP1 activity was inhibited, this short-term enhancement was markedly amplified. We then investigated the effect of this enhancement on Ca(2+) entry during the start of the LTD induction protocol. Enhancement of NMDA receptor-mediated responses was accompanied by an amplification of induction protocol-evoked Ca(2+) transients. Furthermore, this amplification required synaptic activation during the protocol, consistent with an enhancement of Ca(2+) entry mediated by NMDA receptor activation. The sign of NMDA receptor-mediated long-term plasticity, whether potentiation or depression depends on the amplitude of the synaptic Ca(2+) transient during induction. We conclude that short-term phosphorylation-dependent plasticity of the NMDA receptor-mediated EPSCs contributes significantly to the effect of phosphatase inhibition on the subsequent induction of LTD or LTP.


An overview of worldwide and regional time trends in total mercury levels in human blood and breast milk from 1966 to 2015 and their associations with health effects.
Mercury is a pollutant of global concern. To protect human health and environment from mercury pollution, the Minamata Convention on mercury entered into force in 2017. To support a future effectiveness evaluation of the convention, this study assesses worldwide and regional time trends of total mercury levels in human blood and breast milk across different population sub-groups in the last half-century prior to entry-into-force of the Minamata Convention. This study also provides an overview of the epidemiological literature showing evidence of associations between mercury exposure (in terms of total mercury levels in whole blood, cord blood, and breast milk) and human health. We searched electronic databases to identify articles published prior to June 14, 2017 and reported total mercury levels in any of three biological matrices (whole blood, cord blood, or breast milk) and/or associations with human health. Temporal trends of total mercury levels in the selected biological matrices across different population sub-groups were estimated using a linear fit of the log-transformed data. In parallel, statistical methods were employed to assess any possible effect of sources of inhomogeneity (i.e. study and population characteristics such as age, sex, ethnicity, source of exposure, sampling period, and geographical region) in the collected studies. Furthermore, a summary of significant and relevant associations between mercury exposure and human health conditions in children and adults was prepared. We found significant declines in total mercury levels in whole blood, cord blood, and breast milk between 1966 and 2015. A regional overview of total mercury levels in whole blood, cord blood, and breast milk suggests the highest levels in South America, followed by Africa or Asia whereas the population groups from Europe or North America displayed the lowest levels of total mercury in the selected biological matrices. We observed conclusive consistent associations of mercury exposure with selected health conditions, especially neurodevelopment and neurotoxicity in children and adults. For several other health conditions, reported findings in the collected studies do not support conclusive associations. We also found that several studies demonstrated significant associations between mercury exposure below the USEPA reference level and various health conditions. This study provides a worldwide and regional overview of trends in total mercury levels in human blood and breast milk and associated health risks prior to entry-into-force of the Minamata Convention and calls for further epidemiological investigations from across the globe to fully understand the health implications of mercury exposure.


Comparison of lamivudine plus adefovir therapy versus entecavir with or without adefovir therapy for adefovir-resistant chronic hepatitis B.
Data regarding the management of adefovir (ADV) resistance are still limited. The aim of this study is to investigate treatment outcomes of rescue therapy in ADV-resistant chronic hepatitis B (CHB) patients. CHB patients who began rescue therapy due to documented genotypic resistance mutations to ADV between October 2006 and July 2012 were retrospectively reviewed. Sixty-three patients were included in this study. Most patients had history of lamivudine (LAM) resistance. Treatment response was evaluated at 3-month intervals up to 12 months. The cumulative rate of complete virologic response (CVR) in hepatitis B virus (HBV)-infected patients (HBV DNA<60 IU/mL) was 15.9%, 27.2%, 28.9%, and 31.7% after 3, 6, 9, and 12 months of rescue therapy. Thirty-five patients were treated with a combination of LAM plus ADV (LAM+ADV group) and 28 patients were treated with entecavir (ETV)-based therapy (ETV with or without ADV therapy, ETV±ADV group). The cumulative CVR rate was significantly higher in the ETV±ADV group than in the LAM+ADV group at month 12 (46.4% vs. 20.6%, respectively, P=0.040). Multivariate analysis showed that pretreatment serum HBV DNA levels at <6 log10 IU/mL (hazard ratio: 34.109, P=0.001) and type of rescue therapy (hazard ratio: 4.944, P=0.036) were associated with CVR. Lower baseline HBV DNA level and ETV±ADV therapy were the important predictive factors for CVR in ADV-resistant CHB patients. This study suggests the need of early switching to a rescue therapy such as ETV±ADV at the time of low-level viremia.


Simvastatin Efficiently Lowers Small LDL-IgG Immune Complex Levels: A Therapeutic Quality beyond the Lipid-Lowering Effect.
We investigated a polyethylene glycol non-precipitable low-density lipoprotein (LDL) subfraction targeted by IgG and the influence of statin therapy on plasma levels of these small LDL-IgG-immune complexes (LDL-IgG-IC). LDL-subfractions were isolated from 6 atherosclerotic subjects and 3 healthy individuals utilizing iodixanol density gradient ultracentrifugation. Cholesterol, apoB and malondialdehyde (MDA) levels were determined in each fraction by enzymatic testing, dissociation-enhanced lanthanide fluorescence immunoassay and high-performance liquid chromatography, respectively. The levels of LDL-IgG-IC were quantified densitometrically following lipid electrophoresis, particle size distribution was assessed with dynamic light scattering and size exclusion chromatography. The influence of simvastatin (40 mg/day for three months) on small LDL-IgG-IC levels and their distribution among LDL-subfractions (salt gradient separation) were investigated in 11 patients with confirmed coronary artery disease (CAD). We demonstrate that the investigated LDL-IgG-IC are small particles present in atherosclerotic patients and healthy subjects. In vitro assembly of LDL-IgG-IC resulted in particle density shifts indicating a composition of one single molecule of IgG per LDL particle. Normalization on cholesterol levels revealed MDA values twice as high for LDL-subfractions rich in small LDL-IgG-IC if compared to dominant LDL-subfractions. Reactivity of affinity purified small LDL-IgG-IC to monoclonal antibody OB/04 indicates a high degree of modified apoB and oxidative modification. Simvastatin therapy studied in the CAD patients significantly lowered LDL levels and to an even higher extent, small LDL-IgG-IC levels without affecting their distribution. In conclusion simvastatin lowers levels of small LDL-IgG-IC more effectively than LDL-cholesterol and LDL-apoB levels in atherosclerotic patients. This antiatherogenic effect may additionally contribute to the known beneficial effects of this drug in the treatment of atherosclerosis.


Measuring Mosquito-borne Viral Suitability in Myanmar and Implications for Local Zika Virus Transmission.
In South East Asia, mosquito-borne viruses (MBVs) have long been a cause of high disease burden and significant economic costs. While in some SEA countries the epidemiology of MBVs is spatio-temporally well characterised and understood, in others such as Myanmar our understanding is largely incomplete. Here, we use a simple mathematical approach to estimate a climate-driven suitability index aiming to better characterise the intrinsic, spatio-temporal potential of MBVs in Myanmar. Results show that the timing and amplitude of the natural oscillations of our suitability index are highly informative for the temporal patterns of DENV case counts at the country level, and a mosquito-abundance measure at a city level. When projected at fine spatial scales, the suitability index suggests that the time period of highest MBV transmission potential is between June and October independently of geographical location. Higher potential is nonetheless found along the middle axis of the country and in particular in the southern corridor of international borders with Thailand. This research complements and expands our current understanding of MBV transmission potential in Myanmar, by identifying key spatial heterogeneities and temporal windows of importance for surveillance and control. We discuss our findings in the context of Zika virus given its recent worldwide emergence, public health impact, and current lack of information on its epidemiology and transmission potential in Myanmar. The proposed suitability index here demonstrated is applicable to other regions of the world for which surveillance data is missing, either due to lack of resources or absence of an MBV of interest.


Amyloid-beta modulates the association between neurofilament light chain and brain atrophy in Alzheimer's disease.
Neurofilament light chain (NFL) measurement has been gaining strong support as a clinically useful neuronal injury biomarker for various neurodegenerative conditions. However, in Alzheimer's disease (AD), its reflection on regional neuronal injury in the context of amyloid pathology remains unclear. This study included 83 cognitively normal (CN), 160 mild cognitive impairment (MCI), and 73 AD subjects who were further classified based on amyloid-beta (Aβ) status as positive or negative (Aβ+ vs Aβ-). In addition, 13 rats (5 wild type and 8 McGill-R-Thy1-APP transgenic (Tg)) were examined. In the clinical study, reduced precuneus/posterior cingulate cortex and hippocampal grey matter density were significantly associated with increased NFL concentrations in cerebrospinal fluid (CSF) or plasma in MCI Aβ+ and AD Aβ+. Moreover, AD Aβ+ showed a significant association between the reduced grey matter density in the AD-vulnerable regions and increased NFL concentrations in CSF or plasma. Congruently, Tg rats recapitulated and validated the association between CSF NFL and grey matter density in the parietotemporal cortex, entorhinal cortex, and hippocampus in the presence of amyloid pathology. In conclusion, reduced grey matter density and elevated NFL concentrations in CSF and plasma are associated in AD-vulnerable regions in the presence of amyloid positivity in the AD clinical spectrum and amyloid Tg rat model. These findings further support the NFL as a neuronal injury biomarker in the research framework of AD biomarker classification and for the evaluation of therapeutic efficacy in clinical trials.


The Phenomenology of the Diagnostic Process: A Primary Care-Based Survey.
While dichotomous tasks and related cognitive strategies have been extensively researched in cognitive psychology, little is known about how primary care practitioners (general practitioners [GPs]) approach ill-defined or polychotomous tasks and how valid or useful their strategies are. To investigate cognitive strategies used by GPs for making a diagnosis. In a cross-sectional study, we videotaped 282 consultations, irrespective of presenting complaint or final diagnosis. Reflective interviews were performed with GPs after each consultation. Recordings of consultations and GP interviews were transcribed verbatim and analyzed using a coding system that was based on published literature and systematically checked for reliability. In total, 134 consultations included 163 diagnostic episodes. Inductive foraging (i.e., the initial, patient-guided search) could be identified in 91% of consultations. It contributed an average 31% of cues obtained by the GP in 1 consultation. Triggered routines and descriptive questions occurred in 38% and 84% of consultations, respectively. GPs resorted to hypothesis testing, the hallmark of the hypothetico-deductive method, in only 39% of consultations. Video recordings and interviews presumably interfered with GPs' behavior and accounts. GPs might have pursued more hypotheses and collected more information than usual. The testing of specific disease hypotheses seems to play a lesser role than previously thought. Our data from real consultations suggest that GPs organize their search for information in a skillfully adapted way. Inductive foraging, triggered routines, descriptive questions, and hypotheses testing are essential building blocks to make a diagnosis in the generalist setting.


Targeting TRP channels for novel migraine therapeutics.
Migraine is increasingly understood to be a disorder of the brain. In susceptible individuals, a variety of "triggers" may influence altered central excitability, resulting in the activation and sensitization of trigeminal nociceptive afferents surrounding blood vessels (i.e., the trigeminovascular system), leading to migraine pain. Transient receptor potential (TRP) channels are expressed in a subset of dural afferents, including those containing calcitonin gene related peptide (CGRP). Activation of TRP channels promotes excitation of nociceptive afferent fibers and potentially lead to pain. In addition to pain, allodynia to mechanical and cold stimuli can result from sensitization of both peripheral afferents and of central pain pathways. TRP channels respond to a variety of endogenous conditions including chemical mediators and low pH. These channels can be activated by exogenous stimuli including a wide range of chemical and environmental irritants, some of which have been demonstrated to trigger migraine in humans. Activation of TRP channels can elicit CGRP release, and blocking the effects of CGRP through receptor antagonism or antibody strategies has been demonstrated to be effective in the treatment of migraine. Identification of approaches that can prevent activation of TRP channels provides an additional novel strategy for discovery of migraine therapeutics.


Safety, tolerability, and immunogenicity of a recombinant adenovirus type-5 vectored COVID-19 vaccine: a dose-escalation, open-label, non-randomised, first-in-human trial.
A vaccine to protect against COVID-19 is urgently needed. We aimed to assess the safety, tolerability, and immunogenicity of a recombinant adenovirus type-5 (Ad5) vectored COVID-19 vaccine expressing the spike glycoprotein of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strain. We did a dose-escalation, single-centre, open-label, non-randomised, phase 1 trial of an Ad5 vectored COVID-19 vaccine in Wuhan, China. Healthy adults aged between 18 and 60 years were sequentially enrolled and allocated to one of three dose groups (5 × 1010, 1 × 1011, and 1·5 × 1011 viral particles) to receive an intramuscular injection of vaccine. The primary outcome was adverse events in the 7 days post-vaccination. Safety was assessed over 28 days post-vaccination. Specific antibodies were measured with ELISA, and the neutralising antibody responses induced by vaccination were detected with SARS-CoV-2 virus neutralisation and pseudovirus neutralisation tests. T-cell responses were assessed by enzyme-linked immunospot and flow-cytometry assays. This study is registered with ClinicalTrials.gov, NCT04313127. Between March 16 and March 27, 2020, we screened 195 individuals for eligibility. Of them, 108 participants (51% male, 49% female; mean age 36·3 years) were recruited and received the low dose (n=36), middle dose (n=36), or high dose (n=36) of the vaccine. All enrolled participants were included in the analysis. At least one adverse reaction within the first 7 days after the vaccination was reported in 30 (83%) participants in the low dose group, 30 (83%) participants in the middle dose group, and 27 (75%) participants in the high dose group. The most common injection site adverse reaction was pain, which was reported in 58 (54%) vaccine recipients, and the most commonly reported systematic adverse reactions were fever (50 [46%]), fatigue (47 [44%]), headache (42 [39%]), and muscle pain (18 [17%]. Most adverse reactions that were reported in all dose groups were mild or moderate in severity. No serious adverse event was noted within 28 days post-vaccination. ELISA antibodies and neutralising antibodies increased significantly at day 14, and peaked 28 days post-vaccination. Specific T-cell response peaked at day 14 post-vaccination. The Ad5 vectored COVID-19 vaccine is tolerable and immunogenic at 28 days post-vaccination. Humoral responses against SARS-CoV-2 peaked at day 28 post-vaccination in healthy adults, and rapid specific T-cell responses were noted from day 14 post-vaccination. Our findings suggest that the Ad5 vectored COVID-19 vaccine warrants further investigation. National Key R&D Program of China, National Science and Technology Major Project, and CanSino Biologics.


Assembly of turnip yellow mosaic virus replication complexes: interaction between the proteinase and polymerase domains of the replication proteins.
Turnip yellow mosaic virus (TYMV), a positive-strand RNA virus in the alphavirus-like supergroup, encodes two nonstructural replication proteins (140K and 66K), both of which are required for its RNA genome replication. The 140K protein contains domains indicative of methyltransferase, proteinase, and NTPase/helicase activities, while the 66K protein encompasses the RNA-dependent RNA polymerase domain. Recruitment of the 66K protein to the sites of viral replication, located at the periphery of chloroplasts, is dependent upon the expression of the 140K protein. Using antibodies raised against the 140K and 66K proteins and confocal microscopy, we report the colocalization of the TYMV replication proteins at the periphery of chloroplasts in transfected or infected cells. The replication proteins cofractionated in functional replication complexes or with purified chloroplast envelope membranes prepared from infected plants. Using a two-hybrid system and coimmunoprecipitation experiments, we also provide evidence for a physical interaction of the TYMV replication proteins. In contrast to what has been found for other members of the alphavirus-like supergroup, the interaction domains were mapped to the proteinase domain of the 140K protein and to a large region encompassing the core polymerase domain within the 66K protein. Coexpression and colocalization experiments confirmed that the helicase domain of the 140K protein is unnecessary for the proper recruitment of the 66K protein to the chloroplast envelope, while the proteinase domain appears to be essential for that process. These results support a novel model for the interaction of TYMV replication proteins and suggest that viruses in the alphavirus-like supergroup may have selected different pathways to assemble their replication complexes.


Atherogenic normolipidemia - a new phenomenon in the lipoprotein profile of clinically healthy subjects.
The identification of an atherogenic and a non-atherogenic lipoprotein profile, athero phenotype B vs. non-athero phenotype A, in a group of healthy normolipidemic subjects reveals a new clinical phenomenon in lipoprotein profiles, an atherogenic normolipidemia. Individuals with atherogenic normolipidemia are at increased risk to develop premature atherothrombosis and experience a sudden cardiovascular event. A quantitative analysis of non-atherogenic and atherogenic lipoproteins in plasma in a group of healthy normolipidemic volunteers who had no clinical signs of cardiovascular system impairment was performed. An innovative electrophoresis method on polyacrylamide gel (PAG) (Lipoprint LDL System, USA) was used for the analysis of plasma lipoproteins. With regard to lipids, total cholesterol and triglycerides in plasma were analyzed with an enzymatic method, CHOD PAP (Roche Diagnostics, FRG). Prostacyclin and thromboxane A2 were analyzed with an ELISA analysis (DRG USA). A new parameter, the score for anti-atherogenic risk (SAAR), was calculated as the ratio between non-atherogenic to atherogenic plasma lipoproteins in examined subjects. There was a high concentration of LDL3-7 subfraction (p<0.0001) and a slowly increasing triglyceride concentration (p<0.05) in the atherogenic subgroup. The non-atherogenic subgroup of healthy subjects was characterized by high SAAR scores, as well as a low concentration of LDL3-7 subfractions (p<0.0001). Other statistically significant differences between the atherogenic and non-atherogenic subgroup, including total cholesterol, prostanoid parameters (prostacyklin, thromboxane A2), and lipoproteins values, were not confirmed. The advantages of this new method include: (i) identification of an atherogenic and a nonatherogenic lipoprotein profile in an individual's plasma (ii) identification of an atherogenic normolipidemic lipoprotein profile in plasma (iii) introduction of a new risk measure, the score for anti-atherogenic risk (SAAR), for an estimation of a patient's atherogenic risk of atherothrombosis development. (iv) the presence of small dense LDL in plasma is decisive for declaration of an atherogenic lipoprotein profile. It is valid for hyperlipidemia and for normolipidemia as well.


Distinct, developmentally regulated brain mRNAs direct the synthesis of neurotransmitter transporters.
The Xenopus laevis oocyte expression system was utilized to define developmental and structural properties of neurotransmitter transporter mRNAs and the pharmacological characteristics of encoded carriers independent of the complexities of brain tissue preparations. Poly(A)+ RNA from dissected brain regions of neonatal and adult rats was microinjected into Xenopus oocytes and the expression of Na(+)-dependent neurotransmitter transporters determined 48 h later. Transport studies conducted with oocytes injected with RNAs derived from juvenile rat tissues indicate a region- and transporter-specific, postnatal increase in mRNA abundance as a major factor in the developmental changes observed for brain high-affinity amino acid uptake systems. Both L-glutamic acid (Glu) and gamma-aminobutyric acid (GABA) uptake systems were detectable by day 3 in postnatal forebrain mRNA and became progressively enriched during the next 2 weeks of forebrain development. In contrast, brainstem Glu and GABA transporter enrichment was 60-70% of adult values by day 3 and exceeded adult levels by day 10. Parallel determinations of L-glutamic acid decarboxylase mRNA abundance during development argue for distinct regulatory influences on mRNAs directing transmitter synthesis and reuptake. Glycine uptake could not be detected at any point of forebrain development and exhibited a gradual postnatal rise to adult levels over the first 3 postnatal weeks of brainstem development. Uptake studies conducted with well-characterized inhibitors of Glu, GABA, dopamine, and choline transport (D-aspartate, nipecotic acid, nomifensine, and hemicholinium-3, respectively) revealed that oocyte transporters encoded by adult rat brain mRNAs retained antagonist sensitivities exhibited by in vitro brain preparations. In addition, a differential regional sensitivity to the Glu transport antagonist dihydrokainate (1 mM) was observed, lending support to previous reports of region-specific Glu transporter subtypes. To determine the structural diversity present among brain transporter mRNAs, poly(A)+ RNA was size-fractionated on linear (10-31%) sucrose density gradients prior to oocyte injection. These experiments revealed two mRNA size classes (2.4-3.0 kb, 4.0-4.5 kb) independently capable of directing the synthesis of Glu, GABA, and glycine transporters. In regions other than the cerebellum, Glu and GABA transporter activities migrated as single, yet distinct, peaks of 4.0-4.5 kb. In contrast, both Glu and GABA transporters exhibited major peaks of activity at 2.5-3.0 kb with size-fractionated cerebellar mRNA. Brainstem glycine uptake exhibited a broad sedimentation profile, with peaks apparent at 2.4 and 4.0 kb. Taken together, these findings indicate previously unappreciated complexity in mRNA structure and regulation which underlies the expression of amino acid neurotransmitter uptake systems in the rodent CNS.


α-actinin-4 is essential for maintaining the spreading, motility and contractility of fibroblasts.
α-Actinins cross-link actin filaments, with this cross-linking activity regulating the formation of focal adhesions, intracellular tension, and cell migration. Most non-muscle cells such as fibroblasts express two isoforms, α-actinin-1 (ACTN1) and α-actinin-4 (ACTN4). The high homology between these two isoforms would suggest redundancy of their function, but recent studies have suggested different regulatory roles. Interestingly, ACTN4 is phosphorylated upon growth factor stimulation, and this loosens its interaction with actin. Using molecular, biochemical and cellular techniques, we probed the cellular functions of ACTN4 in fibroblasts. Knockdown of ACTN4 expression in murine lung fibroblasts significantly impaired cell migration, spreading, adhesion, and proliferation. Surprisingly, knockdown of ACTN4 enhanced cellular compaction and contraction force, and increased cellular and nuclear cross-sectional area. These results, except the increased contractility, are consistent with a putative role of ACTN4 in cytokinesis. For the transcellular tension, knockdown of ACTN4 significantly increased the expression of myosin light chain 2, a element of the contractility machinery. Re-expression of wild type human ACTN4 in ACTN4 knockdown murine lung fibroblasts reverted cell spreading, cellular and nuclear cross-sectional area, and contractility back towards baseline, demonstrating that the defect was due to absence of ACTN4. These results suggest that ACTN4 is essential for maintaining normal spreading, motility, cellular and nuclear cross-sectional area, and contractility of murine lung fibroblasts by maintaining the balance between transcellular contractility and cell-substratum adhesion.


On the question of proportionality of the count of observed scrapie cases and the size of holding.
The present paper investigates the question of a suitable basic model for the number of scrapie cases in a holding and applications of this knowledge to the estimation of scrapie-affected holding population sizes and adequacy of control measures within holding. Is the number of scrapie cases proportional to the size of the holding in which case it should be incorporated into the parameter of the error distribution for the scrapie counts? Or, is there a different - potentially more complex - relationship between case count and holding size in which case the information about the size of the holding should be better incorporated as a covariate in the modeling? We show that this question can be appropriately addressed via a simple zero-truncated Poisson model in which the hypothesis of proportionality enters as a special offset-model. Model comparisons can be achieved by means of likelihood ratio testing. The procedure is illustrated by means of surveillance data on classical scrapie in Great Britain. Furthermore, the model with the best fit is used to estimate the size of the scrapie-affected holding population in Great Britain by means of two capture-recapture estimators: the Poisson estimator and the generalized Zelterman estimator. No evidence could be found for the hypothesis of proportionality. In fact, there is some evidence that this relationship follows a curved line which increases for small holdings up to a maximum after which it declines again. Furthermore, it is pointed out how crucial the correct model choice is when applied to capture-recapture estimation on the basis of zero-truncated Poisson models as well as on the basis of the generalized Zelterman estimator. Estimators based on the proportionality model return very different and unreasonable estimates for the population sizes. Our results stress the importance of an adequate modelling approach to the association between holding size and the number of cases of classical scrapie within holding. Reporting artefacts and speculative biological effects are hypothesized as the underlying causes of the observed curved relationship. The lack of adjustment for these artefacts might well render ineffective the current strategies for the control of the disease.