Antibody Responses to SARS-CoV-2 After Infection or Vaccination in Children and Young Adults With Inflammatory Bowel Disease.
Characterization of neutralization antibodies to SARS-CoV-2 infection or vaccination in children and young adults with inflammatory bowel disease (IBD) receiving biologic therapies is crucial. We performed a prospective longitudinal cohort study evaluating SARS-CoV-2 spike protein receptor binding domain (S-RBD) IgG positivity along with consistent clinical symptoms in patients with IBD receiving infliximab or vedolizumab. Serum was also obtained following immunization with approved vaccines. The IgG antibody to the spike protein binding domain of SARS-CoV-2 was assayed with a fluorescent bead-based immunoassay that takes advantage of the high dynamic range of fluorescent molecules using flow cytometry. A sensitive and high-throughput neutralization assay that incorporates SARS-CoV-2 spike protein onto a lentivirus and measures pseudoviral entry into ACE2-angiotensin converting enzyme 2 (ACE2) expressing human embryonic kidney 293 (HEK-293) cells was used. There were 436 patients enrolled (mean age, 17 years, range 2-26 years; 58% male; 71% Crohn's disease, 29% ulcerative colitis, IBD-unspecified). Forty-four (10%) of enrolled subjects had SARS-CoV-2 S-RBD IgG antibodies. Compared to non-IBD adults (ambulatory) and hospitalized pediatric patients with PCR documented SARS-CoV-2 infection, S-RBD IgG antibody levels were significantly lower in the IBD cohort and by 6 months post infection most patients lacked neutralizing antibody. Following vaccination (n = 33), patients had a 15-fold higher S-RBD antibody response in comparison with natural infection, and all developed neutralizing antibodies to both wild type and variant SARS-CoV-2. The lower and less durable SARS-CoV-2 S-RBD IgG response to natural infection in IBD patients receiving biologics puts them at risk of reinfection. The robust response to immunization is likely protective.


Xenosurveillance reflects traditional sampling techniques for the identification of human pathogens: A comparative study in West Africa.
Novel surveillance strategies are needed to detect the rapid and continuous emergence of infectious disease agents. Ideally, new sampling strategies should be simple to implement, technologically uncomplicated, and applicable to areas where emergence events are known to occur. To this end, xenosurveillance is a technique that makes use of blood collected by hematophagous arthropods to monitor and identify vertebrate pathogens. Mosquitoes are largely ubiquitous animals that often exist in sizable populations. As well, many domestic or peridomestic species of mosquitoes will preferentially take blood-meals from humans, making them a unique and largely untapped reservoir to collect human blood. We sought to take advantage of this phenomenon by systematically collecting blood-fed mosquitoes during a field trail in Northern Liberia to determine whether pathogen sequences from blood engorged mosquitoes accurately mirror those obtained directly from humans. Specifically, blood was collected from humans via finger-stick and by aspirating bloodfed mosquitoes from the inside of houses. Shotgun metagenomic sequencing of RNA and DNA derived from these specimens was performed to detect pathogen sequences. Samples obtained from xenosurveillance and from finger-stick blood collection produced a similar number and quality of reads aligning to two human viruses, GB virus C and hepatitis B virus. This study represents the first systematic comparison between xenosurveillance and more traditional sampling methodologies, while also demonstrating the viability of xenosurveillance as a tool to sample human blood for circulating pathogens.


Enzymatically active cathepsin D sensitizes breast carcinoma cells to TRAIL.
Cathepsin D (CD), a ubiquitously expressed lysosomal aspartic protease, is upregulated in human breast carcinoma and many other tumor types. CD has been repeatedly reported to act as key mediator of apoptosis induced by various chemotherapeutics. However, there is still controversy over the role of enzymatic/proteolytic versus protein-protein interaction activities of CD in apoptotic signaling. The elucidation of molecular mechanism responsible for the effect of CD in the chemotherapy-induced cell death is crucial for development of an appropriate strategy to target this protease in cancer treatment. Therefore, the objective of this study was to investigate the molecular mechanism behind the CD-mediated regulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced cell death. For this purpose, MDA-MB-231 breast carcinoma cells with an increased level of wt CD (CD) or mutant enzymatically inactive CD (ΔCD) were subjected to TRAIL and the frequency of apoptosis was determined. Our results show that CD facilitates the TRAIL-induced apoptosis of MDA-MB-231 breast cancer cells in enzymatic activity-dependent manner. Moreover, the importance of endosomal/lysosomal acidification in this process was documented. Analysis of the potential substrates specifically cleaved by CD during the TRAIL-induced apoptosis confirmed caspase-8 and Bid proteins as the CD targets. Moreover, in search for protein regulators of apoptosis that can be cleaved by CD at physiologically relevant pH, we identified the Bcl-2 protein as a suitable candidate. The modulatory role of CD in cell response to TRAIL was also confirmed in another breast cancer cell line SKBR3. These experiments identified the CD enzymatic activity as a new factor affecting sensitivity of breast cancer cells to TRAIL.


Predicting tumor-specific survival in patients with spinal metastatic renal cell carcinoma: which scoring system is most accurate?
Although several prognostic scores for spinal metastatic disease have been developed in the past 2 decades, the applicability and validity of these models to specific cancer types are not yet clear. Most of the data used for model formation are from small population sets and have not been updated or externally validated to assess their performance. Developing predictive models is clinically relevant as prognostic assessment is crucial to optimal decision-making, particularly the decision for or against spine surgery. In this study, the authors investigated the performance of various spinal metastatic disease risk models in predicting prognosis for spine surgery to treat metastatic renal cell carcinoma (RCC). Data of patients who underwent surgery for RCC metastatic to the spine at 2 tertiary centers between 2010 and 2019 were retrospectively retrieved. The authors determined the prognostic value associated with the following scoring systems: the Tomita score, original and revised Tokuhashi scores, original and modified Bauer scores, Katagiri score, the Skeletal Oncology Research Group (SORG) classic algorithm and nomogram, and the New England Spinal Metastasis Score (NESMS). Regression analysis of patient variables in association with 1-year survival after surgery was assessed using Cox proportional hazard models. Calibration and time-dependent discrimination analysis were tested to quantify the accuracy of each scoring system at 3 months, 6 months, and 1 year. A total of 86 metastatic RCC patients were included (median age 64 years [range 29-84 years]; 63 males [73.26%]). The 1-year survival rate was 72%. The 1-year survival group had a good performance status (Karnofsky Performance Scale [KPS] score 80%-100%) and an albumin level > 3.5 g/dL (p < 0.05). Multivariable-adjusted Cox regression analysis showed that poor performance status (KPS score < 70%), neurological deficit (Frankel grade A-D), and hypoalbuminemia (< 3.5 g/dL) were associated with a higher risk of death before 1 year (p < 0.05). The SORG nomogram, SORG classic, original Tokuhashi, and original Bauer demonstrated fair performance (0.7 < area under the curve < 0.8). The NESMS differentiates survival among the prognostic categories with the highest accuracy (area under the curve > 0.8). The present study shows that the most cited and commonly used scoring systems have a fair performance predicting survival for patients undergoing spine surgery for metastatic RCC. The NESMS had the best performance at predicting 1-year survival after surgery.


Regulated dynamic subcellular GLUT4 localization revealed by proximal proteome mapping in human muscle cells.
Regulation of glucose transport, which is central for control of whole-body metabolism, is determined by the amount of GLUT4 glucose transporter (also known as SLC2A4) in the plasma membrane (PM) of fat and muscle cells. Physiologic signals [such as activated insulin receptor or AMP-activated protein kinase (AMPK)] increase PM GLUT4. Here, we show that the distribution of GLUT4 between the PM and interior of human muscle cells is dynamically maintained, and that AMPK promotes PM redistribution of GLUT4 by regulating exocytosis and endocytosis. Stimulation of exocytosis by AMPK is mediated by Rab10 and the Rab GTPase-activating protein TBC1D4. APEX2 proximity mapping reveals that GLUT4 traverses both PM-proximal and PM-distal compartments in unstimulated muscle cells, further supporting retention of GLUT4 by a constitutive retrieval mechanism. AMPK-stimulated translocation involves GLUT4 redistribution among the same compartments traversed in unstimulated cells, with a significant recruitment of GLUT4 from the Golgi and trans-Golgi network compartments. Our comprehensive proximal protein mapping provides an integrated, high-density, whole-cell accounting of the localization of GLUT4 at a resolution of ∼20 nm that serves as a structural framework for understanding the molecular mechanisms regulating GLUT4 trafficking downstream of different signaling inputs in a physiologically relevant cell type.


Reversal of myocyte hypertrophy by ventricular unloading: cardiac improvement without adrenergic receptor up-regulation and relocalization.
In previous studies, we found that the improved contractile ability of cardiac myocytes from patients who have had left ventricular assist device (LVAD) support was due to a number of beneficial changes, most notably in calcium handling (increased sarcoplasmic reticulum calcium binding and uptake), improved integrity of cell membranes due to phospholipid reconstruction (reduced lysophospholipid content), and an upregulation of adrenoreceptors (increased adrenoreceptor numbers). However, in the case presented here, there was no increase in adrenoreceptor number, which is something that we usually find in core tissue at the time of LVAD removal or organ transplantation; also, there was no homogeneous postassist device receptor distribution. However, the patient was well maintained for 10 months following LVAD implantation, until a donor organ was available, regardless of the lack of adrenoreceptor improvement. We conclude from these studies that cardiac recovery is the result of the initiation of multiple repair mechanisms, and that the lack of expected changes, in this case increased adrenoreceptors, is not always an accurate indicator of anticipated outcome. We suggest that interventions and strategies have to consider multiple, beneficial changes due to unloading and target a number of biochemical and structural areas to produce improvement, even if not all of these improvements occur.


A new set of BXD recombinant inbred lines from advanced intercross populations in mice.
Recombinant inbred (RI) strains are an important resource for mapping complex traits in many species. While large RI panels are available for Arabidopsis, maize, C. elegans, and Drosophila, mouse RI panels typically consist of fewer than 30 lines. This is a severe constraint on the power and precision of mapping efforts and greatly hampers analysis of epistatic interactions. In order to address these limitations and to provide the community with a more effective collaborative RI mapping panel we generated new BXD RI strains from two independent advanced intercrosses (AI) between C57BL/6J (B6) and DBA/2J (D2) progenitor strains. Progeny were intercrossed for 9 to 14 generations before initiating inbreeding, which is still ongoing for some strains. Since this AI base population is highly recombinant, the 46 advanced recombinant inbred (ARI) strains incorporate approximately twice as many recombinations as standard RI strains, a fraction of which are inevitably shared by descent. When combined with the existing BXD RI strains, the merged BXD strain set triples the number of previously available unique recombinations and quadruples the total number of recombinations in the BXD background. The combined BXD strain set is the largest mouse RI mapping panel. It is a powerful tool for collaborative analysis of quantitative traits and gene function that will be especially useful to study variation in transcriptome and proteome data sets under multiple environments. Additional strains also extend the value of the extensive phenotypic characterization of the previously available strains. A final advantage of expanding the BXD strain set is that both progenitors have been sequenced, and approximately 1.8 million SNPs have been characterized. This provides unprecedented power in screening candidate genes and can reduce the effective length of QTL intervals. It also makes it possible to reverse standard mapping strategies and to explore downstream effects of known sequence variants.


Ratings of perceived exertion throughout an ultramarathon during carbohydrate ingestion.
Ratings of perceived exertion (RPE) and their relation to selected physiological mediators during endurance exercise have been limited to laboratory settings. The present study characterized the pattern of change in perceptual responses and examined the relation between RPE and selected physiological variables during a long competitive sporting event, i.e., an ultramarathon race (68 km). A single-group design was employed in which all of the 28 subjects provided their perceptual ratings 11.9 +/- 0.2) and heart rate (HR) (138 +/- 3) periodically (every 5 km) throughout the ultramarathon, and selected physiological responses were measured before, once during (32 km), and immediately after the race. Runners drank approximately 1,000 ml of carbohydrate beverage each hour (60 gm carbohydrate hr.(-1)) and ate 2 or 3 carbohydrate gel packs per hour (25 gm each(-1)). RPE increased significantly throughout the course of the ultramarathon. No significant correlations were found between RPE and HR at any time throughout the ultramarathon. RPE averaged 10.4 +/- 0.4 at the beginning of the race (6.4 km) and 15.4 +/- 0.4 at the conclusion of the race. Subjects maintained 76.9 +/- 1.1% of maximal heart rate; however, there was a tendency for heart rate to drop significantly after 32 km. Significant time main effects were found for serum glucose, insulin, and cortisol throughout the race. However, no significant correlations were found between RPE and any of these physiological mediators. These data indicate that during an ultramarathon race there is a progressive increase in RPE without an accompanying increase in HR or decrease in blood glucose. Therefore, during competitive self-paced exercise the perceptual responses may be mediated through other neurological and physiological mechanisms.


HOXD4 and regulation of the group 4 paralog genes.
From an evolutionary perspective, it is important to understand the degree of conservation of cis-regulatory mechanisms between paralogous Hox genes. In this study, we have used transgenic analysis of the human HOXD4 locus to identify one neural and two mesodermal 3' enhancers that are capable of mediating the proper anterior limits of expression in the hindbrain and paraxial mesoderm (somites), respectively. In addition to directing expression in the central nervous system (CNS) up to the correct rhombomere 6/7 boundary in the hindbrain, the neural enhancer also mediates a three rhombomere anterior shift from this boundary in response to retinoic acid (RA), mimicking the endogenous Hoxd4 response. We have extended the transgenic analysis to Hoxa4 identifying mesodermal, neural and retinoid responsive components in the 3' flanking region of that gene, which reflect aspects of endogenous Hoxa4 expression. Comparative analysis of the retinoid responses of Hoxd4, Hoxa4 and Hoxb4 reveals that, while they can be rapidly induced by RA, there is a window of competence for this response, which is different to that of more 3' Hox genes. Mesodermal regulation involves multiple regions with overlapping or related activity and is complex, but with respect to neural regulation and response to RA, Hoxb4 and Hoxd4 appear to be more closely related to each other than Hoxa4. These results illustrate that much of the general positioning of 5' and 3' flanking regulatory regions has been conserved between three of the group 4 paralogs during vertebrate evolution, which most likely reflects the original positioning of regulatory regions in the ancestral Hox complex.


Ttk69-dependent repression of lozenge prevents the ectopic development of R7 cells in the Drosophila larval eye disc.
During the development of the Drosophila eye, specific cell types differentiate from an initially equipotent group of uncommitted precursor cells. The lozenge (lz) gene, which is a member of the Runt family of transcriptional regulators, plays a pivotal role in mediating this process through regulating the expression of several fate-specifying transcription factors. However, the regulation of lz, and the control of lz expression levels in different cell types is not fully understood. Here, we show a genetic interaction between Tramtrack69 (Ttk69) a key transcriptional repressor and an inhibitor of neuronal fate specification, and lz, the master patterning gene of cells posterior to the morphogenetic furrow in the Drosophila eye disc. Loss of Ttk69 expression causes the development of ectopic R7 cells in the third instar eye disc, with these cells being dependent upon Lz for their development. Using the binary UAS Gal4 system, we show that overexpression of Ttk69 causes the loss of lz-dependent differentiating cells, and a down-regulation of Lz expression in the developing eye. The loss of lz-dependent cells can be rescued by overexpressing lz via a GMR-lz transgene. We provide additional data showing that factors functioning upstream of Ttk69 in eye development regulate lz in a Ttk69-dependent manner. Our results lead us to conclude that Ttk69 can either directly or indirectly repress lz gene expression to prevent the premature development of R7 precursor cells in the developing eye of Drosophila. We therefore define a mechanism for the tight regulatory control of the master pre-patterning gene, lz, in early Drosophila eye development and provide insight into how differential levels of lz expression can be achieved to effect specific cell fate outcomes.


Activating mutations in RRAS underlie a phenotype within the RASopathy spectrum and contribute to leukaemogenesis.
RASopathies, a family of disorders characterized by cardiac defects, defective growth, facial dysmorphism, variable cognitive deficits and predisposition to certain malignancies, are caused by constitutional dysregulation of RAS signalling predominantly through the RAF/MEK/ERK (MAPK) cascade. We report on two germline mutations (p.Gly39dup and p.Val55Met) in RRAS, a gene encoding a small monomeric GTPase controlling cell adhesion, spreading and migration, underlying a rare (2 subjects among 504 individuals analysed) and variable phenotype with features partially overlapping Noonan syndrome, the most common RASopathy. We also identified somatic RRAS mutations (p.Gly39dup and p.Gln87Leu) in 2 of 110 cases of non-syndromic juvenile myelomonocytic leukaemia, a childhood myeloproliferative/myelodysplastic disease caused by upregulated RAS signalling, defining an atypical form of this haematological disorder rapidly progressing to acute myeloid leukaemia. Two of the three identified mutations affected known oncogenic hotspots of RAS genes and conferred variably enhanced RRAS function and stimulus-dependent MAPK activation. Expression of an RRAS mutant homolog in Caenorhabditis elegans enhanced RAS signalling and engendered protruding vulva, a phenotype previously linked to the RASopathy-causing SHOC2(S2G) mutant. Overall, these findings provide evidence of a functional link between RRAS and MAPK signalling and reveal an unpredicted role of enhanced RRAS function in human disease.


Local specialization at ileocecal junction of the cat and opossum.
In 10 cats and 10 opossums, serial transverse strips of muscularis propria 2 mm wide, from about 1.5 cm above to about 1.5 cm below the ileocecal mucosal junction, were separately stretched to discover the relationship between length and tension and were separately subjected to electrical field stimulation. In both species, ileal strips showed stimulus relaxations, sometimes accompanied by contractions after the stimulus trains (off responses), whereas colonic strips generally did not. These responses were abolished by tetrodotoxin, 3 times 10-7 M in the opossum, 3 times 10-6 M in the cat. Length-tension slopes were greatest just above the ileocecal junction where stimulus relaxation occurred. Tetrodotoxin at 3 times 10-7 M raised basal tension in ileal strips in both species and in colonic strips only in the cat. Muscle of the ileocecal junction resembles that of the esophagogastric and gastroduodenal junctions in showing a relatively high degree of resistance to stretch and prominent neurogenic stimulus relaxation. However, the ileocecal junction, in contrast, also possesses a tonic inhibitory innervation.


Atherosclerosis and cardiovascular disease in systemic lupus erythematosus: effects of in vivo statin treatment.
Statins may have beneficial vascular effects in systemic lupus erythematosus (SLE) beyond their cholesterol-lowering action, although the mechanisms involved are not completely understood. We investigated potential mechanisms involved in the efficacy of fluvastatin in preventing atherothrombosis in SLE. Eighty-five patients with SLE and 62 healthy donors were included in the study. Selected patients (n=27) received 20 mg/day fluvastatin for 1 month. Blood samples were obtained before the start and at the end of treatment. Monocytes from five patients were treated in vitro with fluvastatin. Increased prothrombotic and inflammatory variables were found in patients with SLE. SLE monocytes displayed altered mitochondrial membrane potential and increased oxidative stress. Correlation and association analyses demonstrated a complex interplay among autoimmunity, oxidative stress, inflammation and increased risk of atherothrombosis in SLE. Fluvastatin treatment of patients for 1 month reduced the SLE Disease Activity Index and lipid levels, oxidative status and vascular inflammation. Array studies on monocytes demonstrated differential expression in 799 genes after fluvastatin treatment. Novel target genes and pathways modulated by fluvastatin were uncovered, including gene networks involved in cholesterol and lipid metabolism, inflammation, oxidative stress and mitochondrial activity. Electron microscopy analysis showed increased density volume of mitochondria in monocytes from fluvastatin-treated patients, who also displayed higher expression of genes involved in mitochondrial biogenesis. In vitro treatment of SLE monocytes confirmed the results obtained in the in vivo study. Our overall data suggest that fluvastatin improves the impairment of a redox-sensitive pathway involved in processes that collectively orchestrate the pathophysiology of atherothrombosis in SLE.


Telehealth for "the digital illiterate"--elderly heart failure patients experiences.
Telehealth solutions should be available also for elderly patients with no interest in using, or capacity to use, computers and smartphones. Fourteen elderly, severely ill heart failure patients in home care participated in a telehealth study and used digital pens for daily reporting of their health state--a technology never used before by this patient group. After the study seven patients and two spouses were interviewed face-to-face. A qualitative content analysis of the interview material was performed. The informants had no experience of computers or the Internet and no interest in learning. Still, patients found the digital pen and the health diary form easy to use, thus effortlessly adopting to changes in care provision. They experienced an improved contact with the caregivers and had a sense of increased security despite a multimorbid state. Our study shows that, given that technologies are tailored to specific patient groups, even "the digital illiterate" may use the Internet.


Assessing neuro-systemic & behavioral components in the pathophysiology of blast-related brain injury.
Among the U.S. military personnel, blast injury is among the leading causes of brain injury. During the past decade, it has become apparent that even blast injury as a form of mild traumatic brain injury (mTBI) may lead to multiple different adverse outcomes, such as neuropsychiatric symptoms and long-term cognitive disability. Blast injury is characterized by blast overpressure, blast duration, and blast impulse. While the blast injuries of a victim close to the explosion will be severe, majority of victims are usually at a distance leading to milder form described as mild blast TBI (mbTBI). A major feature of mbTBI is its complex manifestation occurring in concert at different organ levels involving systemic, cerebral, neuronal, and neuropsychiatric responses; some of which are shared with other forms of brain trauma such as acute brain injury and other neuropsychiatric disorders such as post-traumatic stress disorder. The pathophysiology of blast injury exposure involves complex cascades of chronic psychological stress, autonomic dysfunction, and neuro/systemic inflammation. These factors render blast injury as an arduous challenge in terms of diagnosis and treatment as well as identification of sensitive and specific biomarkers distinguishing mTBI from other non-TBI pathologies and from neuropsychiatric disorders with similar symptoms. This is due to the "distinct" but shared and partially identified biochemical pathways and neuro-histopathological changes that might be linked to behavioral deficits observed. Taken together, this article aims to provide an overview of the current status of the cellular and pathological mechanisms involved in blast overpressure injury and argues for the urgent need to identify potential biomarkers that can hint at the different mechanisms involved.


A Computational Modeling and Simulation Approach to Investigate Mechanisms of Subcellular cAMP Compartmentation.
Subcellular compartmentation of the ubiquitous second messenger cAMP has been widely proposed as a mechanism to explain unique receptor-dependent functional responses. How exactly compartmentation is achieved, however, has remained a mystery for more than 40 years. In this study, we developed computational and mathematical models to represent a subcellular sarcomeric space in a cardiac myocyte with varying detail. We then used these models to predict the contributions of various mechanisms that establish subcellular cAMP microdomains. We used the models to test the hypothesis that phosphodiesterases act as functional barriers to diffusion, creating discrete cAMP signaling domains. We also used the models to predict the effect of a range of experimentally measured diffusion rates on cAMP compartmentation. Finally, we modeled the anatomical structures in a cardiac myocyte diad, to predict the effects of anatomical diffusion barriers on cAMP compartmentation. When we incorporated experimentally informed model parameters to reconstruct an in silico subcellular sarcomeric space with spatially distinct cAMP production sites linked to caveloar domains, the models predict that under realistic conditions phosphodiesterases alone were insufficient to generate significant cAMP gradients. This prediction persisted even when combined with slow cAMP diffusion. When we additionally considered the effects of anatomic barriers to diffusion that are expected in the cardiac myocyte dyadic space, cAMP compartmentation did occur, but only when diffusion was slow. Our model simulations suggest that additional mechanisms likely contribute to cAMP gradients occurring in submicroscopic domains. The difference between the physiological and pathological effects resulting from the production of cAMP may be a function of appropriate compartmentation of cAMP signaling. Therefore, understanding the contribution of factors that are responsible for coordinating the spatial and temporal distribution of cAMP at the subcellular level could be important for developing new strategies for the prevention or treatment of unfavorable responses associated with different disease states.


Branching pattern effect and co-assembly with lipids of amphiphilic Janus dendrimersomes.
The influence of the branching patterns on the membrane properties of Janus dendrimers in water has been investigated by dissipative particle dynamics simulations. The hydrophobic fluorinated dendron (RF) contains three types of branching patterns, including 3,4-, 3,5-, and 3,4,5-RF. Consistent with experimental results, the hydrophobic layer thickness (HB) follows the order: 3,5-RF < 3,4-RF < 3,4,5-RF, which can be explained by the extent of interdigitation (Δh): 3,5-RF > 3,4-RF > 3,4,5-RF. Moreover, the 3,4,5-RF membrane shows the highest stretching modulus (KA) and the lowest lateral diffusivity (D). The 3,5-RF membrane is similar to the 3,4-RF membrane but exhibits a higher KA and smaller D. For the nano-sized dendrimersome, its bilayer thickness is less than that of the planar membrane due to its larger extent of interdigitation. The co-assembly of dendrimersomes with lipids has been studied as well. The thickness and the extent of interdigitation of the lipid-rich domain for the hybrid membrane are significantly affected by the lipid concentration (φl) and the branching patterns. As φl increases, the thickness of the lipid-rich domain grows corresponding to the decrease of interdigitation of the lipid-rich domain.


Hypertension: comparison of drug and non-drug treatments.
Thirty-seven reports of the treatment of hypertension by non-pharmacological means were compared with the results of treatment by standard drug regimens. Treatment by drugs produced the greatest lowering of blood pressure. Treatment by weight reduction, yoga, and muscle relaxation each produced smaller, but appreciable, changes in blood pressure biofeedback, and salt restriction were inferior to those of the other regimens and were not significantly different to the effects of placebo treatment. Large comparative trials of pharmacological and non-pharmacological treatments are needed before definite conclusions can be made.


Factors that influence the use of ambulance in acute coronary syndrome.
National guidelines recommend activation of the emergency medical service by patients who have symptoms of acute coronary syndrome (ACS). In spite of this, only 50% to 60% of persons with myocardial infarction initiate care by using the emergency medical service. The aim of this study was to define factors influencing the use of ambulance in ACS. The method used in this study was a national survey comprising intensive cardiac care units at 11 hospitals in Sweden; 1,939 patients with diagnosed ACS and symptom onset outside the hospital completed a questionnaire a few days after admission. Half of the patients went to the hospital by ambulance. Factors associated with ambulance use were knowledge of the importance of quickly seeking medical care and calling for an ambulance when having chest pain (odds ratio [OR] 3.61, 95% CI 2.43-5.45), abrupt onset of pain reaching maximum intensity within minutes (OR 2.08, 1.62-2.69), nausea or cold sweat (OR 2.02, 1.54-2.65), vertigo or near syncope (OR 1.63, 1.21-2.20), ST-elevation ACS (OR 1.58, 1.21-2.06), increasing age (per year) (OR 1.03, 1.02-1.04), previous history of heart failure (OR 2.48, 1.47-4.26), and distance to the hospital of >5 km (OR 2.0, 1.55-2.59). Those who did not call for an ambulance thought self-transport would be faster or did not believe they were sick enough. Symptoms, patient characteristics, ACS characteristics, and perceptions and knowledge were all associated with ambulance use in ACS. The fact that knowledge increases ambulance use and the need for behavioral change pose a challenge for health-care professionals.


How should we best estimate the mean recency duration for the BED method?
BED estimates of HIV incidence from cross-sectional surveys are obtained by restricting, to fixed time T, the period over which incidence is estimated. The appropriate mean recency duration (Ω(T)) then refers to the time where BED optical density (OD) is less than a pre-set cut-off C, given the patient has been HIV positive for at most time T. Five methods, tested using data for postpartum women in Zimbabwe, provided similar estimates of Ω(T) for C = 0.8: i) The ratio (r/s) of the number of BED-recent infections to all seroconversions over T = 365 days: 192 days [95% CI 168-216]. ii) Linear mixed modeling (LMM): 191 days [95% CI 174-208]. iii) Non-linear mixed modeling (NLMM): 196 days [95% CrI 188-204]. iv) Survival analysis (SA): 192 days [95% CI 168-216]. Graphical analysis: 193 days. NLMM estimates of Ω(T)--based on a biologically more appropriate functional relationship than LMM--resulted in best fits to OD data, the smallest variance in estimates of VT, and best correspondence between BED and follow-up estimates of HIV incidence, for the same subjects over the same time period. SA and NLMM produced very similar estimates of Ω(T) but the coefficient of variation of the former was .3 times as high. The r/s method requires uniformly distributed seroconversion events but is useful if data are available only from a single follow-up. The graphical method produces the most variable results, involves unsound methodology and should not be used to provide estimates of Ω(T). False-recent rates increased as a quadratic function of C: for incidence estimation C should thus be chosen as small as possible, consistent with an adequate resultant number of recent cases, and accurate estimation of Ω(T). Inaccuracies in the estimation of Ω(T) should not now provide an impediment to incidence estimation.


Missed opportunities for earlier treatment? A qualitative interview study with parents of children admitted to hospital with serious respiratory tract infections.
To identify potential opportunities for earlier intervention among children who develop a complicated respiratory tract infection (RTI). Qualitative, face-to-face, individual interview study, either in hospital or at home, with parents of children admitted to hospital with a complicated RTI. Participants were recruited from a large UK teaching hospital, and described events (largely) prior to hospital admission. Parents of 22 children (12 with empyema, 8 with pneumonia, 1 with peritonsillar abscess and 1 with mastoiditis). Parents' accounts revealed missed opportunities for timely treatment resulting from parental and health service associated factors. Themes relating to parental factors included problems assessing the severity of the illness (5 parents), beliefs about accessing health services (10 parents; including fear of appearing 'neurotic', belief that their concerns would not be taken seriously, and belief that their child would not be prescribed antibiotics or would be prescribed antibiotics too readily) and feeling powerless to challenge clinical authority (7 parents). Health service associated factors included perceived problems accessing healthcare services (13 parents; including inadequate primary care triage, barriers to accessing timely consultations and past experience of problems accessing healthcare leading to failure to consult) and perceived poor quality clinical encounters (11 parents; including inadequate assessment and communication). Addressing, where appropriate, these parental (skills, fears and beliefs) and health service (access and consultation quality) associated factors may lead to more prompt care for seriously ill children.


National Institutes of Health State-of-the-Science Conference Statement : Preventing Violence and Related Health-Risking, Social Behaviors in Adolescents, October 13-15, 2004.
NIH consensus and state-of-the-science statements are prepared by independent panels of health professionals and public representatives on the basis of (1) the results of a systematic literature review prepared under contract with the Agency for Healthcare Research and Quality (AHRQ), (2) presentations by investigators working in areas relevant to the conference questions during a 2-day public session, (3) questions and statements from conference attendees during open discussion periods that are part of the public session, and (4) closed deliberations by the panel during the remainder of the second day and morning of the third. This statement is an independent report of the panel and is not a policy statement of the NIH or the Federal Government. The statement reflects the panel's assessment of medical knowledge available at the time the statement was written. Thus, it provides a "snapshot in time" of the state of knowledge on the conference topic. When reading the statement, keep in mind that new knowledge is inevitably accumulating through medical and behavioral research.


Protein structure determination from pseudocontact shifts using ROSETTA.
Paramagnetic metal ions generate pseudocontact shifts (PCSs) in nuclear magnetic resonance spectra that are manifested as easily measurable changes in chemical shifts. Metals can be incorporated into proteins through metal binding tags, and PCS data constitute powerful long-range restraints on the positions of nuclear spins relative to the coordinate system of the magnetic susceptibility anisotropy tensor (Δχ-tensor) of the metal ion. We show that three-dimensional structures of proteins can reliably be determined using PCS data from a single metal binding site combined with backbone chemical shifts. The program PCS-ROSETTA automatically determines the Δχ-tensor and metal position from the PCS data during the structure calculations, without any prior knowledge of the protein structure. The program can determine structures accurately for proteins of up to 150 residues, offering a powerful new approach to protein structure determination that relies exclusively on readily measurable backbone chemical shifts and easily discriminates between correctly and incorrectly folded conformations.


Muscle efficiency and elastic storage in the flight motor of Drosophila.
Insects could minimize the high energetic costs of flight in two ways: by employing high-efficiency muscles and by using elastic elements within the thorax to recover energy expended accelerating the wings. However, because muscle efficiency and elastic storage have proven difficult variables to measure, it is not known which of these strategies is actually used. By comparison of mechanical power measurements based on gas exchange with simultaneously measured flight kinematics in Drosophila, a method was developed for determining both the mechanical efficiency and the minimum degree of elastic storage within the flight motor. Muscle efficiency values of 10 percent suggest that insects may minimize energy use in flight by employing an elastic flight motor rather than by using extraordinarily efficient muscles. Further, because of the trade-off between inertial and aerodynamic power throughout the wing stroke, an elastic storage capacity as low as 10 percent may be enough to minimize the energetic costs of flight.


Two-year outcomes of the ARTISAN-SNM study for the treatment of urinary urgency incontinence using the Axonics rechargeable sacral neuromodulation system.
Sacral neuromodulation (SNM) is a guideline-recommended treatment with proven therapeutic benefit for urinary urgency incontinence (UUI) patients. The Axonics® System is the first Food and Drug Administration-approved rechargeable SNM system and is designed to deliver therapy for a minimum of 15 years. The ARTISAN-SNM study was designed to evaluate UUI participants treated with the Axonics System. Two-year follow-up results are presented. One hundred and twenty-nine UUI participants underwent implantation with the Axonics System. Therapeutic response rate, participant quality of life (QoL), and satisfaction were determined using 3-day voiding diaries, ICIQ-OABqol, and satisfaction questionnaires. Participants were considered responders if they had a 50% or greater reduction in UUI episodes post-treatment. As-treated and Completers analyses are presented. At 2 years, 93% of the participants (n = 121 Completers at 2 years) were therapy responders, of which 82% achieved ≥ 75% reduction in UUI episodes and 37% were dry (100% reduction). Daily UUI episodes reduced from 5.6 ± 0.3 at baseline to 1.0 ± 0.2 at 2 years. Statistically significant improvements in ICIQ-OABqol were reported. All participants were able to recharge their device and 94% of participants reported that the recharging frequency and duration were acceptable. Participant demographics nor condition severity were correlated with clinical outcomes or recharging experience. No unanticipated or serious device-related adverse events occurred. At 2 years, participants treated with the Axonics System demonstrated sustained safety and efficacy, high levels of satisfaction with therapy and recharging. Participant-related factors were not associated with efficacy or recharging outcomes, indicating the reported results are applicable to a diverse population.


Serum insulin-like growth factor-I in 1030 healthy children, adolescents, and adults: relation to age, sex, stage of puberty, testicular size, and body mass index.
Serum levels of insulin-like growth factor-I (IGF-I) increase with age and pubertal development. The large variation in circulating IGF-I levels in adolescence makes it difficult to use the IGF-I value of a single child in the assessment of his growth status. In addition, the interference of IGF-binding proteins in many IGF-I assays contributes to this problem. We measured IGF-I in acid-ethanol-extracted serum from 1030 healthy children, adolescents, and adults, employing a RIA that reduces interference of IGF-binding proteins by using monoiodinated Tyr31-[125I]des-(1-3)IGF-I as radioligand. Mean serum IGF-I concentrations increased slowly in prepubertal children from 80-200 micrograms/L with a further steep increase during puberty to approximately 500 micrograms/L. After puberty, a subsequent continuous fall in circulating IGF-I levels was apparent throughout adulthood to a mean of 100 micrograms/L at the age of 80 yr (P < 0.0001). Girls had maximal IGF-I levels at 14.5 yr of age, whereas boys had peak IGF-I levels 1 yr later. This is almost 2 yr later than average peak height velocity. The large variation in serum IGF-I levels during puberty was diminished when data were separated according to sex and Tanner stage of puberty. Interestingly, we found a significant variation with age within the Tanner stages; there was an increase in serum IGF-I concentrations with age in the early pubertal stages and a decrease in the late stages (P < 0.05). Serum IGF-I increased concomitantly with increasing testicular volume. Multiple regression analysis revealed that serum IGF-I levels predicted height velocity in the following year (r = 0.33; P < 0.0001). Body mass index did not correlate significantly with serum IGF-I in prepubertal children in a multiple regression analysis. In conclusion, there was a significant variation in serum IGF-I levels with age within a given Tanner stage of puberty in addition to the well known increase with increasing age or pubertal stage. Accordingly, the effects of sex, age, and puberty on serum IGF-I cannot be separated into simple additive components when studying 1030 children in a cross-sectional design. Thus, the age-, sex-, and puberty-corrected IGF-I values may, in fact, improve the use of serum IGF-I as a diagnostic tool to distinguish between a child with retarded puberty and a GH-deficient individual.


Isatuximab, Carfilzomib, Lenalidomide, and Dexamethasone for the Treatment of High-Risk Newly Diagnosed Multiple Myeloma.
The GMMG-CONCEPT trial investigated isatuximab, carfilzomib, lenalidomide, and dexamethasone (Isa-KRd) in transplant-eligible (TE) and transplant-noneligible (TNE) patients with newly diagnosed multiple myeloma (NDMM) with exclusively high-risk disease for whom prospective trials are limited, aiming to induce minimal residual disease (MRD) negativity. This academic, investigator-initiated, multicenter, phase II trial enrolled patients with high-risk NDMM (HRNDMM) defined by mandatory International Staging System stage II/III combined with del17p, t(4;14), t(14;16), or more than three 1q21 copies as high-risk cytogenetic aberrations (HRCAs). Patients received Isa-KRd induction/consolidation and Isa-KR maintenance. TE patients received high-dose melphalan. TNE patients received two additional Isa-KRd cycles postinduction. This prespecified interim analysis (IA) reports the primary end point, MRD negativity (<10-5, next-generation flow), at the end of consolidation. The secondary end point was progression-free survival (PFS). Among 125 patients with HRNDMM (TE-intention-to-treat [ITT]-IA, 99; TNE-ITT, 26) of the IA population for the primary end point, the median age was 58 (TE-ITT-IA) and 74 (TNE-ITT) years. Del17p was the most common HRCA (TE, 44.4%; TNE, 42.3%); about one third of evaluable TE/TNE patients presented two or more HRCAs, respectively. The trial met its primary end point with MRD negativity rates after consolidation of 67.7% (TE) and 54.2% (TNE) of patients. Eighty-one of 99 TE-ITT-IA patients reached MRD negativity at any time point (81.8%). MRD negativity was sustained for ≥1 year in 62.6% of patients. With a median follow-up of 44 (TE) and 33 (TNE) months, median PFS was not reached in either arm. Isa-KRd effectively induces high rates of sustainable MRD negativity in the difficult-to-treat HRNDMM population, regardless of transplant status, translating into a median PFS that was not yet reached after 44/33 months.


QALYs: is the value of treatment proportional to the size of the health gain?
In societal priority setting between health programs for different patient groups, many people are reluctant to discriminate too strongly between those who can benefit much from treatment and those who can benefit moderately. We suggest that this view of distributive fairness has a counterpart in personal valuations of gains in health. Such valuations may be influenced by psychological reference points and diminishing marginal utility such that the individual utility of care in patient groups with different potentials may be more similar than what conventional QALY estimates suggest. In interviews in three convenience samples, there is some support for the hypothesis. Most respondents do not think that desire for treatment is significantly less in those who stand to gain only moderately compared with those who stand to gain much - even when the treatment is associated with a mortality risk. When stating insurance preferences, a majority of subjects express a greater concern for avoiding the worst states in question than for maximising expected value for money in terms of treatment effects. The tendency applies to outcomes in terms of both quality and quantity of life. Choices between prefixed response options fit well with oral explanations of these choices.


Neonatal Meningococcal Meningitis In France From 2001 To 2013.
Among 831 cases of neonatal bacterial meningitis occurring from 2001 to 2013, Neisseria meningitidis was the third most frequent bacterial species found. All cases occurred only in term neonates and were mainly late onset. Serogroup B accounted for 78% of cases. At diagnosis, 27% of cases had at least one sign of disease severity. All strains were susceptible to cefotaxime, but 12% showed intermediate susceptibility to penicillin G and to aminopenicillin.


Hospital volume and adverse events following esophageal endoscopic submucosal dissection in Japan.
Background and study aims Esophageal endoscopic submucosal dissection (ESD) has gradually acquired popularity as a minimally invasive surgery for early cancers not only in Japan, but also in other countries. However, most reported outcomes have been based on relatively small samples of patients from specialized centers. Therefore, the association between hospital volume and the rate of adverse events following esophageal ESD has been poorly understood. Patients and methods Using a nationwide administrative database in Japan, we identified patients who underwent esophageal ESD between 1 July 2007 and 31 March 2013. Hospital volume was defined as the number of esophageal ESD procedures performed per year at each hospital and was categorized into quartiles. Results In total, 12 899 esophageal ESD procedures at 699 institutions were identified during the study period. Perforation and perforation-related disorders were observed in 422 patients (3.3 %), and one patient died after perforation. There was a significant association between a lower hospital volume and a higher proportion of adverse events following esophageal ESD. Although not statistically significant, a similar tendency was observed in the occurrence of blood transfusion within 1 week after ESD and all-cause in-hospital death. Multivariable logistic regression analysis showed that hospitals with very high case volumes were less likely to experience adverse events following esophageal ESD than hospitals with very low volumes. Conclusions The proportion of perforation and perforation-related disorders following esophageal ESD was permissibly low, and there was a linear association between higher hospital volume and lower rates of adverse events following esophageal ESD.


The immediate effects of private equity acquisition of urology practices on the management of newly diagnosed prostate cancer.
Some worry that physician practices acquired by private equity may increase the use of services to maximize revenue. We assessed the effects of private equity acquisition on spending, use of treatment, and diagnostic testing in men with prostate cancer. We used a 20% sample of national Medicare claims to perform a retrospective cohort study of men with prostate cancer diagnosed from 2014 through 2019. The primary outcome was prostate cancer spending in the first 12 months after diagnosis. Secondary outcomes included the use of treatment and a composite measure of diagnostic testing (e.g., imaging, genomics) in the first 12 months after diagnosis. Multilevel modeling was used to adjust for differences in patient and market characteristics. The effect of practice acquisition on each outcome was assessed using a difference-in-differences design. There were 409 and 4021 men with prostate cancer managed by urologists in acquired and nonacquired practices, respectively. After acquisition, prostate cancer spending was comparable between acquired and nonacquired practices (difference-in-differences estimate $1182, p = 0.36). Acquisition did not affect the use of treatment (difference-in-differences estimate 3.7%, p = 0.30) or the use of diagnostic testing in men who were treated (difference-in-differences -5.5%, p = 0.12) and those managed conservatively (difference-in-differences -2.0%, p = 0.82). In the year following acquisition of urology practices, private equity did not increase prostate cancer spending, the use of treatment or diagnostic testing in men with prostate cancer. Future work should evaluate the effects of private equity acquisition on practice patterns and quality over a longer time horizon.


The synthesis of oligonucleotides that contain 2,4-dinitrophenyl reporter groups.
The synthesis of non-nucleoside-based phosphoramidites that bear the 2,4-dinitrophenyl group is reported. These labelled phosphoramidites, which have been used in solid-phase oligonucleotide synthesis to attach single and multiple dinitrophenyl groups to the 5'-end of oligonucleotides, are entirely compatible with the normal oligonucleotide synthesis cycle. Multiple labelling can be performed readily in high yield and the resulting oligonucleotides can be purified readily by reversed-phase h.p.l.c. The labelled oligonucleotides have been detected using monoclonal and polyclonal anti-dinitrophenyl antibodies.


Brain Structure Covariance Associated With Gait Control in Aging.
Structural and functional brain imaging methods have identified age-related changes in brain structures involved in gait control. This cross-sectional study aims to investigate gray matter networks associated with gait control in aging using structural covariance analysis. Walking speed were measured in 326 nondemented older community-dwellers (age 71.3 ± 4.5; 41.7% female) under three different walking conditions: normal walking and two challenging tasks: motor (ie, fast speed) and an attention-demanding dual task (ie, backward counting). Three main individual gray matter regions were positively correlated with walking speed (ie, slower walking speed was associated with lower brain volumes): right thalamus, right caudate nucleus, and left middle frontal gyrus for normal walking, rapid walking, and dual-task walking condition, respectively. The structural covariance analysis revealed that prefrontal regions were part of the networks associated with every walking condition; the right caudate was associated specifically with the hippocampus, amygdala and insula for the rapid walking condition, and the left middle frontal gyrus with a network involving the cuneus for the dual-task condition. Our results suggest that brain networks associated with gait control vary according to walking speed and depend on each walking condition. Gait control in aging involved a distributed network including regions for emotional control that are recruited in challenging walking conditions.


Highly sensitive detection of nucleic acids using a cascade amplification strategy based on exonuclease III-assisted target recycling and conjugated polyelectrolytes.
In this paper, a novel ratiometric and cascade amplification strategy was developed by combining the unique signal amplification and effective fluorescence resonance energy transfer (FRET) property of conjugated polymers with the Exo III-assisted target recycling method. The target DNA (ssDNAc) could be hybridized with the duplex-stranded probe to trigger the cyclic digestion of the probe strands and lead to the continuous release of fluorescein from the probe. The proposed strategy thus shows enhanced sensitivity toward target DNA with a detection limit of 0.38 nM, which is more sensitive than the previously reported comparable biosensors based on conjugated polyelectrolytes. Furthermore, this method exhibited an improved performance to discriminate single mismatched targets through an efficient FRET-based ratiometric detection method using a conjugated polymer as a donor and an optical transducer. More importantly, this cascade amplification approach offers the advantages of simplicity, which avoids multiple utilization of probes and complex assay steps required in traditional amplification methods.


TRAF2 Cooperates with Focal Adhesion Signaling to Regulate Cancer Cell Susceptibility to Anoikis.
TRAF2, a RING finger adaptor protein, plays an important function in tumor necrosis factor (TNF)- and TNF-like weak inducer of apoptosis (TWEAK)-dependent signaling, in particular during inflammatory and immune responses. We identified a functional interaction of TRAF2 with focal adhesion (FA) signaling involving the focal adhesion kinase (FAK) in the regulation of cell susceptibility to anoikis. Comparison of TRAF2-proficient (TRAF2+/+) versus TRAF2-deficient (TRAF2-/-), and FAK-proficient (FAK+/+) versus FAK-deficient (FAK-/-) mouse embryonic fibroblasts and their matched reconstituted cells demonstrated that TRAF2 interacts physically with the N-terminal portion of FAK and colocalizes to cell membrane protrusions. This interaction was found to be critical for promoting resistance to cell anoikis. Similar results were confirmed in the human breast cancer cell line MDA-MB-231, where TRAF2 and FAK downregulation promoted cell susceptibility to anoikis. In human breast cancer tissues, genomic analysis of The Cancer Genome Atlas database revealed coamplification of TRAF2 and FAK in breast cancer tissues with a predictive value for shorter survival, further supporting a potential role of TRAF2-FAK cooperative signaling in cancer progression.


Clinicopathological features with outcomes of a series of conventional and proximal-type epithelioid sarcomas, diagnosed over a period of 10 years at a tertiary cancer hospital in India.
Epithelioid sarcoma (ES) is an uncommon sarcoma. Lately, its variants, including proximal-type ES, have been recognized. The present study highlights clinicopathological features of 26 (65%) conventional and 14 (35%) cases of proximal-type ES. Thirty-eight percent of cases were seen in 21-30-year age group, including 77.5% cases in men. Extremities were the commonest sites in both the subtypes. Histologically, conventional-type ES displayed nodular tumor aggregates with necrosis, while proximal-type showed solid arrangement of large, "rhabdoid-like" cells. More cases (64.2%) of the proximal type were of grade 3. A range of differential diagnoses was considered. Most important immunohistochemical markers were vimentin, epithelial membrane antigen, cytokeratin, CD34, and desmin. Maximum (72.5%) cases were treated surgically. Recurrences and metastasis were observed more in the proximal type. The 7-year disease-free survival was 19.4% in the conventional and nil in the proximal subtype (p = 0.06). The overall survival rate was also lower in the proximal (31.3%) than conventional type (90.2%; p < 0.001). Other unfavorable parameters were deeper location, larger size, and higher tumor stage. This unusual sarcoma, with characteristic growth patterns, merits a proper histological evaluation, as it has many mimics. Proximal-type ES is rather a morphological subtype, associated with an aggressive course.


A standardized autopsy procurement allows for the comprehensive study of DIPG biology.
Diffuse intrinsic pontine glioma (DIPG) is one of the least understood and most deadly childhood cancers. Historically, there has been a paucity of DIPG specimens for molecular analysis. However, due to the generous participation of DIPG families in programs for postmortem specimen donation, there has been a recent surge in molecular analysis of newly available tumor specimens. Collaborative efforts to share data and tumor specimens have resulted in rapid discoveries in other pediatric brain tumors, such as medulloblastoma, and therefore have the potential to shed light on the biology of DIPG. Given the generous gift of postmortem tissue donation from DIPG patients, there is a need for standardized postmortem specimen accrual to facilitate rapid and effective multi-institutional molecular studies.We developed and implemented an autopsy protocol for rapid procurement, documenting and storing these specimens. Sixteen autopsies were performed throughout the United States and Canada and processed using a standard protocol and inventory method, including specimen imaging, fixation, snap freezing, orthotopic injection, or preservation. This allowed for comparative clinical and biological studies of rare postmortem DIPG tissue specimens, generation of in vivo and in vitro models of DIPG, and detailed records to facilitate collaborative analysis.


Comparing performance of centerline algorithms for quantitative assessment of brain vascular anatomy.
Attributes like length, diameter, and tortuosity of tubular anatomical structures such as blood vessels in medical images can be measured from centerlines. This study develops methods for comparing the accuracy and stability of centerline algorithms. Sample data included numeric phantoms simulating arteries and clinical human brain artery images. Centerlines were calculated from segmented phantoms and arteries with shortest paths centerline algorithms developed with different cost functions. The cost functions were the inverse modified distance from edge (MDFE(i) ), the center of mass (COM), the binary-thinned (BT)-MDFE(i) , and the BT-COM. The accuracy of the centerline algorithms were measured by the root mean square error from known centerlines of phantoms. The stability of the centerlines was measured by starting the centerline tree from different points and measuring the differences between trees. The accuracy and stability of the centerlines were visualized by overlaying centerlines on vasculature images. The BT-COM cost function centerline was the most stable in numeric phantoms and human brain arteries. The MDFE(i) -based centerline was most accurate in the numeric phantoms. The COM-based centerline correctly handled the "kissing" artery in 16 of 16 arteries in eight subjects whereas the BT-COM was correct in 10 of 16 and MDFE(i) was correct in 6 of 16. The COM-based centerline algorithm was selected for future use based on the ability to handle arteries where the initial binary vessels segmentation exhibits closed loops. The selected COM centerline was found to measure numerical phantoms to within 2% of the known length.


Phase I study of YM155, a novel survivin suppressant, in patients with advanced solid tumors.
YM155, a novel molecular targeted agent, suppresses survivin, a member of the inhibitor of apoptosis protein family that is overexpressed in many tumor types. The aim of this study was to determine the maximum tolerated dose (MTD) and to assess the safety, pharmacokinetics, and antitumor activity of YM155 in patients with advanced refractory solid tumors. Patients with advanced refractory solid tumors were treated with escalating doses of YM155 administered by continuous i.v. infusion for 168 hours in 21-day cycles. Of the 34 patients enrolled, 33 (median age, 59 years) received at least 1 dose of YM155 (range, 1-19 cycles). The dose levels studied were 1.8, 3.6, 4.8, 6.0, 8.0, and 10.6 mg/m(2)/d. The MTD was determined to be 8.0 mg/m(2)/d, based on a dose-limiting toxicity of increased blood creatinine observed in 2 patients receiving 10.6 mg/m(2)/d. The most common adverse reactions judged to be related to YM155 were urine microalbumin present; fever; injection-site phlebitis; fatigue; and decreased hemoglobin/anemia, blood albumin, and lymphocyte count. The pharmacokinetic profile was almost linear over the dosing range and was similar between cycles 1 and 2. Urinary excretion of YM155 showed no definite difference among doses. Stable disease was achieved in nine patients. YM155 was safely administered to patients with advanced refractory solid tumors by 168-hour continuous i.v. infusion in 21-day cycles. The MTD was determined to be 8.0 mg/m(2)/d. The safety profile, plasma concentrations achieved, and antitumor activity observed merit further studies with this survivin suppressant, alone and in combination regimens.


A pilot randomized controlled trial of a stepped care intervention package for depression in primary care in Nigeria.
Depression is common in primary care and is often unrecognized and untreated. Studies are needed to demonstrate the feasibility of implementing evidence-based depression care provided by primary health care workers (PHCWs) in sub-Saharan Africa. We carried out a pilot two-parallel arm cluster randomized controlled trial of a package of care for depression in primary care. Six primary health care centers (PHCC) in two Local Government Areas of Oyo State, South West Nigeria were randomized into 3 intervention and 3 control clinics. Three PHCWs were selected for training from each of the participating clinics. The PHCWs from the intervention clinics were trained to deliver a manualized multicomponent stepped care intervention package for depression consisting of psychoeducation, activity scheduling, problem solving treatment and medication for severe depression. Providers from the control clinics delivered care as usual, enhanced by a refresher training on depression diagnosis and management. Outcome measures Patient's Health Questionnaire (PHQ-9), WHO quality of Life instrument (WHOQOL-Bref) and the WHO disability assessment schedule (WHODAS) were administered in the participants' home at baseline, 3 and 6 months. About 98% of the consecutive attendees to the clinics agreed to have the screening interview. Of those screened, 284 (22.7%) were positive (PHQ-9 score ≥ 8) and 234 gave consent for inclusion in the study: 165 from intervention and 69 from control clinics. The rates of eligible and consenting participants were similar in the control and intervention arms. In all 85.9% (92.8% in intervention and 83% in control) of the participants were successfully administered outcome assessments at 6 months. The PHCWs had little difficulty in delivering the intervention package. At 6 months follow up, depression symptoms had improved in 73.0% from the intervention arm compared to 51.6% control. Compared to the mean scores at baseline, there was improvement in the mean scores on all outcome measures in both arms at six months. The results provide support for the feasibility of conducting a fully-powered randomized study in this setting and suggest that the instruments used may have the potential to detect differences between the arms. ISRCTN46754188 (ISRTCN registry at isrtcn.com); registered 23 September 2013, details of the pilot study added 12/02/2015.


The combined impact of maternal age and body mass index on cumulative live birth following in vitro fertilization.
It is critical to evaluate the combined impact of age and body mass index on the cumulative likelihood of live birth following in vitro fertilization, as achieving a lower body mass index before infertility treatment often is recommended for women with overweight and obesity. It is important to consider whether achieving a particular body mass index, thus resulting in an older age at in vitro fertilization cycle start, is beneficial or harmful to the likelihood of live birth. To evaluate the combined impact of age and body mass index on the cumulative live birth rate following in vitro fertilization to inform when delaying in vitro fertilization treatment to achieve a lower body mass index may be beneficial or detrimental to the likelihood of live birth. This is a retrospective study using linked fresh and cryopreserved/frozen cycles from January 2014 to December 2015 from the Society for Reproductive Technology Clinic Outcome Reporting System, representing >90% of in vitro fertilization cycles performed in the United States. The primary outcome was live birth as measured by cumulative live birth rate. Secondary outcomes included implantation rate, clinical pregnancy rate, and miscarriage rate. Poisson and logistic regression were used to calculate risk and odds ratios with 95% confidence intervals to determine differences in implantation, clinical pregnancy, and miscarriage, as appropriate, among first fresh in vitro fertilization cycles compared across age (years) and body mass index (kg/m2) categories. Cox regression was used to calculate hazard ratios with 95% confidence intervals to determine differences in the cumulative live birth rate using fresh plus linked frozen embryo transfer cycles. There were 51,959 first fresh cycles using autologous eggs and 16,067 subsequent frozen embryo transfer cycles. There were 21,395 live births, for an overall cumulative live birth rate of 41.2% per cycle start. The implantation rate, clinical pregnancy rate, and cumulative live birth rate decreased with increasing body mass index and age, and the miscarriage rate increased with increasing body mass index and age (linear trend P<.001 for all). Body mass index had a greater influence on live birth at younger ages as compared with older ages. Age-related decline in fertility has a greater impact than body mass index on the cumulative live birth rate at older ages, suggesting that taking time to achieve lower body mass index before in vitro fertilization may be detrimental for older women with overweight or obesity. Delaying conception to lose weight before in vitro fertilization should be informed by the combination of age and body mass index.


Phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1)-dependent ubiquitination of endogenous Parkin attenuates mitophagy: study in human primary fibroblasts and induced pluripotent stem cell-derived neurons.
Mutations in the E3 ubiquitin ligase Parkin and the mitochondrial PTEN-induced putative kinase 1 (PINK1) have been identified to cause autosomal recessive forms of familial Parkinson disease, with PINK1 functioning upstream of Parkin in a pathway important for the maintenance of mitochondrial function and morphology. Upon the loss of the mitochondrial membrane potential, Parkin translocates to mitochondria in a PINK1-dependent manner to ubiquitinate mitochondrial proteins. Parkin-mediated polyubiquitination of outer mitochondrial membrane (OMM) proteins recruits the ubiquitin- and LC3-binding adaptor protein p62 to mitochondria and induces mitophagy. Although previous studies examined mitophagy in established cell lines through overexpression approaches, there is an imperative to study the role of endogenous Parkin and PINK1 in human-derived and biologically relevant cell models. Here, we demonstrate in human primary fibroblasts and induced pluripotent stem-derived neurons from controls and PINK1 mutation carriers that endogenous levels of Parkin are not sufficient to initiate mitophagy upon loss of the mitochondrial membrane potential, caused by its (self-)ubiquitination, followed by degradation via the ubiquitin proteasome system. Next, we showed differential PINK1-dependent, Parkin-mediated ubiquitination of OMM proteins, which is Parkin dose-dependent and affects primarily OMM proteins of higher molecular mass. In contrast to the situation fibroblasts, we did not detect mitophagy in induced pluripotent stem-derived neurons even upon overexpression of Parkin. Taken together, our data demonstrate that mitophagy differs between human non-neuronal and neuronal cells and between "endogenous" and "Parkin-overexpressing" cellular models.


[A 104-week feeding study of genetically modified soybeans in F344 rats].
A chronic feeding study to evaluate the safety of genetically modified glyphosate-tolerant soybeans (GM soybeans) was conducted using F344 DuCrj rats. The rats were fed diet containing GM soybeans or Non-GM soybeans at the concentration of 30% in basal diet. Non-GM soybeans were a closely related strain to the GM soybeans. These two diets were adjusted to an identical nutrient level. In this study, the influence of GM soybeans in rats was compared with that of the Non-GM soybeans, and furthermore, to assess the effect of soybeans themselves, the groups of rats fed GM and Non-GM soybeans were compared with a group fed commercial diet (CE-2). General conditions were observed daily and body weight and food consumption were recorded. At the termination (104 weeks), animals were subjected to hematology, serum biochemistry, and pathological examinations. There were several differences in animal growth, food intake, organ weights and histological findings between the rats fed the GM and/or Non-GM soybeans and the rats fed CE-2. However, body weight and food intake were similar for the rats fed the GM and Non-GM soybeans. Gross necropsy findings, hematological and serum biochemical parameters, and organ weights showed no meaningful difference between rats fed the GM and Non-GM soybeans. In pathological observation, there was neither an increase in incidence nor any specific type of nonneoplastic or neoplastic lesions in the GM soybeans group in each sex. These results indicate that long-term intake of GM soybeans at the level of 30% in diet has no apparent adverse effect in rats.


How the biotin-streptavidin interaction was made even stronger: investigation via crystallography and a chimaeric tetramer.
The interaction between SA (streptavidin) and biotin is one of the strongest non-covalent interactions in Nature. SA is a widely used tool and a paradigm for protein-ligand interactions. We previously developed a SA mutant, termed Tr (traptavidin), possessing a 10-fold lower off-rate for biotin, with increased mechanical and thermal stability. In the present study, we determined the crystal structures of apo-Tr and biotin-Tr at 1.5 Å resolution. In apo-SA the loop (L3/4), near biotin's valeryl tail, is typically disordered and open, but closes upon biotin binding. In contrast, L3/4 was shut in both apo-Tr and biotin-Tr. The reduced flexibility of L3/4 and decreased conformational change on biotin binding provide an explanation for Tr's reduced biotin off- and on-rates. L3/4 includes Ser45, which forms a hydrogen bond to biotin consistently in Tr, but erratically in SA. Reduced breakage of the biotin-Ser45 hydrogen bond in Tr is likely to inhibit the initiating event in biotin's dissociation pathway. We generated a Tr with a single biotin-binding site rather than four, which showed a simi-larly low off-rate, demonstrating that Tr's low off-rate was governed by intrasubunit effects. Understanding the structural features of this tenacious interaction may assist the design of even stronger affinity tags and inhibitors.


Neural stem cell transcriptional networks highlight genes essential for nervous system development.
Neural stem cells must strike a balance between self-renewal and multipotency, and differentiation. Identification of the transcriptional networks regulating stem cell division is an essential step in understanding how this balance is achieved. We have shown that the homeodomain transcription factor, Prospero, acts to repress self-renewal and promote differentiation. Among its targets are three neural stem cell transcription factors, Asense, Deadpan and Snail, of which Asense and Deadpan are repressed by Prospero. Here, we identify the targets of these three factors throughout the genome. We find a large overlap in their target genes, and indeed with the targets of Prospero, with 245 genomic loci bound by all factors. Many of the genes have been implicated in vertebrate stem cell self-renewal, suggesting that this core set of genes is crucial in the switch between self-renewal and differentiation. We also show that multiply bound loci are enriched for genes previously linked to nervous system phenotypes, thereby providing a shortcut to identifying genes important for nervous system development.


Programmed cell death in mouse primordial germ cells.
In a number of mammalian species, the main events of development of the primordial germ cells (PGCs), the embryonic precursors of the oocytes and spermatozoa, were described during the early twentieth century. Actually, the concept of the origin of germ cells in extragonadal sites before the formation of the gonadal anlagen, was put forward for the human embryo around the first decade of the 1900s (for a review, see De Felici, 2013). PGC development is characterized by two major cellular processes, a movement from the wall of the yolk sac, where the germline is determined, to the gonadal anlagen and an increase in number due to active proliferation. As far as we know, the notion that programmed cell death (PCD) might physiologically occur in mammalian PGCs was for the first time put forward by us in 1993 in the case of the mouse. How we arrived to such a concept and the progress made up to now in the characterization of this process in our and other laboratories mainly in the mouse are the topics of the present review.


Battery-free, fully implantable optofluidic cuff system for wireless optogenetic and pharmacological neuromodulation of peripheral nerves.
Studies of the peripheral nervous system rely on controlled manipulation of neuronal function with pharmacologic and/or optogenetic techniques. Traditional hardware for these purposes can cause notable damage to fragile nerve tissues, create irritation at the biotic/abiotic interface, and alter the natural behaviors of animals. Here, we present a wireless, battery-free device that integrates a microscale inorganic light-emitting diode and an ultralow-power microfluidic system with an electrochemical pumping mechanism in a soft platform that can be mounted onto target peripheral nerves for programmed delivery of light and/or pharmacological agents in freely moving animals. Biocompliant designs lead to minimal effects on overall nerve health and function, even with chronic use in vivo. The small size and light weight construction allow for deployment as fully implantable devices in mice. These features create opportunities for studies of the peripheral nervous system outside of the scope of those possible with existing technologies.


Transgenic mice expressing a human apolipoprotein[a] allele.
The most important determinant of plasma levels of Lp[a] are sequence differences at the highly polymorphic apolipoprotein[a] (apo[a]) locus. To define the sequences that mediate the regulation of apo[a] expression, we cloned a 370 kb DNA fragment that included a 130 kb apo[a] gene, and 40 kb 5'- and 200 kb 3'-flanking region from an individual with high plasma levels of Lp[a] using a YAC vector. This genomic clone was used to generate transgenic mice. In the YAC-apo[a] transgenic mouse, apo[a] was only expressed in the liver, as it is in humans. The mean serum level of apo[a] in 4-week-old YAC-apo[a] transgenic mice was 20 mg/dl. In the female mice the levels of apo[a] varied over a 1.5-fold range during the 4-day estrus cycle and the levels correlated directly with serum progesterone levels. The serum levels of apo[a] decreased to almost undetectable level in male mice after puberty and this decrease was reversed by castration. Ingestion of a high-fat diet resulted in a approximately 100-fold fall in hepatic apo[a] mRNA levels and >60-fold decrease in serum apo[a] levels. To delimit the control elements that mediate tissue-specific and sex hormone-responsive apo[a] transcription, we derived a reporter YAC in which 40 kb of 5' flanking sequences from the cloned apo[a] allele were linked to a luciferase reporter gene. Analysis of four independent transgenic lines revealed no hepatic luciferase expression, suggesting that important cis -acting elements located outside the apo[a] 5'-flanking region are necessary for in vivo expression of apo[a].


Polycystic kidney disease protein fibrocystin localizes to the mitotic spindle and regulates spindle bipolarity.
Autosomal recessive polycystic kidney disease (ARPKD) is a significant hereditary renal disease occurring in infancy and childhood, which presents with greatly enlarged echogenic kidneys, ultimately leading to renal insufficiency and end-stage renal disease. ARPKD is caused by mutations in a single gene PKHD1, which encodes fibrocystin/polyductin (FPC), a large single transmembrane protein generally known to be on the primary cilium, basal body and plasma membrane. Here, using our newly generated antibody raised against the entire C-terminal intracellular cytoplasmic domain (ICD) of FPC, as well as our previously well-characterized antibody against a peptide of ICD, we report for the first time that at least one isoform of FPC is localized to the centrosome and mitotic spindle of dividing cells in multiple cell lines, including MDCK, mIMCD3, LLC-PK1, HEK293, RCTEC and HFCT cells. Using short-hairpin-mediated RNA interference, we show that the inhibition of FPC function in MDCK and mIMCD3 cells leads to centrosome amplification, chromosome lagging and multipolar spindle formation. Consistent with our in vitro findings, we also observed centrosome amplification in the kidneys from human ARPKD patients. These findings demonstrate a novel function of FPC in centrosome duplication and mitotic spindle assembly during cell division. We propose that mitotic defects due to FPC dysfunction contribute to cystogenesis in ARPKD.


Evolution of the genetic code through progressive symmetry breaking.
Evolution of the genetic code in an early RNA world is dependent on the steadily improving specificity of the coevolving protein synthesis machinery for codons, anticodons, tRNAs and amino acids. In the beginning, there is RNA but the machinery does not distinguish yet between the codons, which therefore all encode the same information. Synonymous codons are equivalent under a symmetry group that exchanges (permutes) the codons without affecting the code. The initial group changes any codon into any other by permuting the order of the bases in the triplet as well as by replacing the four RNA bases with each other at every codon position. This group preserves the differences between codons, known as Hamming distances, with a 1-distance corresponding to a single point mutation. Stepwise breaking of the group into subgroups divides the 64 codons into progressively smaller subsets - blocks of equivalent codons under the smaller symmetry groups, with each block able to encode a different message. This formalism prescribes how the evolving machinery increasingly differentiates between codons. The model indicates that primitive ribosomes first identified a unique mRNA reading frame to break the group permuting the order of the bases and subsequently enforced increasingly stringent codon-anticodon basepairing rules to break the subgroups permuting the four bases at each codon position. The modern basepairing rules evolve in five steps and at each step the number of codon blocks doubles. The fourth step generates 16 codon blocks corresponding with the 16 family boxes of the standard code and the last step splits these boxes into 32 blocks of commonly two, but rarely one or three, synonymous codons. The evolving codes transmit at most one message per codon block and as the number of messages increases so does the specificity of the code and of protein synthesis. The selective advantage conferred by better functioning proteins drives the symmetry breaking process. Over time paralogous tRNA evolution expands the anticodon repertoire, which is divided into anticodon blocks matching the codon blocks under the stage-specific ribosomal basepairing rules. Contemporaneously an expanding family of primitive aminoacyl-tRNA synthetases (aaRSs) divides the tRNA diversities into various different and overlapping subsets: each aaRS accepts some tRNAs but rejects all others and several aaRSs may accept the same tRNA species. Selection favoring less ambiguous codes eliminates these overlaps and also imposes the ribosomal anticodon block division as ambiguity arises when different aaRSs accept tRNAs of the same anticodon block. Only when the tRNAs of one or several anticodon blocks are accepted by a unique aaRS does the code become specific. This coding pattern is observed in the standard code and the evolution of amino acid assignments by primitive aaRSs onto tRNAs is traced back via tRNA trees that picture a gradual division of tRNA diversities into blocks with increasingly specific amino acid assignments. Symmetry breaking combined with continuous selection for codes carrying more information evolves increasingly specific codes and efficiently traverses an immense space of all possible codes (>10(84)) to give rise to the standard code.


160-Gb/s optical time division multiplexing and multicasting in parametric amplifiers.
We report the generation of an optical time division multiplexed single data channel at 160 Gb/s using a one-pump fiber-optic parametric amplifier, and its subsequent multicasting. A two-pump fiber optic parametric amplifier was used to perform all-optical multicasting of 160 Gb/s channel to four data streams. New processing scheme combined the increase in signal extinction ratio and low-impairment multicasting using continuous-wave parametric pumps. Selective conjugation of 160 Gb/s was demonstrated for the first time.


Natural selection promotes antigenic evolvability.
The hypothesis that evolvability - the capacity to evolve by natural selection - is itself the object of natural selection is highly intriguing but remains controversial due in large part to a paucity of direct experimental evidence. The antigenic variation mechanisms of microbial pathogens provide an experimentally tractable system to test whether natural selection has favored mechanisms that increase evolvability. Many antigenic variation systems consist of paralogous unexpressed 'cassettes' that recombine into an expression site to rapidly alter the expressed protein. Importantly, the magnitude of antigenic change is a function of the genetic diversity among the unexpressed cassettes. Thus, evidence that selection favors among-cassette diversity is direct evidence that natural selection promotes antigenic evolvability. We used the Lyme disease bacterium, Borrelia burgdorferi, as a model to test the prediction that natural selection favors amino acid diversity among unexpressed vls cassettes and thereby promotes evolvability in a primary surface antigen, VlsE. The hypothesis that diversity among vls cassettes is favored by natural selection was supported in each B. burgdorferi strain analyzed using both classical (dN/dS ratios) and Bayesian population genetic analyses of genetic sequence data. This hypothesis was also supported by the conservation of highly mutable tandem-repeat structures across B. burgdorferi strains despite a near complete absence of sequence conservation. Diversification among vls cassettes due to natural selection and mutable repeat structures promotes long-term antigenic evolvability of VlsE. These findings provide a direct demonstration that molecular mechanisms that enhance evolvability of surface antigens are an evolutionary adaptation. The molecular evolutionary processes identified here can serve as a model for the evolution of antigenic evolvability in many pathogens which utilize similar strategies to establish chronic infections.


Randomized trial of tacrolimus plus mycophenolate mofetil or azathioprine versus cyclosporine oral solution (modified) plus mycophenolate mofetil after cadaveric kidney transplantation: results at 2 years.
A previous report described the 1-year results of a prospective, randomized trial designed to investigate the optimal combination of immunosuppressants in kidney transplantation. Recipients of first cadaveric kidney allografts were treated with tacrolimus+mycophenolate mofetil (MMF), cyclosporine oral solution (modified) (CsA)+MMF, or tacrolimus+azathioprine (AZA). Results at 1 year revealed that optimal efficacy and safety were achieved with a regimen containing tacrolimus+MMF. The present report describes results at 2 years. Two hundred twenty-three recipients of first cadaveric kidney allografts were randomized to receive tacrolimus+MMF, CsA+MMF, or tacrolimus+AZA. All regimens contained corticosteroids, and antibody induction was used only in patients who experienced delayed graft function. Patients were followed up for 2 years. The results at 2 years corroborate and extend the findings of the previous report. Patients randomized to either treatment arm containing tacrolimus experienced improved kidney function. New-onset insulin dependence remained in four, three, and four patients in the tacrolimus+MMF, CsA+MMF, and tacrolimus+AZA treatment arms, respectively. Furthermore, patients with delayed graft function/acute tubular necrosis who were treated with tacrolimus+MMF experienced a 23% increase in allograft survival compared with patients receiving CsA+MMF (P=0.06). Patients randomized to tacrolimus+MMF received significantly lower doses of MMF compared with those administered CsA+MMF. All three immunosuppressive regi-mens provided excellent safety and efficacy. How-ever, the best results overall were achieved with tacrolimus+MMF. The combination may provide particular benefit to kidney allograft recipients who develop delayed graft function/acute tubular necrosis. Renal function at 2 years was better in the tacrolimus treatment groups compared with the CsA group.


2-Azo-, 2-diazocine-thiazols and 2-azo-imidazoles as photoswitchable kinase inhibitors: limitations and pitfalls of the photoswitchable inhibitor approach.
In photopharmacology, photoswitchable compounds including azobenzene or other diarylazo moieties exhibit bioactivity against a target protein typically in the slender E-configuration, whereas the rather bulky Z-configuration usually is pharmacologically less potent. Herein we report the design, synthesis and photochemical/inhibitory characterization of new photoswitchable kinase inhibitors targeting p38α MAPK and CK1δ. A well characterized inhibitor scaffold was used to attach arylazo- and diazocine moieties. When the isolated isomers, or the photostationary state (PSS) of isomers, were tested in commonly used in vitro kinase assays, however, only small differences in activity were observed. X-ray analyses of ligand-bound p38α MAPK and CK1δ complexes revealed dynamic conformational adaptations of the protein with respect to both isomers. More importantly, irreversible reduction of the azo group to the corresponding hydrazine was observed. Independent experiments revealed that reducing agents such as DTT (dithiothreitol) and GSH (glutathione) that are typically used for protein stabilization in biological assays were responsible. Two further sources of error are the concentration dependence of the E-Z-switching efficiency and artefacts due to incomplete exclusion of light during testing. Our findings may also apply to a number of previously investigated azobenzene-based photoswitchable inhibitors.


Epigenetic regulation of RELN and GAD1 in the frontal cortex (FC) of autism spectrum disorder (ASD) subjects.
Both Reelin (RELN) and glutamate decarboxylase 67 (GAD1) have been implicated in the pathophysiology of Autism Spectrum Disorders (ASD). We have previously shown that both mRNAs are reduced in the cerebella (CB) of ASD subjects through a mechanism that involves increases in the amounts of MECP2 binding to the corresponding promoters. In the current study, we examined the expression of RELN, GAD1, GAD2, and several other mRNAs implicated in this disorder in the frontal cortices (FC) of ASD and CON subjects. We also focused on the role that epigenetic processes play in the regulation of these genes in ASD brain. Our goal is to better understand the molecular basis for the down-regulation of genes expressed in GABAergic neurons in ASD brains. We measured mRNA levels corresponding to selected GABAergic genes using qRT-PCR in RNA isolated from both ASD and CON groups. We determined the extent of binding of MECP2 and DNMT1 repressor proteins by chromatin immunoprecipitation (ChIP) assays. The amount of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) present in the promoters of the target genes was quantified by methyl DNA immunoprecipitation (MeDIP) and hydroxyl MeDIP (hMeDIP). We detected significant reductions in the mRNAs associated with RELN and GAD1 and significant increases in mRNAs encoding the Ten-eleven Translocation (TET) enzymes 1, 2, and 3. We also detected increased MECP2 and DNMT1 binding to the corresponding promoter regions of GAD1, RELN, and GAD2. Interestingly, there were decreased amounts of 5mC at both promoters and little change in 5hmC content in these same DNA fragments. Our data demonstrate that RELN, GAD1, and several other genes selectively expressed in GABAergic neurons, are down-regulated in post-mortem ASD FC. In addition, we observed increased DNMT1 and MECP2 binding at the corresponding promoters of these genes. The finding of increased MECP2 binding to the RELN, GAD1 and GAD2 promoters, with reduced amounts of 5mC and unchanged amounts of 5hmC present in these regions, suggests the possibility that DNMT1 interacts with and alters MECP2 binding properties to selected promoters. Comparisons between data obtained from the FC with CB studies showed some common themes between brain regions which are discussed.


Inhibition of the Fe(4)S(4)-cluster-containing protein IspH (LytB): electron paramagnetic resonance, metallacycles, and mechanisms.
We report the inhibition of the Aquifex aeolicus IspH enzyme (LytB, (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate reductase, EC 184.108.40.206) by a series of diphosphates and bisphosphonates. The most active species was an alkynyl diphosphate having IC(50) = 0.45 microM (K(i) approximately 60 nM), which generated a very large change in the 9 GHz EPR spectrum of the reduced protein. On the basis of previous work on organometallic complexes, together with computational docking and quantum chemical calculations, we propose a model for alkyne inhibition involving pi (or pi/sigma) "metallacycle" complex formation with the unique fourth Fe in the Fe(4)S(4) cluster. Aromatic species had less activity, and for these we propose an inhibition model based on an electrostatic interaction with the active site E126. Overall, the results are of broad general interest since they not only represent the first potent IspH inhibitors but also suggest a conceptually new approach to inhibiting other Fe(4)S(4)-cluster-containing proteins that are of interest as drug and herbicide targets.


How pervasive is biotic homogenization in human-modified tropical forest landscapes?
Land-cover change and ecosystem degradation may lead to biotic homogenization, yet our understanding of this phenomenon over large spatial scales and different biotic groups remains weak. We used a multi-taxa dataset from 335 sites and 36 heterogeneous landscapes in the Brazilian Amazon to examine the potential for landscape-scale processes to modulate the cumulative effects of local disturbances. Biotic homogenization was high in production areas but much less in disturbed and regenerating forests, where high levels of among-site and among-landscape β-diversity appeared to attenuate species loss at larger scales. We found consistently high levels of β-diversity among landscapes for all land cover classes, providing support for landscape-scale divergence in species composition. Our findings support concerns that β-diversity has been underestimated as a driver of biodiversity change and underscore the importance of maintaining a distributed network of reserves, including remaining areas of undisturbed primary forest, but also disturbed and regenerating forests, to conserve regional biota.


Hydrogen sulfide and nitric oxide interactions in inflammation.
Together with carbon monoxide (CO), nitric oxide (NO) and hydrogen sulfide (H2S) form a group of physiologically important gaseous transmitters, sometimes referred to as the "gaseous triumvirate". The three molecules share a wide range of physical and physiological properties: they are small gaseous molecules, able to freely penetrate cellular membranes; they are all produced endogenously in the body and they seem to exert similar biological functions. In the cardiovascular system, for example, they are all vasodilators, promote angiogenesis and protect tissues against damage (e.g. ischemia-reperfusion injury). In addition, they have complex roles in inflammation, with both pro- and anti-inflammatory effects reported. Researchers have focused their efforts in understanding and describing the roles of each of these molecules in different physiological systems, and in the past years attention has also been given to the gases interaction or "cross-talk". This review will focus on the role of NO and H2S in inflammation and will give an overview of the evidence collected so far suggesting the importance of their cross-talk in inflammatory processes.


[External evaluation of serology results in blood banks in Colombia].
To analyze the serological results found in Colombian blood banks that participate in the external quality program (EQP) of that country's National Institute of Health, in order to improve the quality of the screening of blood for the main serological markers of transfusion-transmitted infectious diseases. Each blood bank received a panel of six sera with different reactivity and positivity to hepatitis B surface antigen (HBsAg), as well as to antibodies to HIV 1-2, Trypanosoma cruzi (the causative agent of Chagas' disease), Treponema pallidum (the causative agent of syphilis), hepatitis B core (HBc) antigen, hepatitis C virus (HCV), and human T-lymphotropic virus (HTLV). The screening techniques used were enzyme-linked immunosorbent assay (ELISA), microparticle enzyme immunoassay (MEIA), and hemagglutination. With the panel sera, the participating blood banks were asked to apply the same tests that they use on a daily basis to screen blood units and to send their results to the National Blood Banks Unit of the Colombian National Institute of Health. Of the 46 blood banks participating in the EQP, 43 of them (93%) returned their results within the requested timeframe. The ELISA test was the one that was used most often (83.0%). There were a total of 49 (5%) false positive results and 12 (3%) false negative results. Of those 12 false negative results, 6 of them corresponded to the detection of syphilis, 2 to Chagas' disease, 2 to anti-HBc antibodies, 1 to anti-HCV antibodies, and 1 to HBsAg. Eighty percent of the discordant results came from 23 blood banks that each collected fewer than 6 000 units of blood per year, and 15% came from 5 blood banks that collected 6 000 to 12 000 units per year. One of the blood banks that collected more than 12 000 units annually had three false positive results, and none of those larger blood banks had any false positive results. The percentage of false negative results (3%) found during the EQP can be considered high, since tests that are negative during blood screening are not repeated, and the decision to declare a unit of blood suitable for transfusion is based on that single result. There is a need to thoroughly review the procedures for screening blood in Colombia, particularly at the centers that performed poorly in this EQP exercise.