Novel mutations mapping to the fourth sodium channel domain of Nav1.7 result in variable clinical manifestations of primary erythromelalgia.
We identified and clinically investigated two patients with primary erythromelalgia mutations (PEM), which are the first reported to map to the fourth domain of Nav1.7 (DIV). The identified mutations (A1746G and W1538R) were cloned and transfected to cell cultures followed by electrophysiological analysis in whole-cell configuration. The investigated patients presented with PEM, while age of onset was very different (3 vs. 61 years of age). Electrophysiological characterization revealed that the early onset A1746G mutation leads to a marked hyperpolarizing shift in voltage dependence of steady-state activation, larger window currents, faster activation kinetics (time-to-peak current) and recovery from steady-state inactivation compared to wild-type Nav1.7, indicating a pronounced gain-of-function. Furthermore, we found a hyperpolarizing shift in voltage dependence of slow inactivation, which is another feature commonly found in Nav1.7 mutations associated with PEM. In silico neuron simulation revealed reduced firing thresholds and increased repetitive firing, both indicating hyperexcitability. The late-onset W1538R mutation also revealed gain-of-function properties, although to a lesser extent. Our findings demonstrate that mutations encoding for DIV of Nav1.7 can not only be linked to congenital insensitivity to pain or paroxysmal extreme pain disorder but can also be causative of PEM, if voltage dependency of channel activation is affected. This supports the view that the degree of biophysical property changes caused by a mutation may have an impact on age of clinical manifestation of PEM. In summary, these findings extent the genotype-phenotype correlation profile for SCN9A and highlight a new region of Nav1.7 that is implicated in PEM.


Long-term outcomes of acute treatment with cognitive therapy v. interpersonal psychotherapy for adult depression: follow-up of a randomized controlled trial.
Although equally efficacious in the acute phase, it is not known how cognitive therapy (CT) and interpersonal psychotherapy (IPT) for major depressive disorder (MDD) compare in the long run. This study examined the long-term outcomes of CT v. IPT for MDD. One hundred thirty-four adult (18-65) depressed outpatients who were treated with CT (n = 69) or IPT (n = 65) in a large open-label randomized controlled trial (parallel group design; computer-generated block randomization) were monitored across a 17-month follow-up phase. Mixed regression was used to determine the course of self-reported depressive symptom severity (Beck Depression Inventory II; BDI-II) after treatment termination, and to test whether CT and IPT differed throughout the follow-up phase. Analyses were conducted for the total sample (n = 134) and for the subsample of treatment responders (n = 85). Furthermore, for treatment responders, rates of relapse and sustained response were examined for self-reported (BDI-II) and clinician-rated (Longitudinal Interval Follow-up Evaluation; LIFE) depression using Cox regression. On average, the symptom reduction achieved during the 7-month treatment phase was maintained across follow-up (7-24 months) for CT and IPT, both in the total sample and in the responder sample. Two-thirds (67%) of the treatment responders did not relapse across the follow-up period on the BDI-II. Relapse rates assessed with the LIFE were somewhat lower. No differential effects between conditions were found. Patients who responded to IPT were no more likely to relapse following treatment termination than patients who responded to CT. Given that CT appears to have a prophylactic effect following successful treatment, our findings suggest that IPT might have a prophylactic effect as well.


A toxicokinetic model for thiamethoxam in rats: implications for higher-tier risk assessment.
Risk assessment for mammals is currently based on external exposure measurements, but effects of toxicants are better correlated with the systemically available dose than with the external administered dose. So for risk assessment of pesticides, toxicokinetics should be interpreted in the context of potential exposure in the field taking account of the timescale of exposure and individual patterns of feeding. Internal concentration is the net result of absorption, distribution, metabolism and excretion (ADME). We present a case study for thiamethoxam to show how data from ADME study on rats can be used to parameterize a body burden model which predicts body residue levels after exposures to LD50 dose either as a bolus or eaten at different feeding rates. Kinetic parameters were determined in male and female rats after an intravenous and oral administration of (14)C labelled by fitting one-compartment models to measured pesticide concentrations in blood for each individual separately. The concentration of thiamethoxam in blood over time correlated closely with concentrations in other tissues and so was considered representative of pesticide concentration in the whole body. Body burden model simulations showed that maximum body weight-normalized doses of thiamethoxam were lower if the same external dose was ingested normally than if it was force fed in a single bolus dose. This indicates lower risk to rats through dietary exposure than would be estimated from the bolus LD50. The importance of key questions that should be answered before using the body burden approach in risk assessment, data requirements and assumptions made in this study are discussed in detail.


Activin, BMP and FGF pathways cooperate to promote endoderm and pancreatic lineage cell differentiation from human embryonic stem cells.
The study of how human embryonic stem cells (hESCs) differentiate into insulin-producing beta cells has twofold significance: first, it provides an in vitro model system for the study of human pancreatic development, and second, it serves as a platform for the ultimate production of beta cells for transplantation into patients with diabetes. The delineation of growth factor interactions regulating pancreas specification from hESCs in vitro is critical to achieving these goals. In this study, we describe the roles of growth factors bFGF, BMP4 and Activin A in early hESC fate determination. The entire differentiation process is carried out in serum-free chemically-defined media (CDM) and results in reliable and robust induction of pancreatic endoderm cells, marked by PDX1, and cell clusters co-expressing markers characteristic of beta cells, including PDX1 and insulin/C-peptide. Varying the combinations of growth factors, we found that treatment of hESCs with bFGF, Activin A and BMP4 (FAB) together for 3-4days resulted in strong induction of primitive-streak and definitive endoderm-associated genes, including MIXL1, GSC, SOX17 and FOXA2. Early proliferative foregut endoderm and pancreatic lineage cells marked by PDX1, FOXA2 and SOX9 expression are specified in EBs made from FAB-treated hESCs, but not from Activin A alone treated cells. Our results suggest that important tissue interactions occur in EB-based suspension culture that contribute to the complete induction of definitive endoderm and pancreas progenitors. Further differentiation occurs after EBs are embedded in Matrigel and cultured in serum-free media containing insulin, transferrin, selenium, FGF7, nicotinamide, islet neogenesis associated peptide (INGAP) and exendin-4, a long acting GLP-1 agonist. 21-28days after embedding, PDX1 gene expression levels are comparable to those of human islets used for transplantation, and many PDX1(+) clusters are formed. Almost all cells in PDX1(+) clusters co-express FOXA2, HNF1ß, HNF6 and SOX9 proteins, and many cells also express CPA1, NKX6.1 and PTF1a. If cells are then switched to medium containing B27 and nicotinamide for 7-14days, then the number of insulin(+) cells increases markedly. Our study identifies a new chemically defined culture protocol for inducing endoderm- and pancreas-committed cells from hESCs and reveals an interplay between FGF, Activin A and BMP signaling in early hESC fate determination.


A cobalt(ii) spin-crossover compound with partially charged TCNQ radicals and an anomalous conducting behavior.
The bifunctional salt [Co(terpy)2](TCNQ)3·CH3CN (terpy = 2,2';6',2''-terpyridine, TCNQ = 7,7,8,8-tetracyanoquino-dimethane) exhibits a high room temperature conductivity of 0.13 S cm-1 and an anomaly in conductivity at ∼190 K as evidenced by variable temperature structural, magnetic and conductivity studies. The anomaly in the conductivity at 190 K has been correlated with the temperature dependent structural breathing and Jahn-Teller distortion of the low spin state of the SCO units, as well as the charge fluctuations and supramolecular π-stacking interactions of partially charged TCNQ radicals. The modular synthetic approach leads to an accessible source of partially charged TCNQ radicals for the facile preparation of bifunctional molecular materials with high electrical conductivity.


Temporally precise control of single-neuron spiking by juxtacellular nanostimulation.
Temporal patterns of action potentials influence a variety of activity-dependent intra- and intercellular processes and play an important role in theories of neural coding. Elucidating the mechanisms underlying these phenomena requires imposing spike trains with precisely defined patterns, but this has been challenging due to the limitations of existing stimulation techniques. Here we present a new nanostimulation method providing control over the action potential output of individual cortical neurons. Spikes are elicited through the juxtacellular application of short-duration fluctuating currents ("kurzpulses"), allowing for the sub-millisecond precise and reproducible induction of arbitrary patterns of action potentials at all physiologically relevant firing frequencies (<120 Hz), including minute-long spike trains recorded in freely moving animals. We systematically compared our method to whole cell current injection, as well as optogenetic stimulation, and show that nanostimulation performance compares favorably with these techniques. This new nanostimulation approach is easily applied, can be readily performed in awake behaving animals, and thus promises to be a powerful tool for systematic investigations into the temporal elements of neural codes, as well as the mechanisms underlying a wide variety of activity-dependent cellular processes.NEW & NOTEWORTHY Assessing the impact of temporal features of neuronal spike trains requires imposing arbitrary patterns of spiking on individual neurons during behavior, but this has been difficult to achieve due to limitations of existing stimulation methods. We present a technique that overcomes these limitations by using carefully designed short-duration fluctuating juxtacellular current injections, which allow for the precise and reliable evocation of arbitrary patterns of neuronal spikes in single neurons in vivo.


Program for the epidemiological evaluation of stroke in Tandil, Argentina (PREVISTA) study: rationale and design.
The availability of population-based epidemiological data on the incident risk of stroke is very scarce in Argentina and other Latin American countries. In response to the priorities established by the World Health Organization and the United Nations, PREVISTA was envisaged as a population-based program to determine the risk of first-ever and recurrent stroke and transient ischemic attack incidence and mortality in Tandil, Buenos Aires, Argentina. The study will be conducted according to Standardized Tools for Stroke Surveillance (STEPS Stroke) methodology and will enroll all new (incident) and recurrent consecutive cases of stroke and transient ischemic attack in the City of Tandil between May 1st, 2013 and April 30, 2015. The study will include patients with ischemic stroke, non-traumatic primary intracerebral hemorrhage, subarachnoid hemorrhage, and transient ischemic attack. To ensure the inclusion of every cerebrovascular event during an observation period of two years, we will instrument an 'intensive screening program', consisting of a comprehensive daily tracking of every potential event of stroke or transient ischemic attack using multiple overlapping sources. Mortality would be determined during follow-up for every enrolled patient. Also, fatal community events would be screened daily through revision of death certificates at funeral homes and local offices of vital statistics. All causes of death will be adjudicated by an ad-hoc committee. The close population of Tandil is representative of a large proportion of Latin-American countries with low- and middle-income economies. The findings and conclusions of PREVISTA may provide data that could support future health policy decision-making in the region.


Simulated data sets for single molecule kinetics: some limitations and complications of data analysis.
When the fluorescence intensity of a chromophore attached to or bound in an enzyme relates to a specific reactive step in the enzymatic reaction, a single molecule fluorescence study of the process reveals a time sequence in the fluorescence emission that can be analyzed to derive kinetic and mechanistic information. Reports of various experimental results and corresponding theoretical studies have provided a basis for interpreting these data and understanding the methodology. We have found it useful to parallel experiments with Monte Carlo simulations of potential models hypothesized to describe the reaction kinetics. The simulations can be adapted to include experimental limitations, such as limited data sets, and complexities such as dynamic disorder, where reaction rates appear to change over time. By using models that are known a priori, the simulations reveal some of the challenges of interpreting finite single-molecule data sets by employing various statistical signatures that have been identified.


Quinone formation as a chemoprevention strategy for hybrid drugs: balancing cytotoxicity and cytoprotection.
Cellular defense mechanisms that respond to damage from oxidative and electrophilic stress, such as from quinones, represent a target for chemopreventive agents. Drugs bioactivated to quinones have the potential to activate antioxidant/electrophile responsive element (ARE) transcription of genes for cytoprotective phase 2 enzymes such as NAD(P)H-dependent quinone oxidoreductase (NQO1) but can also cause cellular damage. Two isomeric families of compounds were prepared, including the NO-NSAIDs (NO-donating nonsteroidal anti-inflammatory drugs) NCX 4040 and NCX 4016; one family was postulated to release a quinone methide on esterase bioactivation. The study of reactivity and GSH conjugation in model and cell systems confirmed the postulate. The quinone-forming family, including NCX 4040 and conisogenic bromides and mesylate, was rapidly bioactivated to a quinone, which gave activation of ARE and consequent induction of NQO1 in liver cells. Although the control family, including NCX 4016 and conisogenic bromides and mesylates, cannot form a quinone, ARE activation and NQO1 induction were observed, compatible with slower SN2 reactions with thiol sensor proteins, and consequent ARE-luciferase and NQO1 induction. Using a Chemoprevention Index estimate, the quinone-forming compounds suffered because of high cytoxicity and were more compatible with cancer therapy than chemoprevention. In the Comet assay, NCX 4040 was highly genotoxic relative to NCX 4016. There was no evidence that NO contributes to the observed biological activity and no evidence that NCX 4040 is an NO donor, instead, rapidly releasing NO3- and quinone. These results indicate a strategy for studying the quinone biological activity and reinforce the therapeutic attributes of NO-ASA through structural elements other than NO and ASA.


[Neuropsychological testing in medicolegal cases].
Because the assessment of cognitive function is among the most important diagnostic evaluations in medicolegal cases, neuropsychological testing, the optimal scientific method available to determine the manifestation of brain dysfunction, is a potentially very strong tool in those cases. However, the use of neuropsychological testing in a medicolegal setting should be done with caution. Firstly, the nature of any neuropsychological testing is complex, and cannot be simplified as "Test A measures Function A' ". Oversimplified explanation of a test is misleading, resulting in serious misjudgment in the legal procedure. Secondly, the result of the test shows someone's cognitive function at the time of the test, not the time of the act in question (i. e., a crime). Finally and most importantly, the cognitive function measured by neuropsychological testing does not directly correspond to the legal questions being asked, even when the terms used in the fields of law and neuroscience are similar. In determining whether an individual meets a specified legal standard (e. g., criminal responsibility), the results of neuropsychological testing are far from sufficient and substantial additional information is required. With these caveats in mind, an expert witness should carefully choose the neuropsychological testing battery in each case, which may greatly help to understand the individual's mental state at the time of the act in question.


The CHANGE program: comparing an interactive versus prescriptive obesity intervention on university students' self-esteem and quality of life.
Previous studies incorporating Motivational Interviewing administered via Co-Active Life Coaching tools (MI-via-CALC) have elicited positive results among adults with obesity. However, there is a paucity of this research that includes sufficient power and a comparison group. This study's purpose was to compare MI-via-CALC with a validated obesity intervention among university students. Participants (n = 45) were randomised to either a telephone-based 12-week: (a) MI-via-CALC program whereby a certified coach worked with subjects to achieve goals through dialogue; or (b) lifestyle modification treatment following the LEARN Program for Weight Management. Participants completed the Rosenberg Self-Esteem Scale and Short Form Functional Health Status Scale (SF-36) at baseline, mid-, and post-treatment, and 3 and 6 months following the program. Analyses revealed that both conditions elicited significant time effects between baseline and 6 months for self-esteem and all dimensions of the SF-36 (e.g. overall health). MI-via-CALC compares favorably with LEARN as an obesity treatment. Given that self-esteem and quality of life are essential for promoting behavior change among individuals with obesity, this study offers unique insights into their change processes. Future research should provide both treatments and allow participants to choose based on their personal preferences, learning styles, and needs.


Insertion mutagenesis of XpsD, an outer-membrane protein involved in extracellular protein secretion in Xanthomonas campestris pv. campestris.
XpsD is an outer-membrane protein required for extracellular protein secretion in Xanthomonas campestris pv. campestris. Cross-linking and gelfiltration chromatography analyses have suggested that it forms a multimer. To determine its structure-function relationship, linker-insertion mutants were constructed in an xpsD gene carried on a plasmid. To assay for secretion function, each mutant gene was introduced into an xpsD::Tn5 mutant strain (XC1708) and assayed for alpha-amylase secretion on starch plates. To test whether the mutant genes exerted a dominant-negative effect, each was introduced into the parental strain XC1701 and examined for secretion interference. Nine functional, one semi-functional and eleven non-functional mutants were obtained. All the non-functional mutants, except two for which the mutant proteins were undetectable on immunoblots, showed interference of normal secretion. The insertion sites in the different mutant proteins are randomly distributed throughout the entire sequence of the XpsD protein. All the permissive insertion sites are located where beta-turn or coiled secondary structure is predicted. Over half of the non-permissive sites are located within predicted helical or beta-sheet regions. By pretreating total membranes of XC1701 in SDS at 50 degrees C, an immunoreactive band with high molecular mass (HMM) could be detected that remained in the stacking gel during SDS-PAGE. The semi-functional and all functional mutant proteins formed HMM complexes that were as SDS-resistant as those of the wild-type, whereas all except three of the non-functional mutant proteins formed HMM structures that were less resistant to SDS than the wild-type. By analysing the appearance of SDS-resistant HMM complexes, we were able to detect conformational alterations in XpsD that are too subtle to be detected by other assays.


The effect of 100% oxygen on the propagation of tracheobronchial injury during high-frequency and conventional mechanical ventilation.
We compared the histologic alterations in the tracheae and bronchi of 30 premature baboons that were ventilated with either 100% or prn (as needed) oxygen (the fraction of inspired oxygen necessary to maintain the PaO2 between 50 and 80 mm Hg). The baboons were treated with either conventional positive-pressure ventilation (7 were treated with 100%; 7, prn) or high-frequency oscillatory ventilation (8 were treated with 100%; 8, prn). We used a semiquantitative scoring system to grade tissue changes in the trachea, carina, and main-stem bronchi. The fraction of inspired oxygen for all prn animals fell to a plateau of approximately 0.28 after 36 hours, where it remained for the duration of the study. The 15 100% baboons were ventilated for a mean of 139 hours, while the 15 prn baboons were ventilated for a mean of 151 hours. The findings in all conventional and oscillator-ventilated animals were similar and characterized by squamous metaplasia, cilia loss, and goblet cell loss. For both methods of ventilation, there were no differences in the injury scores between 100% and prn oxygen-treated animals. We concluded that there were no additional tracheobronchial histologic changes with 100% oxygen compared with prn oxygen.


Prokaryotic suppression subtractive hybridization PCR cDNA subtraction, a targeted method to identify differentially expressed genes.
Molecular biology tools can be used to monitor and optimize biological treatment systems, but the application of nucleic acid-based tools has been hindered by the lack of available sequences for environmentally relevant biodegradation genes. The objective of our work was to extend an existing molecular method for eukaryotes to prokaryotes, allowing us to rapidly identify differentially expressed genes for subsequent sequencing. Suppression subtractive hybridization (SSH) PCR cDNA subtraction is a technique that can be used to identify genes that are expressed under specific conditions (e.g., growth on a given pollutant). While excellent methods for eukaryotic SSH PCR cDNA subtraction are available, to our knowledge, no methods previously existed for prokaryotes. This work describes our methodology for prokaryotic SSH PCR cDNA subtraction, which we validated using a model system: Pseudomonas putida mt-2 degrading toluene. cDNA from P. putida mt-2 grown on toluene (model pollutant) or acetate (control substrate) was subjected to our prokaryotic SSH PCR cDNA subtraction protocol to generate subtraction clone libraries. Over 90% of the sequenced clones contained gene fragments encoding toluene-related enzymes, and 20 distinct toluene-related genes from three key operons were sequenced. Based on these results, prokaryotic SSH PCR cDNA subtraction shows promise as a targeted method for gene identification.


Agonist-induced desensitization of beta-adrenoceptors of bovine bronchial epithelial cells.
1. Cultured bovine bronchial epithelial cells express a large number of beta-adrenoceptors of the beta 2 subtype which are coupled to activation of adenylate cyclase and synthesis of cyclic AMP. The mechanisms involved in agonist-induced desensitization of beta-adrenoceptor function in these cells were investigated. 2. Preincubation of cells with the beta-adrenoceptor agonist isoprenaline induced a rapid desensitization of adenylate cyclase activity stimulated by isoprenaline (40% at 30 min) with little or no decrease in forskolin stimulation or in the number of beta-adrenoceptors detected by radioligand binding, indicating that uncoupling of beta-adrenoceptors from adenylate cyclase had occurred. 3. Uncoupling of beta-adrenoceptors from the stimulatory guanine-nucleotide-binding protein Gs was indicated by the loss of GTP effects on agonist competition for radioligand binding. Results from subcellular fractionation by sucrose density gradient centrifugation suggest that rapid internalization or sequestration of beta-adrenoceptors occurred along with the desensitization. 4. Preincubation with isoprenaline for longer times led to a marked decrease in total receptor number (85% at 18 h), indicating that receptor down-regulation also occurs. Recovery of receptors after down-regulation was prevented by cycloheximide and by actinomycin D, suggesting that degradation of both receptor protein and receptor mRNA may be involved in down-regulation.


Trait anhedonia is associated with reduced reactivity and connectivity of mesolimbic and paralimbic reward pathways.
Anhedonia is the inability to experience pleasure from normally pleasant stimuli. Although anhedonia is a prominent feature of many psychiatric disorders, trait anhedonia is also observed dimensionally in healthy individuals. Currently, the neurobiological basis of anhedonia is poorly understood because it has been mainly investigated in patients with psychiatric disorders. Thus, previous studies have not been able to adequately disentangle the neural correlates of anhedonia from other clinical symptoms. In this study, trait anhedonia was assessed in well-characterized healthy participants with no history of Axis I psychiatric illness. Functional magnetic resonance imaging with musical stimuli was used to examine brain responses and effective connectivity in relation to individual differences in anhedonia. We found that trait anhedonia was negatively correlated with pleasantness ratings of music stimuli and with activation of key brain structures involved in reward processing, including nucleus accumbens (NAc), basal forebrain and hypothalamus which are linked by the medial forebrain bundle to the ventral tegmental area (VTA). Brain regions important for processing salient emotional stimuli, including anterior insula and orbitofrontal cortex were also negatively correlated with trait anhedonia. Furthermore, effective connectivity between NAc, VTA and paralimbic areas, that regulate emotional reactivity to hedonic stimuli, was negatively correlated with trait anhedonia. Our results indicate that trait anhedonia is associated with reduced reactivity and connectivity of mesolimbic and related limbic and paralimbic systems involved in reward processing. Critically, this association can be detected even in individuals without psychiatric illness. Our findings have important implications both for understanding the neurobiological basis of anhedonia and for the treatment of anhedonia in psychiatric disorders.


The health impacts of preventive cardiovascular medication reduction on older populations: protocol for a systematic review and meta-analysis.
Polypharmacy is inevitable and appropriate for many conditions, but in some cases, it can be problematic resulting in an increased risk of harm and reduced quality of life. There has been an increasing interest to reduce cardioprotective medications in older adults to potentially reduce the risk of harm due to treatment; however, there is no evidence on safety and efficacy to support this practice currently. This paper describes a protocol for a systematic review on the safety and efficacy of reducing cardioprotective medication in older populations. MEDLINE (PubMed), Embase (Ovid), and CENTRAL (Cochrane Central Register of Controlled Trials) will be searched from their inception onwards for relevant studies. Randomised controlled trials and non-randomised studies on interventions (prospective, retrospective cohort, case-control) conducted in older adults (75 years or older) examining reduction of cardioprotective medications will be included. The primary outcome of this study will be all-cause hospitalisation. Secondary outcome variables of interest are all-cause hospitalisation, mortality, quality of life, serious adverse events, major adverse cardiovascular events, falls, fractures, cognitive functioning, bleeding events, renal functioning, medication burden, drug reinstatement, time-in-hospital, and frailty status. Two reviewers will independently screen all citations, full-text articles, and extract data. Confidence in cumulative evidence will be assessed using the GRADE approach; the risk of bias will be assessed by the RoB-II tool for randomised controlled studies and ROBINS-I for non-randomised studies. Where sufficient data are available, we will conduct a random effects meta-analysis by combining the outcomes of the included studies. Sub-group analysis and meta-regression are planned to assess the potential harms and risks of different drug classes and the impacts in different patient populations (e.g. sex, cognitive status, renal status, and age). The study will be a comprehensive review on all published articles identified using our search strategy on the safety and efficacy of cardioprotective medication reduction in the older population. The findings will be crucial to inform clinicians on potential health outcomes of reducing cardiovascular medication in the elderly. PROSPERO CRD42020208223.


Complete plastid genomes from Ophioglossum californicum, Psilotum nudum, and Equisetum hyemale reveal an ancestral land plant genome structure and resolve the position of Equisetales among monilophytes.
Plastid genome structure and content is remarkably conserved in land plants. This widespread conservation has facilitated taxon-rich phylogenetic analyses that have resolved organismal relationships among many land plant groups. However, the relationships among major fern lineages, especially the placement of Equisetales, remain enigmatic. In order to understand the evolution of plastid genomes and to establish phylogenetic relationships among ferns, we sequenced the plastid genomes from three early diverging species: Equisetum hyemale (Equisetales), Ophioglossum californicum (Ophioglossales), and Psilotum nudum (Psilotales). A comparison of fern plastid genomes showed that some lineages have retained inverted repeat (IR) boundaries originating from the common ancestor of land plants, while other lineages have experienced multiple IR changes including expansions and inversions. Genome content has remained stable throughout ferns, except for a few lineage-specific losses of genes and introns. Notably, the losses of the rps16 gene and the rps12i346 intron are shared among Psilotales, Ophioglossales, and Equisetales, while the gain of a mitochondrial atp1 intron is shared between Marattiales and Polypodiopsida. These genomic structural changes support the placement of Equisetales as sister to Ophioglossales + Psilotales and Marattiales as sister to Polypodiopsida. This result is augmented by some molecular phylogenetic analyses that recover the same relationships, whereas others suggest a relationship between Equisetales and Polypodiopsida. Although molecular analyses were inconsistent with respect to the position of Marattiales and Equisetales, several genomic structural changes have for the first time provided a clear placement of these lineages within the ferns. These results further demonstrate the power of using rare genomic structural changes in cases where molecular data fail to provide strong phylogenetic resolution.


Urticaria and bacterial infections.
The association between urticaria and infectious diseases has been discussed for >100 years. However, a causal relationship with underlying or precipitating infection is difficult to establish. The purpose of this work was to perform a systematic analysis of the published cases of urticaria associated with bacterial infections. We give an umbrella breakdown of up-to-date systematic reviews and other important publications on the complex association of urticaria and bacterial infections. We did a Medline search, for English language articles published until January 2014, using the key words "urticaria" and "bacteria/bacterial disease"; a second analysis was performed in groups of bacteria and using each germ name as a key word. Many bacterial infections have been associated with urticaria manifestation, such as Helicobacter pylori, Streptococcus, Staphylococcus, Mycoplasma pneumonia, Salmonella, Brucella, Mycobacterium leprae, Borrelia, Chlamydia pneumonia, and Yersinia enterocolitica. In some cases the skin manifestations, described as urticaria, could be caused by the presence of the microorganism in the skin, or for the action of their toxins, or to the complement activation mediated by circulating immune complexes. Although only a weak association with urticaria of unclear pathogenesis exists, clinicians should consider these bacterial agents in the workup of the patients with urticaria. The eradication of the infection could, in fact, lead to the resolution of urticaria. Prospective studies and well-structured research are obviously needed to better clarify the real role of bacteria in the pathogenesis of urticaria and their relative prevalence.


The study of familial hypercholesterolemia in Italy: A narrative review.
In this review we outline our experience in the clinical and molecular diagnosis of familial hypercholesterolemia (FH), built up over more than three decades. We started our work by selecting FH patients on the basis of stringent clinical criteria, including extensive family studies. In most patients we confirmed the clinical diagnosis by showing a reduced LDLR activity in skin fibroblasts. After the isolation of LDLR cDNA and the characterization of the corresponding gene by the Dallas group, we started a systematic molecular investigation of our patients first using Southern blotting, and, subsequently Sanger sequencing. Up to now we have been able to identify 260 mutations of LDLR gene in more than 1000 genotyped FH patients, including 68 homozygotes. During this survey we identified 13 mutation clusters located in different geographical districts, which gave us the chance to compare the phenotype of patients carrying the most common mutations. We also found that mutations in APOB and PCSK9 genes were a rare cause of FH in our cohort. Despite our efforts, we failed to identify mutations in candidate genes in ∼20% of cases of definite FH. An exome-wide study, conducted within the context of an international collaboration, excluded the presence of other major genes in our unexplained FH cases. Recently, we have adopted sequencing technology of the next generation (NGS) with the parallel sequencing of a panel of FH targeted genes as a way of obtaining a more comprehensive picture of the gene variants potentially involved in the disease.


Effects of recombinant human growth hormone on muscle protein turnover in malnourished hemodialysis patients.
To assess the effect of recombinant human growth hormone (rhGH) on muscle protein metabolism in uremic patients with malnutrition, forearm [3H]phenylalanine kinetics were evaluated in six chronically wasted (body weight 79% of ideal weight) hemodialysis (HD) patients in a self-controlled, crossover study. Forearm protein dynamics were evaluated before, after a 6-wk course of rhGH (5 mg thrice weekly) and after a 6-wk washout period. After rhGH: (a) forearm phenylalanine net balance--the difference between phenylalanine incorporation into and phenylalanine release from muscle proteins--decreased by 46% (-8+/-2 vs. -15+/-2 nmol/min x 100 ml at the baseline and -11+/-2 after washout, P < 0.02); (b) phenylalanine rate of disposal, an index of protein synthesis, increased by 25% (25+/-5 vs. 20+/-5 at the baseline and 20+/-4 after washout, P < 0.03); (c) phenylalanine rate of appearance, an index of protein degradation, was unchanged (33+/-5 vs. 35+/-5 at the baseline and 31+/-4 after washout); (d) forearm potassium release declined (0.24+/-0.13 vs. 0.60+/-0.15 microeq/min at the baseline, and 0.42+/-0.20 microeq/min after washout P < 0.03); (e) changes in the insulin-like growth factor binding protein (IGFBP)-1 levels and insulin-like growth factor-I (IGF-I)/IGFBP-3 ratios accounted for 15.1% and 47.1% of the percent variations in forearm net phenylalanine balance, respectively. Together, these two factors accounted for 62.2% of variations in forearm net phenylalanine balance during and after rhGH administration. These data indicate: (a) that rhGH administration in malnourished hemodialysis patients is followed by an increase in muscle protein synthesis and by a decrease in the negative muscle protein balance observed in the postabsorptive state; and (b) that the reduction in net protein catabolism obtained with rhGH can be accounted for by the associated changes in circulating free, but not total, IGF-I levels.


Evidence-based fitness promotion in an afterschool setting: implementation fidelity and its policy implications.
Little is known about how the adoption of evidence-based physical activity (PA) curricula by out-of-school time (OST) programs affects children's physical fitness, and there are no clear guidelines of what constitutes reasonable gains given the types of PA instruction currently offered in these programs. Using a three-wave, quasi-experimental, naturalistic observation design, this study evaluated the implementation of an evidence-based PA instruction curriculum (Sports, Play, and Active Recreation for Kids [SPARK]) and examined whether the potential health benefits of evidence-based PA instruction can be replicated in this context when compared to OST programs that do not use evidence-based PA curricula. Quality of PA instruction and SPARK implementation fidelity were also assessed. Results indicated that children in the non-evidence-based/standard PA instruction programs engaged in higher levels of moderate-to-vigorous PA (MVPA) and showed greater improvements in fitness levels over time. The findings from this chapter suggest that while it is generally accepted that evidence-based approaches yield higher levels of PA when implemented by researchers under controlled conditions, findings are inconsistent when evidence-based PA instruction is implemented in the field, under presumably less controlled conditions. It appears that when it comes to PA instruction in afterschool, either less structured activities or well-implemented evidence-based practices could be the key to promoting higher PA levels and greater health and fitness for school-aged children.


Yeast intrachromosomal recombination: long gene conversion tracts are preferentially associated with reciprocal exchange and require the RAD1 and RAD3 gene products.
A yeast intrachromosomal recombination system based on an inverted repeat has been designed to examine mitotic gene conversion tract length and the association of crossing over with gene conversion as a function of the conversion tract length. Short conversion tracts are found to be preferentially noncrossover while conversion tracts longer than 1.16 kb show a 50% association with crossover. Mutation in the excision repair gene RAD1 leads to a reduction in conversion tracts of at least 1.16 kb and a reduction in crossovers associated with conversion, regardless of the length of the conversion tract. Mutation in the excision repair gene RAD3, which encodes a DNA helicase, also leads to a reduction in conversion tracts of at least 1.16 kb, but has no effect on the frequency of associated crossovers. The roles of RAD1 and RAD3 in recombination are discussed.


Sexually dimorphic genome-wide binding of retinoid X receptor alpha (RXRα) determines male-female differences in the expression of hepatic lipid processing genes in mice.
Many hepatic functions including lipid metabolism, drug metabolism, and inflammatory responses are regulated in a sex-specific manner due to distinct patterns of hepatic gene expression between males and females. Regulation for the majority of these genes is under control of Nuclear Receptors (NRs). Retinoid X Receptor alpha (RXRα) is an obligate partner for multiple NRs and considered a master regulator of hepatic gene expression, yet the full extent of RXRα chromatin binding in male and female livers is unclear. ChIP-Seq analysis of RXRα and RNA Polymerase2 (Pol2) binding was performed livers of both genders and combined with microarray analysis. Mice were gavage-fed with the RXR ligand LG268 for 5 days (30 mg/kg/day) and RXRα-binding and RNA levels were determined by ChIP-qPCR and qPCR, respectively. ChIP-Seq revealed 47,845 (male) and 46,877 (female) RXRα binding sites (BS), associated with ∼12,700 unique genes in livers of both genders, with 91% shared between sexes. RXRα-binding showed significant enrichment for 2227 and 1498 unique genes in male and female livers, respectively. Correlating RXRα binding strength with Pol2-binding revealed 44 genes being male-dominant and 43 female-dominant, many previously unknown to be sexually-dimorphic. Surprisingly, genes fundamental to lipid metabolism, including Scd1, Fasn, Elovl6, and Pnpla3-implicated in Fatty Liver Disease pathogenesis, were predominant in females. RXRα activation using LG268 confirmed RXRα-binding was 2-3 fold increased in female livers at multiple newly identified RXRα BS including for Pnpla3 and Elovl6, with corresponding ∼10-fold and ∼2-fold increases in Pnpla3 and Elovl6 RNA respectively in LG268-treated female livers, supporting a role for RXRα regulation of sexually-dimorphic responses for these genes. RXRα appears to be one of the most widely distributed transcriptional regulators in mouse liver and is engaged in determining sexually-dimorphic expression of key lipid-processing genes, suggesting novel gender- and gene-specific responses to NR-based treatments for lipid-related liver diseases.


Severe and multiple trauma in older patients; incidence and mortality.
To examine the differences between severely injured older patients (aged over 65 years) compared with similarly injured younger adults in terms of incidence, inpatient mortality and factors predicting outcome. Data prospectively entered into the Trauma Audit and Research Network (TARN) database from our level I trauma unit over a 5-year period were retrospectively examined, with 3172 patients included in the final analysis. Older patients accounted for 13.8% of those with severe injuries (Injury Severity Score 16 or more) and almost 2% of our trauma admissions overall. High energy injuries were responsible for the majority of these injuries though relatively minor trauma became increasingly important in older patients. Mortality rates in the older patients were more than twice those seen in the adult population (19% in the under 40's to almost 50% in the over 75's). Age, Injury Severity Score and Glasgow Coma Score continued to be predictive of mortality in older patients but other factors relevant in younger adults were not. Patients in the older group without physiological derangement on admission were still at a relatively high risk of inpatient mortality. This was in contrast to the younger patients, suggesting that it might be more difficult to predict which older patients might benefit from more aggressive monitoring or treatment. Despite increased mortality in older patients, significant survival rates were achieved even in the oldest. Active treatment should not be withdrawn on the basis of age alone.


DNA replication-dependent intranuclear relocation of double minute chromatin.
Double minutes (DMs) seen in a substantial fraction of human tumors are the cytogenetic manifestation of gene amplification which renders the tumor cells advantageous for growth and survival. DMs are acentric and atelomeric chromatin composed of circular DNA. In this study, we found they showed a remarkable relocation inside the nucleus which was spatially and temporally coupled to DNA replication. Using the human COLO 320DM tumor line, we detected DMs by fluorescence in situ hybridization followed by confocal examination. The location of multi-copy DMs was evaluated statistically by an easy method developed in this study. By examination of a synchronized culture, we found that DMs preferentially located at the nuclear periphery during G1-phase of the cell cycle, which is consistent with the location at M-phase. The peripheral DMs were in contact with the nuclear lamina which was shown by the simultaneous detection of DMs and lamin protein. The peripheral location persisted until the cells reached the G1/S-boundary, then the DMs relocated promptly to inward once the DNA replication started. The relocation was obvious using two different probes that detect DMs, or using two different methods for the cell fixation. Furthermore, the simultaneous detection of DMs and the site of DNA replication suggested that the inward relocation of peripheral DMs initiated just prior to the onset of DNA replication at the periphery. On the other hand, if the same amplified sequences were placed in a chromosome as an homogeneously staining region, they did not show any significant relocation during S-phase. From these and reported results, there may exist a generalized inward motion of some kind of chromatin that precedes the replication of their DNA. DMs might magnify the motion by their acentric, atelomeric or small circular nature.


Seeing the unexpected: how sex differences in stress responses may provide a new perspective on the manifestation of psychiatric disorders.
In this report, the authors propose that underlying sex differences in the biobehavioral response to stress may contribute to the variance in prevalence of some psychiatric disorders based on sex. The authors begin with a discussion of stress physiology and review a new theory on sex differences in stress responses (ie, the "tend-and-befriend" response), which may provide a recent framework for considering sex differences in the manifestation of some psychiatric illnesses. The authors then move to a discussion of major depression and attention deficit hyperactivity disorder as examples of how sex differences in stress responses may influence the behavioral symptoms of psychiatric disorders that are more often diagnosed in one sex compared with another. The authors conclude with a brief discussion of the implications of this new perspective on treatment approaches and encourage further inquiry into the importance of sex-based differences in the behavioral manifestation of some psychiatric illnesses.


Septal-lateral annular cinching ('SLAC) reduces mitral annular size without perturbing normal annular dynamics.
Septal-lateral (S-L) mitral annular diameter reduction is thought to be central to the efficacy of ring annuloplasty in correcting functional mitral regurgitation (MR), but rings perturb mitral annulus (MA) dynamic motion and limit posterior leaflet excursion. The effects of S-L annular cinching ('SLAC'), a novel method for mitral annular reduction, were investigated. Eight adult sheep had multiple radioopaque markers placed on the left ventricle, leaflet edges, and around the MA. The S-L trans-annular suture was anchored to the mid-septal MA and externalized through the mid-lateral MA and left ventricular wall. Animals were studied immediately postoperatively with biplane videofluoroscopy before and after suture cinching to reduce annular size. MA area (MAA) and S-L dimension were calculated throughout the cardiac cycle from the annular marker coordinates. MAA contraction (AMAA) was expressed as percentage decrease from maximum to minimum MAA. Anterior (AML) and posterior (PML) leaflet angular excursion were calculated as the change in angle between each leaflet edge marker and the S-L annular dimension during the cardiac cycle. MA folding was calculated as the change in distance during systole of the mid-septal annular marker from a plane fitted to the lateral MA markers. SLAC reduced end-diastolic (ED) S-L diameter (21.6+/-2.8 versus 17.1+/-2.6 mm; p = 0.0005) and ED MAA (618+/-126 versus 525+/-114 mm2; p = 0.0004), but did not perturb normal AMAA (15.8+/-4.1 versus 15.1+/-4.8%; p = 0.4), annular flexion (2.0+/-0.7 versus 1.8+/-0.7 mm; p = 0.3) or AML excursion (55+/-7 versus 53+/-7 degrees; p = 0.1). PML excursion was decreased only slightly (52+/-11 versus 44+/-12 degrees; p = 0.002). SLAC substantially reduced S-L annular size, but without perturbing normal MA contraction dynamics, MA flexion, or anterior leaflet excursion. This novel surgical method might represent an alternative to mitral annuloplasty for patients with certain types of mitral pathology.


Quantifying cell-surface biomarker expression in thick tissues with ratiometric three-dimensional microscopy.
The burgeoning fields of in vivo three-dimensional (3D) microscopy and endomicroscopy, as well as ex vivo tissue cytometry have introduced new challenges for tissue preparation and staining with exogenous molecular contrast agents. These challenges include effective delivery of the agents, and once delivered, distinguishing between bound verses unbound molecular probes. If applied topically, there are additional issues with rinsing off unbound probe, which can be nonuniform and inefficient in thick tissues, thus leading to ambiguous contrast and a large nonspecific background that may obscure molecule-specific staining. Therefore, we have developed a ratiometric 3D microscopy scheme that not only reduces the effects of nonspecific sources of contrast, but also enables quantification of the relative binding affinity of imaging probes to their biomarker targets. Here we demonstrate this ratiometric approach by simultaneously imaging a HER2/neu (erbB2)-targeted monoclonal antibody labeled with one fluorophore and an isotype-matched negative control antibody labeled with another fluorophore. By taking a pixel-by-pixel calibrated ratio between the signals from each fluorescent image channel, accurate quantification of specific versus nonspecific binding affinity is achieved with cultured cells, yielding data that are in agreement with analyses via flow cytometry. We also demonstrate quantitative 3D microscopic imaging of biomarker expression in tissue models and in thick human biopsy samples of normal, HER2-negative, and HER2-positive breast tumors. This strategy enables rapid, quantitative, and unambiguous volumetric microscopy of biomarker expression in thick tissues, including whole biopsies, and will enable real-time optical assessment of disease markers in the living body.


Atomic-scale sensing of the magnetic dipolar field from single atoms.
Spin resonance provides the high-energy resolution needed to determine biological and material structures by sensing weak magnetic interactions. In recent years, there have been notable achievements in detecting and coherently controlling individual atomic-scale spin centres for sensitive local magnetometry. However, positioning the spin sensor and characterizing spin-spin interactions with sub-nanometre precision have remained outstanding challenges. Here, we use individual Fe atoms as an electron spin resonance (ESR) sensor in a scanning tunnelling microscope to measure the magnetic field emanating from nearby spins with atomic-scale precision. On artificially built assemblies of magnetic atoms (Fe and Co) on a magnesium oxide surface, we measure that the interaction energy between the ESR sensor and an adatom shows an inverse-cube distance dependence (r-3.01±0.04). This demonstrates that the atoms are predominantly coupled by the magnetic dipole-dipole interaction, which, according to our observations, dominates for atom separations greater than 1 nm. This dipolar sensor can determine the magnetic moments of individual adatoms with high accuracy. The achieved atomic-scale spatial resolution in remote sensing of spins may ultimately allow the structural imaging of individual magnetic molecules, nanostructures and spin-labelled biomolecules.


The non-targeted effects of radiation are perpetuated by exosomes.
Exosomes contain cargo material from endosomes, cytosol, plasma membrane and microRNA molecules, they are released by a number of non-cancer and cancer cells into both the extracellular microenvironment and body fluids such as blood plasma. Recently we demonstrated radiation-induced non-targeted effects [NTE: genomic instability (GI) and bystander effects (BE)] are partially mediated by exosomes, particularly the RNA content. However the mechanistic role of exosomes in NTE is yet to be fully understood. The present study used MCF7 cells to characterise the longevity of exosome-induced activity in the progeny of irradiated and unirradiated bystander cells. Exosomes extracted from conditioned media of irradiated and bystander progeny were added to unirradiated cells. Analysis was carried out at 1 and 20/24 population doublings following medium/exosome transfer for DNA/chromosomal damage. Results confirmed exosomes play a significant role in mediating NTE of ionising radiation (IR). This effect was remarkably persistent, observed >20 doublings post-irradiation in the progeny of bystander cells. Additionally, cell progeny undergoing a BE were themselves capable of inducing BE in other cells via exosomes they released. Furthermore we investigated the role of exosome cargo. Culture media from cells exposed to 2 Gy X-rays was subjected to ultracentrifugation and four inoculants prepared, (a) supernatants with exosomes removed, and pellets with (b) exosome proteins denatured, (c) RNA degraded, and (d) a combination of protein-RNA inactivation. These were added to separate populations of unirradiated cells. The BE was partially inhibited when either exosome protein or exosome RNA were inactivated separately, whilst combined RNA-protein inhibition significantly reduced or eliminated the BE. These results demonstrate that exosomes are associated with long-lived signalling of the NTE of IR. Both RNA and protein molecules of exosomes work in a synergistic manner to initiate NTE, spread these effects to naïve cells, and perpetuate GI in the affected cells.


Impact of neighborhood resources on cardiovascular disease: a nationwide six-year follow-up.
Living in a socially deprived neighborhood is associated with lifestyle risk factors, e.g., smoking, physical inactivity and unhealthy diet, as well as an increased risk of cardiovascular disease, i.e., coronary heart disease and stroke. The aim was to study whether the odds of cardiovascular disease vary with the neighbourhood availability of potentially health-damaging and health-promoting resources. A nationwide sample of 2 040 826 men and 2 153 426 women aged 35-80 years were followed for six years for first hospitalization of coronary heart disease or stroke. Neighborhood availability of health-damaging resources (i.e., fast-food restaurants and bars/pubs) and health-promoting resources (i.e., health care facilities and physical activity facilities) were determined by use of geographic information systems (GIS). We found small or modestly increased odds ratios (ORs) for both coronary heart disease and stroke, related to the availability of both health-damaging and health-promoting resources. For example, in women, the unadjusted OR (95 % confidence interval) for stroke in relation to availability of fast-food restaurants was 1.18 (1.15-1.21). Similar patterns were observed in men, with an OR = 1.08 (1.05-1.10). However, the associations became weaker or disappeared after adjustment for neighborhood-level deprivation and individual-level age and income. This six year follow-up study shows that neighborhood availability of potentially health-damaging as well as health-promoting resources may make a small contribution to the risk of coronary heart disease and stroke. However, most of these associations were attenuated or disappeared after adjustment for neighborhood-level deprivation and individual-level age and income. Future studies are needed to further examine factors in the causal pathway between neighborhood deprivation and cardiovascular disease.


Evidence for tetrasomic inheritance in a tetraploid Solanum commersonii (+) S. tuberosum somatic hybrid through the use of molecular markers.
In order to assess the potential for interspecific recombination between the cultivated Solanum tuberosum (tbr) and the sexually isolated wild species Solanum commersonii(cmm), genetic analysis of a F(2) progeny obtained by selfing one tetraploid cmm (+) tbr somatic hybrid was performed through molecular markers. For this purpose, the extent of disomic and/or tetrasomic inheritance of species-specific RAPD and AFLP markers was determined by following their segregation in a 90-genotype progeny, and testing all the possible segregation ratios in a selfed tetraploid progeny. The RAPD analysis performed using 16 primers revealed that the cmm-specific RAPDs were mainly (93.7%) duplex markers and were equally distributed between loci with a disomic (46.7%) and tetrasomic (53.3%) inheritance. The AFLP analysis led to the identification of 272 (58%) informative AFLPs, which were either cmm- or tbr-specific markers. About 63% of cmm-specific AFLPs were duplex loci, most of which (92.6%) were inherited as tetrasomic loci. As regards the tbr-specific AFLPs, the percentage of simplex loci (52.9%) was higher than that of duplex loci (32.6%), and among the latter most (88.5%) were inherited as tetrasomic loci. Overall, 130 duplex markers were found, of which 53.1% were cmm-specific and 46.9% were tbr-specific. Out of 130 markers, 18 (13.8%) were inherited as disomic, and 112 (86.2%) as tetrasomic, loci. This implies that the majority of duplex markers were located on chromosomes which at meiosis tend to randomly pair as bivalents or to form tetravalents. The total number of simplex loci was 119, and most of them (82.3%) were tbr-specific loci. In some cases the observed segregation ratios even allowed us to clearly determine whether a random chromosome or chromatid segregation was detected. This was the case of three cmm-specific RAPDs, 19 cmm- and 25 tbr-specific AFLPs, which fit a 20.8:1 or 2.5:1 ratio, both cases for which a clear random chromatid segregation can be assumed, since they represent the limit of segregation expected when the distance between the locus and the centromere always leads to a cross-over event. The percentage of ascertained crossing-over events was around 37% out of the tetrasomically inherited loci clearly identified (128 loci), a value indicating that the flow of genes from the sexually isolated S. commersonii to the cultivated potato is possible, for at least a large proportion of genes.


Environmental risk factors for autism: do they help cause de novo genetic mutations that contribute to the disorder?
Recent research has discovered that a number of genetic risk factors for autism are de novo mutations. Advanced parental age at the time of conception is associated with increased risk for both autism and de novo mutations. We investigated the hypothesis that other environmental factors associated with increased risk for autism might also be mutagenic and contribute to autism by causing de novo mutations. A survey of the research literature identified 9 environmental factors for which increased pre-conceptual exposure appears to be associated with increased risk for autism. Five of these factors--mercury, cadmium, nickel, trichloroethylene, and vinyl chloride--are established mutagens. Another four--including residence in regions that are urbanized, located at higher latitudes, or experience high levels of precipitation--are associated with decreased sun exposure and increased risk for vitamin D deficiency. Vitamin D plays important roles in repairing DNA damage and protecting against oxidative stress--a key cause of DNA damage. Factors associated with vitamin D deficiency will thus contribute to higher mutation rates and impaired repair of DNA. We note how de novo mutations may also help explain why the concordance rate for autism is so markedly higher in monozygotic than dizygotic twins. De novo mutations may also explain in part why the prevalence of autism is so remarkably high, given the evidence for a strong role of genetic factors and the low fertility of individuals with autism--and resultant selection pressure against autism susceptibility genes. These several lines of evidence provide support for the hypothesis, and warrant new research approaches--which we suggest--to address limitations in existing studies. The hypothesis has implications for understanding possible etiologic roles of de novo mutations in autism, and it suggests possible approaches to primary prevention of the disorder, such as addressing widespread vitamin D deficiency and exposure to known mutagens.


Male breast cancer: a 30-year experience in South Australia.
Male breast cancer (MBC) is an uncommon disease with a paucity of information in the literature. The treatment of MBC has traditionally been extrapolated from experience with female breast cancer. This study reports on the treatment and outcomes of this disease in South Australia over a 30-year period. From 1977 to 2007 63 patients with a median age of 62 years (range 33-85 years) were identified and treated for MBC. Data obtained, included initial stage, pathological features, treatment and outcomes. With a median follow up of 4.9 years (range 2 months to 19 years) the 5-year overall survival (OS) rate was 85% with median survival of 5.5 years. In all, 18 (29%) were diagnosed with recurrent disease, while 45 (72%) remained disease free. The median time to recurrence was 2.5 years. One patient failed locally, three (4%) had locoregional recurrence and distant recurrence was noted in 14 patients (22%). Disease stage at presentation was a significant predictor of 5-year OS and recurrence (P = 0.012 and P = 0.0001). Tumor diameter was also a significant predictor of 5-year OS and recurrence (P = 0.006 and P = 0.021). This retrospective series has a 5-year OS that compares favorably with other published series of MBC. The positive findings may help change the misperception that MBC is an inherently aggressive disease process with a poor clinical outcome. Further studies are needed to carefully and thoroughly investigate this rare but treatable disease.


Missed opportunities for vaccination in Peru 2010-2020: A study of socioeconomic inequalities.
Missed Opportunities for Vaccination (MOV) represent a major risk in the re-emergence of immunopreventable diseases. However, in the region, there are few published studies on MOVs using national databases such as demographic and health surveys (DHS). This study aims to describe the frequency and trends of MOVs for the first dose of vaccines against the leading causes of infant morbidity and mortality, their complete vaccination coverage, and trends in socioeconomic inequalities at the national and departmental levels for an 11-years period. Using DHS data from an 11-year period (2010-2020), we calculated frequencies and trends in MOVs of vaccines for the leading causes of child morbidity and mortality, estimated inequalities in MOVs using the Slope Inequality Index (SII) and conducted a spatial autocorrelation test to identify clusters of higher or lower inequality in MOVs at the national level. We found that, at the national level, greater inequality was concentrated in the wealthiest categories of each socioeconomic variable. We identified that departments with higher poverty rates concentrated higher levels of inequality in the MOVs in the lowest strata of the socioeconomic variables. In addition, we found that some departments with similar geographic and socioeconomic characteristics had spatially correlated levels of inequality on MOVs. These findings can help to identify the heterogeneity that exists in the distribution of MOVs among departments and socioeconomic strata, which would help to prioritize specific areas and subpopulations for national immunization strategies. No additional funding source was required for this study.


Regional differences in retinoid release from embryonic neural tissue detected by an in vitro reporter assay.
Retinoic acid and related retinoids have been suggested to contribute to the pattern of cell differentiation during vertebrate embryonic development. To identify cell groups that release morphogenetically active retinoids, we have developed a reporter assay that makes use of a retinoic acid inducible response element (RARE) to drive lacZ or luciferase reporter genes in stably transfected cell lines. This reporter gene assay allows detection of retinoids released from embryonic tissues over a range equivalent to that induced by femtomole amounts of retinoic acid. We have used this assay first to determine whether the floor plate, a cell group that has polarizing properties in neural tube and limb bud differentiation, is a local source of retinoids within the spinal cord. We have also examined whether the effects of exogenously administered retinoic acid on anteroposterior patterning of cells in the developing central nervous system correlate with differences in retinoid release from anterior and posterior neural tissue. We find that the release of morphogenetically active retinoids from the floor plate is only about 1.5-fold that of the dorsal spinal cord, which does not have neural tube or limb polarizing activity. These results suggest that the spatial distribution of retinoid release from spinal cord tissues differs from that of the neural and limb polarizing activity. This assay has also shown that retinoids are released from the embryonic spinal cord at much greater levels than from the forebrain. This result, together with previous observations that the development of forebrain structures is suppressed by low concentrations of retinoic acid, suggest that the normal development of forebrain structures is dependent on the maintenance of low concentrations of retinoids in anterior regions of the embryonic axis. This assay has also provided initial evidence that other embryonic tissues with polarizing properties in vivo release retinoids in vitro.


Temporal genetic variation of the red fox, Vulpes vulpes, across western Europe and the British Isles.
Quaternary climatic fluctuations have had profound effects on the phylogeographic structure of many species. Classically, species were thought to have become isolated in peninsular refugia, but there is limited evidence that large, non-polar species survived outside traditional refugial areas. We examined the phylogeographic structure of the red fox (Vulpes vulpes), a species that shows high ecological adaptability in the western Palaearctic region. We compared mitochondrial DNA sequences (cytochrome b and control region) from 399 modern and 31 ancient individuals from across Europe. Our objective was to test whether red foxes colonised the British Isles from mainland Europe in the late Pleistocene, or whether there is evidence that they persisted in the region through the Last Glacial Maximum. We found red foxes to show a high degree of phylogeographic structuring across Europe and, consistent with palaeontological and ancient DNA evidence, confirmed via phylogenetic indicators that red foxes were persistent in areas outside peninsular refugia during the last ice age. Bayesian analyses and tests of neutrality indicated population expansion. We conclude that there is evidence that red foxes from the British Isles derived from central European populations that became isolated after the closure of the landbridge with Europe.


An fNIRS-Based Motor Imagery BCI for ALS: A Subject-Specific Data-Driven Approach.
Functional near-infrared spectroscopy (fNIRS) has recently gained momentum in research on motor-imagery (MI)-based brain-computer interfaces (BCIs). However, strikingly, most of the research effort is primarily devoted to enhancing fNIRS-based BCIs for healthy individuals. The ability of patients with amyotrophic lateral sclerosis (ALS), among the main BCI end-users to utilize fNIRS-based hemodynamic responses to efficiently control an MI-based BCI, has not yet been explored. This study aims to quantify subject-specific spatio-temporal characteristics of ALS patients' hemodynamic responses to MI tasks, and to investigate the feasibility of using these responses as a means of communication to control a binary BCI. Hemodynamic responses were recorded using fNIRS from eight patients with ALS while performing MI-Rest tasks. The generalized linear model (GLM) analysis was conducted to statistically estimate and evaluate individualized spatial activation. Selected channel sets were statistically optimized for classification. Subject-specific discriminative features, including a proposed data-driven estimated coefficient obtained from GLM, and optimized classification parameters were identified and used to further evaluate the performance using a linear support vector machine (SVM) classifier. Inter-subject variations were observed in spatio-temporal characteristics of patients' hemodynamic responses. Using optimized classification parameters and feature sets, all subjects could successfully use their MI hemodynamic responses to control a BCI with an average classification accuracy of 85.4% ± 9.8%. Our results indicate a promising application of fNIRS-based MI hemodynamic responses to control a binary BCI by ALS patients. These findings highlight the importance of subject-specific data-driven approaches for identifying discriminative spatio-temporal characteristics for an optimized BCI performance.


Patient medication knowledge and adherence to asthma pharmacotherapy: a pilot study in rural Australia.
Asthma is a chronic disease with both inflammatory and bronchoconstrictive elements and often requires multiple medications. Most asthma regimens include medications with different therapeutic modes of action and a number of different medication delivery devices. To effectively participate in their asthma management, patients need to recognize each of their medication types, understand their purpose, adhere to their treatment regimen, and be proficient in using the required delivery devices. This study evaluated patient knowledge of asthma pharmacotherapy and adherence. An interview study was undertaken in two rural locations, in Australia, to elicit participants' knowledge, use, and inhalation device technique. Of participants, 75.9% used preventer medication and the remaining 24.1% used reliever medication only. Of those using preventer medication, 82.5% could distinguish their preventer from a range of asthma medicines. Metered dose inhalers (MDIs) were used by 80% of participants; 23% used a Turbuhaler(R); 24% used an Accuhaler(R); and 5% used an MDI with a spacer device. The study established poor medication knowledge, suboptimal device technique, and disturbing levels of adherence with management recommendations. Asthma education strategies need to be modified to engage patients with low asthma knowledge to achieve improved patient outcomes. Further, strategies need to motivate patients to use preventer medication during times when they feel well.


The electronic structure of perfluorodecalin studied by soft X-ray spectroscopy and electronic structure calculations.
Fluorine and carbon K absorption and emission spectra of liquid perfluorodecalin are presented and analyzed in terms of density functional calculations-configuration interaction. A comprehensive view of the electronic structure is given, and site-specific intramolecular interactions are investigated in detail. It is found that, while the outer fluorine atoms have excess charge in the ground state, the lowest excitations must be associated with charge transfer towards the inner carbon atoms.


From single biobanks to international networks: developing e-governance.
The future holds the possibility to link and network biobanks, existing biorepositories and reference databases for research purposes in ways that have not been possible before. There is the potential to develop 'research portals' that will enable researchers to access these research resources that are located around the globe with the click of a mouse. In this paper, I will argue that our current governance system for research is unable to provide all of the oversight and accountability mechanisms that are required for this new way of doing research that is based upon flows of data across international borders. For example, our current governance framework for research is nationally based, with a complex system of laws, policies and practice that can be unique to a jurisdiction. It is also evident that many of the nationally based governance bodies in this field do not have the legal powers or expertise to adjudicate on the complex issues, such as privacy and disclosure risks that are raised by cross-border data sharing. In addition, the conceptual underpinning of this research governance structure is based on the "one researcher, one project, one jurisdiction" model. In the conclusion of this paper, I lay out some preliminary ideas as to how this system has to change to accommodate research that is based on networks. I suggest that a move to digital governance mechanisms might be a start to making research governance systems more appropriate for the 21st century.


Feasibility of Using Distal Endpoints for In-room PET Range Verification of Proton Therapy.
In an effort to verify the dose delivery in proton therapy, Positron Emission Tomography (PET) scans have been employed to measure the distribution of β+ radioactivity produced from nuclear reactions of the protons with native nuclei. Because the dose and PET distributions are difficult to compare directly, the range verification is currently carried out by comparing measured and Monte Carlo (MC) simulation predicted PET distributions. In order to reduce the reliance on MC, simulated PET (simPET) and dose distal endpoints were compared to explore the feasibility of using distal endpoints for in-room PET range verification. MC simulations were generated for six head and neck patients with corrections for radiological decay, biological washout, and PET resolution. One-dimensional profiles of the dose and simPET were examined along the direction of the beam and covering the cross section of the beam. The chosen endpoints of the simPET (x-intercept of the linear fit to the distal falloff) and planned dose (20-50% of maximum dose) correspond to where most of the protons are below the threshold energy for the nuclear reactions. The difference in endpoint range between the distal surfaces of the dose and MC-PET were compared and the spread of range differences were assessed. Among the six patients, the mean difference between MC-PET and dose depth was found to be -1.6 mm to +0.5 mm between patients, with a standard deviation of 1.1 to 4.0 mm across the individual beams. In clinical practice, regions with deviations beyond the safety margin need to be examined more closely and can potentially lead to adjustments to the treatment plan.


Investigating the impact of captivity and domestication on limb bone cortical morphology: an experimental approach using a wild boar model.
The lack of bone morphological markers associated with the human control of wild animals has prevented the documentation of incipient animal domestication in archaeology. Here, we assess whether direct environmental changes (i.e. mobility reduction) could immediately affect ontogenetic changes in long bone structure, providing a skeletal marker of early domestication. We relied on a wild boar experimental model, analysing 24 wild-born specimens raised in captivity from 6 months to 2 years old. The shaft cortical thickness of their humerus was measured using a 3D morphometric mapping approach and compared with 23 free-ranging wild boars and 22 pigs from different breeds, taking into account sex, mass and muscle force differences. In wild boars we found that captivity induced an increase in cortical bone volume and muscle force, and a topographic change of cortical thickness associated with muscular expression along a phenotypic trajectory that differed from the divergence induced by selective breeding. These results provide an experimental proof of concept that changes in locomotor behaviour and selective breeding might be inferred from long bones morphology in the fossil and archaeological record. These trends need to be explored in the archaeological record and further studies are required to explore the developmental changes behind these plastic responses.


A hyphenated optical trap capillary electrophoresis laser induced native fluorescence system for single-cell chemical analysis.
Single-cell measurements allow a unique glimpse into cell-to-cell heterogeneity; even small changes in selected cells can have a profound impact on an organism's physiology. Here an integrated approach to single-cell chemical sampling and assay are described. Capillary electrophoresis (CE) with laser-induced native fluorescence (LINF) has the sensitivity to characterize natively fluorescent indoles and catechols within individual cells. While the separation and detection approaches are well established, the sampling and injection of individually selected cells requires new approaches. We describe an optimized system that interfaces a single-beam optical trap with CE and multichannel LINF detection. A cell is localized within the trap and then the capillary inlet is positioned near the cell using a computer-controlled micromanipulator. Hydrodynamic injection allows cell lysis to occur within the capillary inlet, followed by the CE separation and LINF detection. The use of multiple emission wavelengths allows improved analyte identification based on differences in analyte fluorescence emission profiles and migration time. The system enables injections of individual rat pinealocytes and quantification of their endogenous indoles, including serotonin, N-acetyl-serotonin, 5-hydroxyindole-3-acetic acid, tryptophol and others. The amounts detected in individual cells incubated in 5-hydroxytryptophan ranged from 10(-14) mol to 10(-16) mol, an order of magnitude higher than observed in untreated pinealocytes.


The Michaelis Complex of Arginine Kinase Samples the Transition State at a Frequency That Matches the Catalytic Rate.
Arginine kinase (AK), which is a member of the phosphagen kinase family, serves as a model system for studying the structural and dynamic determinants of biomolecular enzyme catalysis of all major states involved of the enzymatic cycle. These states are the apo state (substrate free), the Michaelis complex analogue AK:Arg:Mg·AMPPNP (MCA), a product complex analogue AK:pAIE:Mg·ADP (PCA), and the transition state analogue AK:Arg:Mg·ADP:NO3- (TSA). The conformational dynamics of these states have been studied by NMR relaxation dispersion measurements of the methyl groups of the Ile, Leu, and Val residues at two static magnetic fields. Although all states undergo significant amounts of μs-ms time scale dynamics, only the MCA samples a dominant excited state that resembles the TSA, as evidenced by the strong correlation between the relaxation dispersion derived chemical shift differences Δω and the equilibrium chemical shift differences Δδ of these states. The average lifetime of the MCA is 36 ms and the free energy difference to the TSA-like form is 8.5 kJ/mol. It is shown that the conformational energy landscape of the Michaelis complex analogue is shaped in a way that at room temperature it channels passage to the transition state, thereby determining the rate-limiting step of the phosphorylation reaction of arginine. Conversely, relaxation dispersion experiments of the TSA reveal that it samples the structures of the Michaelis complex analogue or the apo state as its dominant excited state. This reciprocal behavior shows that the free energy of the TSA, with all ligands bound, is lower by only about 8.9 kJ/mol than that of the Michaelis or apo complex conformations with the TSA ligands present.


Alkaline Earth Metal and Lanthanide(III) Complexes of Ligands Based upon 1,4,7,10-Tetraazacyclododecane-1,7-bis(acetic acid).
The macrocyclic ligand DO2A (1,4,7,10-tetraazacyclododecane-1,7-bis(acetic acid)) was prepared and used as a building block for four new macrocyclic ligands having mixed side-chain chelating groups. These ligands and their complexes with Mg(II), Ca(II), and Ln(III) were studied extensively by potentiometry, high-resolution NMR, and water proton relaxivity measurements. The protonation constants of all compounds compared well with those of other cyclen-based macrocyclic ligands. All Ca(II) complexes were found to be more stable than the corresponding Mg(II) complexes. Trends for the stabilities of the Ln(III) complexes are discussed and compared with literature data, incorporating the effects of water coordination numbers, Ln(III) contraction, and the nature of the side chains and the steric hindrance between them. (1)H NMR titrations of DO2A revealed that the first and second protonations take place preferentially at the secondary ring nitrogens, while the third and fourth involved protonation of the acetates. (17)O NMR shifts showed that the DyDO2A(+) complex had two inner-sphere water molecules. Water proton spin-lattice relaxation rates for the GdDO2A(+) complex were also consistent with water exchange between bulk water and two inner-sphere Gd(III) coordination positions. Upon formation of the diamagnetic complexes of DO2A (Ca(II), Mg(II), La(III), and Lu(III)), all of the macrocyclic ring protons became nonequivalent due to slow conformational rearrangements, while the signals for the acetate CH(2) protons remained a singlet.


Changes in embryonic stem cell colony morphology and early differentiation markers driven by colloidal crystal topographical cues.
The use of materials properties to guide cell behaviour is an attractive option for regenerative medicine, where controlling stem cell behaviour is important for the establishment of a functioning cell population. A wide range of materials properties have been shown to influence many types of cells but little is known about the effects of topography on embryonic stem cells (ESCs). In order to advance this knowledge, we synthesised and characterised substrates formed of silica colloidal crystal (SCC) microspheres to present highly ordered and reproducible topographical features from 120-600 nm in diameter. We found that, compared to cells cultured on flat glass, cells cultured on the SCC substrates retained transcription of stem cell (Dppa5a, Nanog, and Pou5f1) and endoderm (Afp, Gata4, Sox17, and Foxa2) markers more similar to undifferentiated ESCs, suggesting the substrates are restricting differentiation, particularly towards the endoderm lineage. Additionally, five days after seeding, we observed strikingly different colony morphology, with cells on the SCC substrates growing in spherical colonies approximately ten cells thick, while cells on glass were growing in flat monolayers. Colonies on the SCC substrates developed a central pit, which was never observed in cells cultured on glass, and expressed proteins related to epithelialisation. Together, these data demonstrate the potential of using topographical cues to control stem cell behaviour in vitro.


Maspin inhibits cell migration in the absence of protease inhibitory activity.
Maspin is a member of the serpin family of protease inhibitors and is a tumor suppressor gene acting at the level of tumor invasion and metastasis. This in vivo activity correlates with the ability of maspin to inhibit cell migration in vitro. This behavior suggests that maspin inhibits matrix-degrading proteases, such as those of the plasminogen activation system, in a similar manner to the serpin PAI-1. However, there is controversy concerning the protease inhibitory activity of maspin. It is devoid of activity against a wide range of proteases, in common with other non-inhibitory serpins, but has recently been reported to inhibit plasminogen activators associated with cells and other biological surfaces (Sheng, S. J., Truong, B., Fredrickson, D., Wu, R. L., Pardee, A. B., and Sager, R. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 499-504; McGowen, R., Biliran, H., Jr., Sager, R., and Sheng, S. (2000) Cancer Res. 60, 4771-4778). We have compared the effects of maspin with those of PAI-1 in a range of situations in which plasminogen activation is potentiated, reflecting the biological context of this proteolytic system: urokinase-type plasminogen activator bound to its receptor on the surface of tumor cells, tissue-type plasminogen activator specifically bound to vascular smooth muscle cells, fibrin, and the prion protein. Maspin was found to have no inhibitory effect in any of these situations, in contrast to the efficient inhibition observed with PAI-1, but nevertheless maspin inhibited the migration of both tumor and vascular smooth muscle cells. We conclude that maspin is a non-inhibitory serpin and that protease inhibition does not account for its activity as a tumor suppressor.


Association between depression and anxiety scores and inflammation in patients with isolated coronary artery ectasia.
Depression and anxiety disorders are frequently found in combination with obstructive coronary artery disease. Coronary artery ectasia (CAE) is an atypical form of coronary artery disease, the etiology of which has not yet been clearly defined. The aim of this study was to assess the existence of a relationship between anxiety/depression and CAE. A CAE group (n=41; mean age: 58.9±9.0 years) and a control group (n=42; mean age: 58.0±9.6 years) were compared. The anxiety and depression status of patients was evaluated using the Hospital Anxiety and Depression Scale (HADS) questionnaire. Age, sex, ejection fraction, and cardiovascular risk factor data were similar in both groups. The serum Creactive protein (CRP) and uric acid levels as well as the leukocyte count were significantly higher in the CAE group (p<0.05). The HADS anxiety score was higher in the CAE group, but without statistical significance (p=0.23). The HADS depression score and total HADS score was significantly higher in the CAE group (p<0.001 and p<0.001). The total HADS score and the HADS depression score were correlated with the serum CRP level (r=0.489; p<0.001 and r=0.543; p<0.001, respectively), whereas the anxiety score was not correlated with CRP (r=0.85; p=0.23). The depression score, CRP, and uric acid levels were greater in patients with isolated CAE compared with those of patients with normal coronaries. The anxiety score did not demonstrate a relationship to CAE; however, there was an association between the depression score and CRP, which is an inflammatory marker.


Potential role for ligand-gated ion channels after seizure-induced neurogenesis.
Epileptic seizures result in an increased generation of new neurons in the dentate gyrus of the adult mammalian hippocampus. The role of these seizure-induced newborn neurons in the process of epileptogenesis remains largely unknown. Recent work, however, suggests an aberrant incorporation of newborn cells into the existing hippocampal network in such a way that they promote hippocampal hyperexcitability. In the present review, we discuss current knowledge about the possible role of seizure-induced newly generated neurons and the putative involvement of ligand-gated ion channels in the process of epileptogenesis.


Role of Vitamins B, C, and D in the fight against tuberculosis.
Worldwide, tuberculosis (TB) is still a serious and significant health concern, more so with the emergence of multidrug-resistant-TB. The inability of mankind to control this infection stems from the fact that the vaccines and drugs that were once effective against TB are no longer efficacious. This has led to a search for new antituberculous agents and adjuvant therapy. Vitamins are being revisited for their role in pathogenicity as well as for their antimycobacterial properties. Vitamins such as biotin and thiamin are essential for Mycobacterium tuberculosis and are required for establishment of infection. On the other hand, vitamins such as Vitamin C and Vitamin D have been shown to possess antimycobacterial properties. To combat M. tuberculosis, innovative strategies need to be devised, keeping in mind the efficacy of the agent to be used. Vitamins can prove to be useful agents capable of modifying the life cycle and biology of M. tuberculosis. We present here a brief overview of the available knowledge on thiamin, biotin, Vitamin C, and Vitamin D, keeping TB treatment and control in perspective.


Possible association of mitochondrial transcription factor A (TFAM) genotype with sporadic Alzheimer disease.
Mitochondrial transcription factor A (TFAM) is essential for transcription and replication of mammalian mitochondrial DNA (mtDNA). Disturbance of maintenance of mtDNA integrity or mitochondrial function may underlay neurodegenerative disorders such as Alzheimer disease (AD). TFAM, the gene encoding TFAM maps to chromosome 10q21.1, a region that showed linkage to late-onset AD in several study samples. We screened TFAM for single nucleotide polymorphisms (SNPs) and genotyped the G>C SNP rs1937, coding for S12T in mitochondrial signal sequence of TFAM, and the A>G SNP rs2306604 (IVS4+113A>G) in 372 AD patients and 295 nondemented control subjects. There was an association of genotype rs1937G/G with AD in females and an association of a TFAM haplotype with AD both in the whole sample and in females. The findings suggest that a TFAM haplotype containing rs1937 G (for S12) may be a moderate risk factor for AD.


How dependent is the alcohol industry on heavy drinking in England?
To understand the extent of the alcohol industry's financial dependence on drinking above government low-risk guidelines in England. Scenario modelling using descriptive analyses of pooled data from the 2013 and 2014 waves of two nationally representative surveys: the Health Survey for England (HSE) and the Living Costs and Food Survey (LCF). We estimated the proportion of alcohol sales revenue accounted for by drinkers above guideline levels, and how this varies between different beverage and retailer types. We then estimated the impact on sales revenue if the entire population reduced their drinking to within guideline levels, as well as the average price increases necessary to compensate for such a loss of revenue. England. A total of 16 872 individual (HSE) and 9975 household (LCF) survey respondents. Transaction-level estimates of volume of alcohol purchased and price paid by beverage type and trade sector. Those drinking above guideline levels are estimated to account for 68% of total alcohol sales revenue in 2013/14: 81% of off-trade revenue and 60% of on-trade revenue. This represents 77% of beer, 70% of cider, 66% of wine and 50% of spirits sales value. The heaviest drinking 4% of the population account for 30% of all consumption and 23% of all industry revenue. If all consumers reduced their drinking to within guideline levels, alcohol sales revenue could decline by 38% (£13 billion). To mitigate this loss, average prices paid would have to rise substantially-for example, by £2.64 for a pint of on-trade beer or £12.25 for a 70 cl bottle of off-trade spirits. In England, the alcohol industry appears to be highly financially dependent upon heavy drinking, and might face significant financial losses were consumers to drink within guideline levels.


Antibiotic-induced release of endotoxin from bacteria in vitro.
The ability of cefotaxime, ciprofloxacin, piperacillin and tobramycin to cause release of endotoxin was examined in vitro with cultures of Enterobacter cloacae and Escherichia coli. Endotoxin was measured by a quantitative limulus amoebocyte lysate assay and its presence was confirmed by silver staining of the lipopolysaccharide moiety following SDS-PAGE. The morphology of the bacteria during antibiotic exposure was examined by scanning electronmicroscopy. Cefotaxime, ciprofloxacin and piperacillin caused significant endotoxin release, correlating with their ability to affect cell-wall morphology, causing filamentation, wall breakage and cell lysis. In contrast, little endotoxin was released when bacteria were exposed to tobramycin and no morphological changes were observed when bacteria were exposed to bactericidal concentrations of this aminoglycoside. Its antimicrobial spectrum and bactericidal activity make tobramycin an appropriate agent for treatment of sepsis caused by gram-negative bacteria and its lack of propensity to elicit excessive release of endotoxin may avoid exacerbation of endotoxin-related shock in sepsis.


Uncovering genomic causes of co-morbidity in epilepsy: gene-driven phenotypic characterization of rare microdeletions.
Patients with epilepsy often suffer from other important conditions. The existence of such co-morbidities is frequently not recognized and their relationship with epilepsy usually remains unexplained. We describe three patients with common, sporadic, non-syndromic epilepsies in whom large genomic microdeletions were found during a study of genetic susceptibility to epilepsy. We performed detailed gene-driven clinical investigations in each patient. Disruption of the function of genes in the deleted regions can explain co-morbidities in these patients. Co-morbidities in patients with epilepsy can be part of a genomic abnormality even in the absence of (known) congenital malformations or intellectual disabilities. Gene-driven phenotype examination can also reveal clinically significant unsuspected condition.


Marine sponge-derived polymeric alkylpyridinium salts as a novel tumor chemotherapeutic targeting the cholinergic system in lung tumors.
Previous studies have shown that the cholinergic system plays a pivotal rule in small cell lung cancer (SCLC) cell growth through an autocrine loop that activates the nicotinic cholinergic receptor, which together with the activation of this receptor by nicotine links SCLC evolution with tobacco use. Non-small cell lung cancer (NSCLC) is the most common form of lung cancer and is also linked to tobacco use. Here we describe the presence of molecules of the cholinergic system in NSCLC samples and cell lines and investigate the implications of the cholinergic system in cell growth regulation. Cholino-acetyltransferase (ChAT), vesicular acetylcholine transporter (VAChT) and acetylcholinesterase (AChE) were observed in NSCLC tumor biopsies and in NSCLC cell lines. Polymeric alkylpyridinium salts (poly-APS) are AChE inhibitors isolated from the crude extract of the marine sponge, Reniera sarai. These metabolites were characterized as a mixture of two polymers of 3-octylpyridinium, including 29 and 99 monomeric units. Exposure of normal lung fibroblast and NSCLC cell lines to poly-APS revealed a selective cytotoxicity for cancer cells as compared to the normal fibroblast cell lines. FACS analysis indicated poly-APS induced apoptosis in NSCLC cells but not in normal lymphocytes. Non-toxic doses of poly-APS also potently reduced NSCLC cell-cell adhesion in suspension cultures. The limited toxicity of poly-APS on normal cells was confirmed by injection in the caudal vein of mice. No overt effects on health parameters, such as weight gain and physical behavior, were observed, and histological analysis of major organs did not reveal differences between the treated animals as compared to controls. These data demonstrate that NSCLC cells express cholinergic molecules that may be involved in cell growth regulation and that the cholinesterase inhibitor, poly-APS, shows selective toxicity toward NSCLC cells while having no apparent toxicity towards normal cells and tissue in vitro and in vivo.


Modulated Electrohyperthermia in Integrative Cancer Treatment for Relapsed Malignant Glioblastoma and Astrocytoma: Retrospective Multicenter Controlled Study.
There are interesting studies on glioma therapy with modulated electrohyperthermia (mEHT), which combines heat therapy with an electric field. Clinical researchers not only found the mEHT method feasible for palliation but also reported evidence of therapeutic response. To study the efficacy and safety of mEHT for the treatment of relapsed malignant glioma and astrocytoma versus best supportive care (BSC). We collected data retrospectively on 149 patients affected by malignant glioma and astrocytoma. Inclusion criteria were informed consent signed; >18 years old; histological diagnosis of malignant glioma or astrocytoma; relapsed after surgery, adjuvant temozolomide-based chemotherapy, and radiotherapy; and indication for treatment with mEHT in palliative setting. mEHT was performed with capacitive coupling technique keeping the skin surface at 26°C and the tumor temperature at 40°C to 42.5°C for > 90% of treatment duration (20-60 minutes). The applied power was 40 to 150 W using a step-up heating protocol. Results from patients treated with mEHT were compared with those treated with BSC. A total of 149 consecutive patients were enrolled in the study, 111 (74%) had glioblastoma multiforme (GBM), and 38 (26%) had astrocytoma (AST). mEHT was performed for 28 (25%) of GBM and 24 (63%) of AST patients. Tumor response at the 3-month follow-up was observed in 29% and 48% of GBM and AST patients after mEHT, and in 4% and 10% of GBM and AST patients after BSC, respectively. The survival rate at first and second year in the mEHT group was 77.3% and 40.9% for AST, and 61% and 29% for GBM, respectively. The 5-year overall survival of AST was 83% after mEHT versus 25% after BSC and 3.5% after mEHT versus 1.2% after BSC for GBM. The median overall survival of mEHT was 14 months (range 2-108 months) for GBM and 16.5 months (range 3-156 months) for the AST group. We observed 4 long-term survivors in the AST and 2 in the GBM group. Two of the long survivors in AST and 1 in GBM group were treated by mEHT. mEHT in integrative therapy may have a promising role in the treatment and palliation of relapsed GBM and AST.