[ADRB2 Gene Knockout in Human Primary T Cells by Multiple sgRNAs Construced using CRISPR/Cas9 Technology].
To knockout ADRB2 gene rapidly and efficiently in human primary T cells by using CRISPR/Cas9 technology and multiple sgRNAs strategy. Six paired-sgRNAs, which were designed to target the 5' constitutive coding exons of ADRB2 gene, were cloned into pGL3-U6-sgRNA-PGK-Puro vector separately. The expre-ssion vectors containing the single sgRNAs were constructed and transiently co-transfected into HEK-293T cell line with Cas9 expression vector. The sgRNA-mediated cleavage efficiency was tested by T7EN I digestion assay. Concatenating four highly efficient paired sgRNAs were cloned into pGL3-U6-sgRNA-ccdB-EF1α-Puro expression vector. The reco-mbinant plasmid allows the cells to express 4 sgRNAs, which target different sites on the ADRB2 genomic locus. The cleavage efficiency and mutation model were tested by T7EN I digest assay and T-A cloning technique. Multiple sgRNAs plasmid and Cas9 plasmid was transiently transferred into human primary T cells by electroporation. Flow cytometry (FCM) was used to detect the knockout efficiency of β2 adrenergic receptor (β2-AR). The results of T7EN I digestion and TA cloning sequencing showed that the multiple sgRNAs strategy could obtain more abundant mutation types and higher gene editing efficiency than single sgRNA. In addition to the deletion and insertion of bases, large fragment DNA deletions and inversions could be observed. All of the random 10 TA clones for detection were genetically modified, thus the mutation efficiency was as high as 100%. FCM assay showed that 43.09% of the cells in the control T cells were β2-AR positive, but the proportion of β2-AR positive cells in the multiple sgRNAs electrotransformed T cells decreased to 25.61%. A method, which is simple and operable, for knocking out β2-AR in human primary T cells has been established preliminarily. The results are helpful for the further study of the role of β2-AR in human T cells.


Superpose3D: a local structural comparison program that allows for user-defined structure representations.
Local structural comparison methods can be used to find structural similarities involving functional protein patches such as enzyme active sites and ligand binding sites. The outcome of such analyses is critically dependent on the representation used to describe the structure. Indeed different categories of functional sites may require the comparison program to focus on different characteristics of the protein residues. We have therefore developed superpose3D, a novel structural comparison software that lets users specify, with a powerful and flexible syntax, the structure description most suited to the requirements of their analysis. Input proteins are processed according to the user's directives and the program identifies sets of residues (or groups of atoms) that have a similar 3D position in the two structures. The advantages of using such a general purpose program are demonstrated with several examples. These test cases show that no single representation is appropriate for every analysis, hence the usefulness of having a flexible program that can be tailored to different needs. Moreover we also discuss how to interpret the results of a database screening where a known structural motif is searched against a large ensemble of structures. The software is written in C++ and is released under the open source GPL license. Superpose3D does not require any external library, runs on Linux, Mac OSX, Windows and is available at http://cbm.bio.uniroma2.it/superpose3D.


Comparison of two instruments for measurement of quality of life in clinical practice--a qualitative study.
The study aimed to investigate the meaning patients assign to two measures of quality of life: the Schedule for Evaluation of Individual Quality of Life Direct Weighting (SEIQoL-DW) and the SEIQoL-DW Disease Related (DR) version, in a clinical oncology setting. Even though the use of quality of life assessments has increased during the past decades, uncertainty regarding how to choose the most suitable measure remains. SEIQoL-DW versions assesses the individual's perception of his or her present quality of life by allowing the individual to nominate the domains to be evaluated followed by a weighting procedure resulting in qualitative (domains) as well as quantitative outcomes (index score). The study applied a cross-sectional design with a qualitative approach and collected data from a purposeful sample of 40 patients with gastrointestinal cancer. Patients were asked to complete two measures, SEIQoL-DW and the SEIQoL-DR, to assess quality of life. This included nomination of the areas in life considered most important and rating of these areas; after completion patients participated in cognitive interviews around their selections of areas. Interviews were audiotaped and transcribed verbatim which was followed by analysis using a phenomenographic approach. The analyses of nominated areas of the two measures resulted in 11 domains reflecting what patients perceived had greatest impact on their quality of life. Analysis of the cognitive interviews resulted in 16 thematic categories explaining the nominated domains. How patients reflected around their quality of life appeared to differ by version (DW vs. DR). The DW version more often related to positive aspects in life while the DR version more often related to negative changes in life due to having cancer. The two SEIQoL versions tap into different concepts; health-related quality of life, addressing losses and problems related to having cancer and, quality of life, more associated with aspects perceived as positive in life. The SEIQoL-DR and the SEIQoL-DW are recommended in clinical practice to take both negative and positive aspects into account and acting on the problems of greatest importance to the patient.


[Once again the so-called Testament of Hippocrates].
This study is dealing with the so-called Testament of Hippocrates, a short ethic about the doctor's background, knowledge, character, appearance and behaviour towards the patient. Along with the three well-known Greek manuscripts the author is looking at two other by now unknown codices, so that the two Greek versions are accompanied by a third and larger version. After a careful consideration of the relations between the different versions he is discussing all readings of the codices explaining the terms within the background of the history of medicine. At the end he is presenting a reconstruction of the Greek original together with a German translation.


Plastic polymers for efficient DNA microarray hybridization: application to microbiological diagnostics.
Fabrication of microarray devices using traditional glass slides is not easily adaptable to integration into microfluidic systems. There is thus a need for the development of polymeric materials showing a high hybridization signal-to-background ratio, enabling sensitive detection of microbial pathogens. We have developed such plastic supports suitable for highly sensitive DNA microarray hybridizations. The proof of concept of this microarray technology was done through the detection of four human respiratory viruses that were amplified and labeled with a fluorescent dye via a sensitive reverse transcriptase PCR (RT-PCR) assay. The performance of the microarray hybridization with plastic supports made of PMMA [poly(methylmethacrylate)]-VSUVT or Zeonor 1060R was compared to that with high-quality glass slide microarrays by using both passive and microfluidic hybridization systems. Specific hybridization signal-to-background ratios comparable to that obtained with high-quality commercial glass slides were achieved with both polymeric substrates. Microarray hybridizations demonstrated an analytical sensitivity equivalent to approximately 100 viral genome copies per RT-PCR, which is at least 100-fold higher than the sensitivities of previously reported DNA hybridizations on plastic supports. Testing of these plastic polymers using a microfluidic microarray hybridization platform also showed results that were comparable to those with glass supports. In conclusion, PMMA-VSUVT and Zeonor 1060R are both suitable for highly sensitive microarray hybridizations.


Tracing Back the Evolutionary Route of Enteroinvasive Escherichia coli (EIEC) and Shigella Through the Example of the Highly Pathogenic O96:H19 EIEC Clone.
Enteroinvasive Escherichia coli (EIEC) cause intestinal illness through the same pathogenic mechanism used by Shigella spp. The latter species can be typed through genomic and phenotypic methods used for E. coli and have been proposed for reclassification within E. coli species. Recently the first appearance of a highly pathogenic EIEC O96:H19 was described in Europe as the causative agent of two large outbreaks that occurred in Italy and in the United Kingdom. In contrast to Shigella spp and to the majority of EIEC strains, EIEC O96:H19 fermented lactose, lacked pathoadaptive mutations, and showed good fitness in extracellular environment, similarly to non-pathogenic E. coli, suggesting they have emerged following acquisition of the invasion plasmid by a non-pathogenic E. coli. Here we describe the whole genome comparison of two EIEC O96:H19 strains isolated from severe cases of diarrhea in Uruguay in 2014 with the sequences of EIEC O96:H19 available in the public domain. The phylogenetic comparison grouped all the O96:H19 strains in a single cluster, while reference EIEC strains branched into different clades with Shigella strains occupying apical positions. The comparison of the virulence plasmids showed the presence of a complete conjugation region in at least one O96:H19 EIEC. Reverse Transcriptase Real Time PCR experiments confirmed in this strain the expression of the pilin-encoding gene and conjugation experiments suggested its ability to mobilize an accessory plasmid in a recipient strain. Noteworthy, the tra region was comprised between two reversely oriented IS600 elements, which were also found as remnants in another EIEC O96:H19 plasmid lacking the tra locus. We hypothesize that an IS-mediated recombination mechanism may have caused the loss of the conjugation region commonly observed in EIEC and Shigella virulence plasmids. The results of this study support the hypothesis of EIEC originating from non-pathogenic E. coli through the acquisition of the virulence plasmid via conjugation. Remarkably, this study showed the ability of a circulating EIEC strain to mobilize plasmids through conjugation, suggesting a mechanism for the emergence of novel EIEC clones.


[Obesity and hypertension].
A significant association between hypertension and obesity has been noted in several epidemiological studies. In particular, a progressive increase has been demonstrated, both in men and women, in the prevalence of hypertension in relation to the different measures of obesity. Such association is independent of age, gender, and also probably the ethnic group. It has also been shown that obesity may antedate and predict the development of hypertension. Even among subjects with normal or optimal blood pressure, the obese subjects are more likely to develop high blood pressure levels in the following years. The opposite can also be true. Hypertensive subjects are more likely to develop obesity than normotensive ones. It has been suggested that initial sympathetic hyperactivity may lead to high blood pressure and progressive hyperstimulation, followed by down-regulation, of beta-adrenergic receptors, with subsequent development of obesity because of the lesser beta-adrenoceptor that induces dissipation of calories. Visceral obesity seems to be more important than subcutaneous obesity with respect to high blood pressure. Finally, in addition to the well established relation between low weight at birth and adult hypertension, it has also been shown that any transient increases in body weight may be paralleled by increases in blood pressure at any time in life. Obesity may be associated with hypertension through an increased sympathetic tone, increased fluid retention and insulin resistance. Animal models of hypertension associated with obesity may be suitable to investigate in detail the pathophysiological mechanisms of such association. In this setting decreased nitric oxide production and resistance to leptin have been identified as important determinants of obesity in hypertension. From a therapeutic standpoint, it is well known that weight reduction is associated with a drop in blood pressure. Unfortunately, however, obese hypertensive subjects who loose weight in the short term often do not succeed to maintain the weight loss, with consequent new gain in body weight and blood pressure. Thus, weight reduction should be maintained in the long term to elicit a sustained and effective antihypertensive efficacy.


Rate of reinfection with S. mansoni following treatment in two newly reclaimed areas of Egypt.
This paper reports an on-going study in two newly reclaimed areas in Ismailia Governorate, El-Manayef, 10 km south-west of the city of Ismailia (area 1) and the other in Siani Peninsula, just east of the Bitter Lakes (area 2). In he baseline survey, the prevalence of infection of 49.9% and 40.1% and high intensity infection of 21.8% and 15.7% were found for S. mansoni in areas 1 and 2 respectively. Geometric mean egg counts for those found positive were 101.2 and 75.9. In a subsequent survey, a year after treatment of positives, the overall prevalence was 30.2% and 30.5% and the prevalence of high intensity infection was 8.6% and 10.5% in the two areas. The prevalence among those found positive and then treated during the first round (reinfection rate) was 31.7% and 19.2%. Geometric mean egg counts had declined from 141.3 to 63.1 and from 133.6 to 59 among those who were reinfected. The occurrence of reinfection was strongly associated with younger age in both areas. Rates of reinfection showed a strong but not significant association with male sex in area 2 (p = 0.087), but no association in area 1. These high rates of reinfection demonstrate the importance of promotion of hygiene behaviors as well as ensuring access to chemotherapy.


Determination of cephalosporins in raw bovine milk by high-performance liquid chromatography.
A high-performance liquid chromatographic (HPLC) method based on solid-phase extraction was developed for the determination of cefazolin, cefoperazone, cefquinome and ceftiofur in raw bovine milk. The milk fat was removed by centrifugation and the cephalosporins were extracted in acetonitrile. The extract was cleaned up by solid-phase extraction on an octadecyl sorbent. The compounds were separated by ion-paired gradient HPLC on a phenyl column with ultraviolet detection at 270 nm. The limits of detection estimated by a conservative model were 11 microg/kg for cefazolin and cefoperazone and 7 microg/kg for cequinome and ceftiofur. The mean recoveries were 86-88% for cefazolin, 91-93% for cefoperazone, 69-72% for cefquinome and 84-88% for ceftiofur in the concentration range 20-200 microg/kg.


It takes two to tango: NAD+ and sirtuins in aging/longevity control.
The coupling of nicotinamide adenine dinucleotide (NAD+) breakdown and protein deacylation is a unique feature of the family of proteins called 'sirtuins.' This intimate connection between NAD+ and sirtuins has an ancient origin and provides a mechanistic foundation that translates the regulation of energy metabolism into aging and longevity control in diverse organisms. Although the field of sirtuin research went through intensive controversies, an increasing number of recent studies have put those controversies to rest and fully established the significance of sirtuins as an evolutionarily conserved aging/longevity regulator. The tight connection between NAD+ and sirtuins is regulated at several different levels, adding further complexity to their coordination in metabolic and aging/longevity control. Interestingly, it has been demonstrated that NAD+ availability decreases over age, reducing sirtuin activities and affecting the communication between the nucleus and mitochondria at a cellular level and also between the hypothalamus and adipose tissue at a systemic level. These dynamic cellular and systemic processes likely contribute to the development of age-associated functional decline and the pathogenesis of diseases of aging. To mitigate these age-associated problems, supplementation of key NAD+ intermediates is currently drawing significant attention. In this review article, we will summarize these important aspects of the intimate connection between NAD+ and sirtuins in aging/longevity control.


Hysteresis at low Reynolds number: onset of two-dimensional vortex shedding
Hysteresis has been observed in a study of the transition between laminar flow and vortex shedding in a quasi-two-dimensional system. The system is a vertical, rapidly flowing soap film which is penetrated by a rod oriented perpendicular to the film plane. Our experiments show that the transition from laminar flow to a periodic von Karman vortex street can be hysteretic, i.e., vortices can survive at velocities lower than the velocity needed to generate them.


Medical Malpractice in Orthopedic Surgery: A Westlaw-Based Demographic Analysis.
A recent study that evaluated the risk of facing a malpractice claim by physician specialty found that orthopedic surgeons were at a significantly greater risk of being sued than other medical specialists. To date, no studies have characterized trends in orthopedic surgery malpractice claims. The Westlaw legal database was used to locate state and federal jury verdicts and settlements related to medical malpractice and orthopedic surgery from 2010 to 2016. Eighty-one cases were analyzed. The mean age of the affected patients and/or plaintiffs was 53.4 years. Spine surgery (21 cases; 25.9%), knee surgery (17 cases; 21.0%), and hip surgery (11 cases; 13.6%) were litigated most often. Procedural error (71 cases; 87.7%) and negligence (58 cases; 71.6%) were the 2 most commonly cited reasons for litigation. The jury found in favor of the defendant in most (50 cases; 61.7%) of the cases. The mean plaintiff (17 cases; 21.0%) verdict payout was $3,015,872, and the mean settlement (13 cases; 16.0%) value was $1,570,833. Unnecessary surgery (odds ratio [OR], 12.29; 95% confidence interval [CI], 1.91-108.46; P=.040) and surgery resulting in death (OR, 26.26; 95% CI, 2.55-497.42; P=.040) were significant predictors of a verdict in favor of the plaintiff. Patient death (OR, 0.05; 95% CI, 0.01-0.38; P=.021) and male patient sex (OR, 0.26; 95% CI, 0.09-0.71; P=.033) were significant negative predictors of a verdict in favor of the defendant. The jury found in favor of the defendant orthopedic surgeon in most cases. Procedural error and/or negligence were cited most commonly by the plaintiffs as the bases for the claims. Verdicts in favor of the plaintiffs resulted in payouts nearly double those of settlements. [Orthopedics. 2018; 41(5):e615-e620.].


Desmoglein 2, but not desmocollin 2, protects intestinal epithelia from injury.
Desmosomes are the least understood intercellular junctions in the intestinal epithelia and provide cell-cell adhesion via the cadherins desmoglein (Dsg)2 and desmocollin (Dsc)2. We studied these cadherins in Crohn's disease (CD) patients and in newly generated conditional villin-Cre DSG2 and DSC2 knockout mice (DSG2ΔIEC; DSC2ΔIEC). CD patients exhibited altered desmosomes and reduced Dsg2/Dsc2 levels. The intestines of both transgenic animal lines were histopathologically inconspicuous. However, DSG2ΔIEC, but not DSC2ΔIEC mice displayed an increased intestinal permeability, a wider desmosomal space as well as alterations in desmosomal and tight junction components. After dextran sodium sulfate (DSS) treatment and Citrobacter rodentium exposure, DSG2ΔIEC mice developed a more-pronounced colitis, an enhanced intestinal epithelial barrier disruption, leading to a stronger inflammation and activation of epithelial pSTAT3 signaling. No susceptibility to DSS-induced intestinal injury was noted in DSC2ΔIEC animals. Dsg2 interacted with the cytoprotective chaperone Hsp70. Accordingly, DSG2ΔIEC mice had lower Hsp70 levels in the plasma membrane compartment, whereas DSC2ΔIEC mice displayed a compensatory recruitment of galectin 3, a junction-tightening protein. Our results demonstrate that Dsg2, but not Dsc2 is required for the integrity of the intestinal epithelial barrier in vivo.


[Transcatheter percutaneous commissurotomy in rheumatic mitral stenosis].
Percutaneous transcatheter balloon mitral commissurotomy was performed in an 11 year old girl. The atrial septum was perforated and an 8 mm angioplasty balloon was advanced the atrial septal perforation was then dilated to allow passage of 25 mm balloon valvuloplasty catheter across the mitral annulus. Transmitral balloon inflation acutely decreased the end diastolic transmitral gradient (23 mm Hg to 2 mm Hg). Immediate gradient reduction was associated with increases in cardiac output (3.4 to 5.3 L/min). Murmur intensity diminished immediately post-commissurotomy. Balloon commissurotomy did not produced mitral regurgitation. Five weeks later, follow-up catheterization was performed and did not show change in mitral valvular gradient (2 mmHg). Clinical improvement was evident.


Delays during PBMC isolation have a moderate effect on yield, but severly compromise cell viability.
Peripheral blood mononuclear cells (PBMCs) are a versatile material for clinical routine as well as for research projects. However, their isolation via density gradient centrifugation is still time-consuming. When samples are taken beyond usual laboratory handling times, it may sometimes be necessary to pause the isolation process. Our aim was to evaluate the impact of delays up to 48 h after the density gradient centrifugation on PBMC yield, purity and viability. PBMCs were isolated from samples of 20 donors, either with BD Vacutainer CPT tubes (CPT) or with the standard Ficoll method. Isolation was paused after initial density gradient centrifugation for 0, 24, or 48 h. PBMC yield (% output/input), purity (% PBMCs/total cells) and viability (% Annexin V-/propidium iodide-) were compared. The yield did not change significantly over time when CPT were used (55%/52%/47%), but did after isolation with the standard method (62%/40%[p<0.0001]/53%[p<0.01]). Purity was marginally affected if CPT were used (95%/93%[p=n.s./92%[p<0.05] vs. 97% for all time points with standard method). Whereas viable PBMCs decreased steadily for CPT isolates (62%/51%[p<0.001]/36%[p<0.0001]), after standard Ficoll gradient isolation, cell apoptosis was more pronounced already after 24 h delay, and viability did not further decrease after 48 h (64%/44%[p<0.0001]/40%[p<0.0001]). In conclusion, our findings suggest that while post-centrifugation delays ≥24 h might have only a minor effect on cell yield and purity, their impact on cell viability is substantial, even when CPT are used.


Cardiovascular health impacts of wildfire smoke exposure.
In recent years, wildland fires have occurred more frequently and with increased intensity in many fire-prone areas. In addition to the direct life and economic losses attributable to wildfires, the emitted smoke is a major contributor to ambient air pollution, leading to significant public health impacts. Wildfire smoke is a complex mixture of particulate matter (PM), gases such as carbon monoxide, nitrogen oxide, and volatile and semi-volatile organic compounds. PM from wildfire smoke has a high content of elemental carbon and organic carbon, with lesser amounts of metal compounds. Epidemiological studies have consistently found an association between exposure to wildfire smoke (typically monitored as the PM concentration) and increased respiratory morbidity and mortality. However, previous reviews of the health effects of wildfire smoke exposure have not established a conclusive link between wildfire smoke exposure and adverse cardiovascular effects. In this review, we systematically evaluate published epidemiological observations, controlled clinical exposure studies, and toxicological studies focusing on evidence of wildfire smoke exposure and cardiovascular effects, and identify knowledge gaps. Improving exposure assessment and identifying sensitive cardiovascular endpoints will serve to better understand the association between exposure to wildfire smoke and cardiovascular effects and the mechanisms involved. Similarly, filling the knowledge gaps identified in this review will better define adverse cardiovascular health effects of exposure to wildfire smoke, thus informing risk assessments and potentially leading to the development of targeted interventional strategies to mitigate the health impacts of wildfire smoke.


[Optical density and surface tension in amniotic fluid in the late pregnancy (author's transl)].
113 amniotic fluid samples from pregnant women between the 28th and the 41st week of gestation were examined for optical density and surface tension. Surface tension measurement was done by means of the Wilhelmy-balance which shows a very good correlation with the L/S-ratio. Optical density was measured at 405, 580, 650, 750 and 900 nm wave length. For the comparison with the surface tension optical 650 nm was used, since at this wave length only minimal absorbance of known biologic pigments occurs. Pulmonary maturity was diagnosed when the surface of the hysteresis area (dynamic surface tension measurement) was more than 20 cm2 or when optical density at 650 nm was false positive in 3,7% and false negative in 40,7%. The same percentage was found in the predication of fetal lung maturity by this method after fetal outcome. The measurement of the optical density is a rapid and simple method, but because of the high number of false negative results it is advised to use one of the more sophisticated methods for the prediction of fetal lung maturity in cases of a negative optical density reading at 650 nm.


Kallikrein-gene expression in the rat gastrointestinal tract.
The serine proteinase glandular kallikrein has been demonstrated in the gastrointestinal tract, although there is some doubt as to whether it is synthesized there or derives from exocrine-gland secretions. Using a rat pancreatic kallikrein cRNA probe we have demonstrated kallikrein-like gene expression in the corpus, duodenum, jejunum, ileum, caecum and colon, and compared the pattern of expression with that of the gastrointestinal peptides somatostatin, gastrin and glucagon. In addition, using a panel of oligonucleotide probes specific for various members of the rat kallikrein-gene family, we have shown that the kallikrein-like gene expressed appears to be expressed as true kallikrein.


Predicting restenosis of drug-eluting stents placed in real-world clinical practice: derivation and validation of a risk model from the EVENT registry.
Prediction of restenosis after percutaneous coronary intervention (PCI) remains challenging, and existing risk assessment algorithms were developed before the widespread adoption of drug-eluting stents (DES). We used data from the EVENT registry to develop a risk model for predicting target lesion revascularization (TLR) in 8829 unselected patients undergoing DES implantation between 2004 and 2007. Using a split-sample validation technique, predictors of TLR at 1 year were identified from two thirds of the subjects (derivation cohort) using multiple logistic regression. Integer point values were created for each predictor, and the summed risk score (range, 0 to 10) was applied to the remaining sample (validation cohort). At 1 year, TLR occurred in 4.2% of patients, and after excluding stent thrombosis and early mechanical complications, the incidence of late TLR (more likely representing restenosis-related TLR) was 3.6%. Predictors of TLR were age <60, prior PCI, unprotected left main PCI, saphenous vein graft PCI, minimum stent diameter < or =2.5 mm, and total stent length > or =40 mm. Comparison of observed versus predicted rates of TLR according to risk score demonstrated good model fit in the validation set. There was more than a 3-fold difference in TLR rates between the lowest risk category (score=0; TLR rate, 2.2%) and the highest risk category (score > or =5; TLR rate, 7.5%). The overall incidence of TLR remains low among unselected patients receiving DES in routine clinical practice. A simple risk model incorporating 6 readily available clinical and angiographic variables helps identify individuals at extremely low (<2%) and modestly increased (>7%) risk of TLR after DES implantation.


Fine-tuning of triarylamine-based photosensitizers for dye-sensitized solar cells.
The synthesis of a series of novel organic photosensitizers based on modified triarylamine motives has been achieved. The new "push-pull" dyes, bearing one to three naphthyl units in place of phenyl rings, have been successfully obtained following original synthetic routes. The structural, optical and electrochemical properties of these chromophores were studied by combining crystallographic, experimental and theoretical data. We further present a systematic study of this series of dyes. When embedded in dye-sensitized solar cell devices, the new photosensitizers showed good spectral response with IPCE greater than 85% from 470 to 580 nm and overall power conversion efficiencies reaching 6.6%. One of these new sensitizers, containing one naphthyl attached to the π-conjugated linker and having phenyl rings on the outside, led to the highest molar extinction coefficient and energy conversion efficiency of the series while exhibiting higher electron lifetime.


First Versus Second Order Latent Growth Curve Models: Some Insights From Latent State-Trait Theory.
First order latent growth curve models (FGMs) estimate change based on a single observed variable and are widely used in longitudinal research. Despite significant advantages, second order latent growth curve models (SGMs), which use multiple indicators, are rarely used in practice, and not all aspects of these models are widely understood. In this article, our goal is to contribute to a deeper understanding of theoretical and practical differences between FGMs and SGMs. We define the latent variables in FGMs and SGMs explicitly on the basis of latent state-trait (LST) theory and discuss insights that arise from this approach. We show that FGMs imply a strict trait-like conception of the construct under study, whereas SGMs allow for both trait and state components. Based on a simulation study and empirical applications to the CES-D depression scale (Radloff, 1977) we illustrate that, as an important practical consequence, FGMs yield biased reliability estimates whenever constructs contain state components, whereas reliability estimates based on SGMs were found to be accurate. Implications of the state-trait distinction for the measurement of change via latent growth curve models are discussed.


Accounting for frailty when treating chronic diseases.
Chronic diseases are considered to be major determinants of frailty and it could be hypothesized that their treatment may counteract the development of frailty. However, the hypothesis that intensive treatment of chronic diseases might reduce the progression of frailty is poorly supported by existing studies. In contrast, some evidence suggests that intensive treatment of chronic diseases may increase negative health outcomes in frail older adults. In particular, if treatment of symptoms related to chronic diseases (i.e. pain in osteoarthritis, dyspnoea in respiratory disease, motor symptoms in Parkinson disease) might potentially reverse frailty, the benefits related to preventive pharmacological treatment of chronic diseases (i.e. antihypertensive treatment) in patients with prevalent frailty is not certain. In particular, several factors might alter the risk/benefit ratio of a given treatment in persons with frailty. These include: exclusion of frail persons from clinical studies, reduced life expectancy in frail persons, increased susceptibility to iatrogenic events, and functional deficits associated with frailty. Therefore, frailty acts as an effect modifier, by modifying the risks and benefits of chronic disease treatments. This hypothesis must be considered and tested in future clinical intervention studies and clinical guidelines should provide specific recommendations for the treatment of frail people, underlining the pros and the cons of pharmacological treatment and possible targets for therapy in this population. Meanwhile, in older patients, the prescribing process should be individualized and flexible.


Structural and functional properties of human multidrug resistance protein 1 (MRP1/ABCC1).
Multidrug ABC transporters such as P-glycoprotein (P-gp/MDR1/ABCB1) and multidrug resistance protein 1 (MRP1/ABCC1) play an important role in the extrusion of drugs from the cell and their overexpression can be a cause of failure of anticancer and antimicrobial chemotherapy. Recently, the mouse P-gp/Abcb1a structure has been determined and this has significantly enhanced our understanding of the structure-activity relationship (SAR) of mammalian ABC transporters. This paper highlights our current knowledge on the structural and functional properties and the SAR of human MRP1/ABCC1. Although the crystal structure of MRP1/ABCC1 has yet to be resolved, the current topological model of MRP1/ABCC1 contains two transmembrane domains (TMD1 and TMD2) each followed by a nucleotide binding domain (NBD) plus a third NH2-terminal TMD0. MRP1/ABCC1 is expressed in the liver, kidney, intestine, brain and other tissues. MRP1/ABCC1 transports a structurally diverse array of important endogenous substances (e.g. leukotrienes and estrogen conjugates) and xenobiotics and their metabolites, including various conjugates, anticancer drugs, heavy metals, organic anions and lipids. Cells that highly express MRP1/ABCC1 confer resistance to a variety of natural product anticancer drugs such as vinca alkaloids (e.g. vincristine), anthracyclines (e.g. etoposide) and epipodophyllotoxins (e.g. doxorubicin and mitoxantrone). MRP1/ABCC1 is associated with tumor resistance which is often caused by an increased efflux and decreased intracellular accumulation of natural product anticancer drugs and other anticancer agents. However, most compounds that efficiently reverse P-gp/ABCB1-mediated multidrug resistance have only low affinity for MRP1/ABCC1 and there are only a few effective and relatively specific MRP1/ABCC1 inhibitors available. A number of site-directed mutagenesis studies, biophysical and photolabeling studies, SAR and QSAR, molecular docking and homology modeling studies have documented the role of multiple residues in determining the substrate specificity and inhibitor selectivity of MRP1/ABCC1. Most of these residues are located in the TMs of TMD1 and TMD2, in particular TMs 4, 6, 7, 8, 10, 11, 14, 16, and 17, or in close proximity to the membrane/cytosol interface of MRP1/ABCC1. The exact transporting mechanism of MRP1/ABCC1 is unclear. MRP1/ABCC1 and other multidrug transporters are front-line mediators of drug resistance in cancers and represent important therapeutic targets in future chemotherapy. The crystal structure of human MRP1/ABCC1 is expected to be resolved in the near future and this will provide an insight into the SAR of MRP1/ABCC1 and allow for rational design of anticancer drugs and potent and selective MRP1/ABCC1 inhibitors.


Single molecule analysis by surfaced-enhanced Raman scattering.
Our main objective in this tutorial review is to provide insight into some of the questions surrounding single molecule detection (SMD) using surface-enhanced Raman scattering (SERS) and surface-enhanced resonance Raman scattering (SERRS). Discovered thirty years ago, SERS is now a powerful analytical tool, strongly tied to plasmonics, a field that encompasses and profits from the optical enhancement found in nanostructures that support localized plasmon excitations. The spectrum of the single molecule carries the quantum fingerprints of the system modulated by the molecule-nanostructure interactions and the electronic resonances that may result under laser excitation. This information is embedded in vibrational band parameters. The dynamics and the molecular environment will affect the bandwidth of the observed Raman bands. In addition, the localized surface plasmon resonances (LSPR) empower the nanostructure with a number of optical properties that will also leave their mark on the observed inelastic scattering process. Therefore, controlling size, shape and the formation of the aggregation state (or fractality) of certain metallic nanostructures becomes a main task for experimental SERS/SERRS. This molecule-nanostructure coupling may, inevitably, lead to spectral fluctuations, increase photobleaching or photochemistry. An attempt is made here to guide the interpretation of this wealth of information when approaching the single molecule regime.


Regulation of phosphodiesterase 3 and inducible cAMP early repressor in the heart.
Growing evidence suggests that multiple spatially, temporally, and functionally distinct pools of cyclic nucleotides exist and regulate cardiac performance, from acute myocardial contractility to chronic gene expression and cardiac structural remodeling. Cyclic nucleotide phosphodiesterases (PDEs), by hydrolyzing cAMP and cyclic GMP, regulate the amplitude, duration, and compartmentation of cyclic nucleotide-mediated signaling. In particular, PDE3 enzymes play a major role in regulating cAMP metabolism in the cardiovascular system. PDE3 inhibitors, by raising cAMP content, have acute inotropic and vasodilatory effects in treating congestive heart failure but have increased mortality in long-term therapy. PDE3A expression is downregulated in human and animal failing hearts. In vitro, inhibition of PDE3A function is associated with myocyte apoptosis through sustained induction of a transcriptional repressor ICER (inducible cAMP early repressor) and thereby inhibition of antiapoptotic molecule Bcl-2 expression. Sustained induction of ICER may also cause the change of other protein expression implicated in human and animal failing hearts. These data suggest that the downregulation of PDE3A observed in failing hearts may play a causative role in the progression of heart failure, in part, by inducing ICER and promoting cardiac myocyte dysfunction. Hence, strategies that maintain PDE3A function may represent an attractive approach to circumvent myocyte apoptosis and cardiac dysfunction.


Combined metabolomics and network pharmacology to elucidate the mechanisms of Dracorhodin Perchlorate in treating diabetic foot ulcer rats.
Background: Diabetic foot ulcer (DFU) is a severe chronic complication of diabetes, that can result in disability or death. Dracorhodin Perchlorate (DP) is effective for treating DFU, but the potential mechanisms need to be investigated. We aimed to explore the mechanisms underlying the acceleration of wound healing in DFU by the topical application of DP through the combination of metabolomics and network pharmacology. Methods: A DFU rat model was established, and the rate of ulcer wound healing was assessed. Different metabolites were found in the skin tissues of each group, and MetaboAnalyst was performed to analyse metabolic pathways. The candidate targets of DP in the treatment of DFU were screened using network pharmacology. Cytoscape was applied to construct an integrated network of metabolomics and network pharmacology. Moreover, the obtained hub targets were validated using molecular docking. After the topical application of DP, blood glucose, the rate of wound healing and pro-inflammatory cytokine levels were assessed. Results: The levels of IL-1, hs-CRP and TNF-α of the Adm group were significantly downregulated. A total of 114 metabolites were identified. These could be important to the therapeutic effects of DP in the treatment of DFU. Based on the network pharmacology, seven hub genes were found, which were partially consistent with the metabolomics results. We focused on four hub targets by further integrated analysis, namely, PAH, GSTM1, DHFR and CAT, and the crucial metabolites and pathways. Molecular docking results demonstrated that DP was well combined with the hub targets. Conclusion: Our research based on metabolomics and network pharmacology demonstrated that DP improves wound healing in DFU through multiple targets and pathways, and it can potentially be used for DFU treatment.


Nerve-muscle interactions regulate motor terminal growth and myoblast distribution during muscle development.
Interactions between motoneurons and muscles influence many aspects of neuromuscular development in all animals. These interactions can be readily investigated during adult muscle development in holometabolous insects. In this study, the development of the dorsolongitudinal flight muscle (DLM) and its innervation is investigated in the moth, Manduca sexta, to address the specificity of neuromuscular interactions. The DLM develops from an anlage containing both regressed larval template fibers and imaginal myoblasts. In the adult, each fiber bundle (DLM1-5) is innervated by a single motoneuron (MN1-MN5), with the dorsal-most fiber bundle (DLM5) innervated by a mesothoracic motoneuron (MN5). The DLM failed to develop following complete denervation because myoblasts failed to accumulate in the DLM anlage. After lesioning MN1-4, MN5 retained its specificity for the DLM5 region of the anlage and failed to rescue DLM1-4. Thus specific innervation of the DLM fiber bundles does not depend on interactions among motoneurons. Myoblast accumulation, but not myonuclear proliferation, increased around the MN5 terminals, producing a hypertrophied adult DLM5. Therefore, motoneurons compete for uncommitted myoblasts. MN5 terminals subsequently grew more rapidly over the hypertrophied DLM5 anlage, indicating that motoneuron terminal expansion is regulated by the size of the target muscle anlage.


Management of severe polyuria in idiopathic Fanconi syndrome.
Polyuria is a common problem in patients with tubular diseases, especially for those with CKD and high-output Fanconi syndrome. There are currently no guidelines on how to treat debilitating polyuria, in children or adults, and vasopressin is usually not effective. A 13-year-old female with idiopathic Fanconi syndrome and an eGFR of 69 mL/min/1.73 m2 was severely affected by polyuria of 5 L per day (voiding at least 11 times during the day and up to 8 times at night), impacting her mood (measured by the RCADS-child) and academic performance at school. In the absence of guidelines and with literature discouraging the use of indomethacin in this condition, we attempted indomethacin treatment at a dose of 2 mg/kg divided in two doses with substantial success. Urine output dropped to 2.5L and this was accompanied by a substantial decrease of her sodium wasting from 24.6 to 7.7 mmol/kg/day. Over the course of 18 months, the patient's eGFR dropped temporarily to 60 mL/min/1.73 m2 and was 68 mL/min/1.73 m2 at last follow-up. However, a sodium-23 (23Na) MRI of her thigh revealed ongoing moderate sodium decrease in her skin and substantial Na+ decrease in her muscle when compared to age-matched peers with normal kidney function. Indomethacin may be a safe and effective treatment option for polyuria in idiopathic Fanconi syndrome.


Mimics and chameleons in stroke.
Ischaemic stroke is a treatable medical emergency. In an era of time-dependent reperfusion techniques, it is crucial that an accurate and prompt diagnosis is made. Approximately 30% of patients admitted to hyperacute stroke units are subsequently found not to have a final diagnosis of acute stroke although some of these patients do have incidental or previously symptomatic cerebrovascular disease. These patients do not benefit from thrombolysis and may require the input of other specialists or treatments. Meanwhile, a proportion of patients with acute stroke have unusual presentations and are sometimes initially admitted to general medical admissions units prior to accessing stroke unit care. It is important that atypical presentations of stroke are recognised so that patients are not denied the benefits of stroke unit care and secondary prevention. This article describes some characteristics of common stroke mimics and chameleons, considers how to avoid diagnostic mistakes and discusses the contributory role of imaging.


Adolescent suicide attempts: risks and protectors.
In 1997, suicide was the third leading cause of death among 10- to 19-year-olds in the United States, with the greatest increases in suicide rates in the previous decade experienced by black and other minority youth. The purpose of this study was to identify risk and protective factors for suicide attempts among black, Hispanic, and white male and female adolescents. We used data from the National Longitudinal Study of Adolescent Health, conducted in 1995 and 1996. A nationally representative sample of 13 110 students in grades 7 through 12 completed 2 in-home interviews, an average of 11 months apart. We examined Time 1 factors at the individual, family, and community level that predicted or protected against Time 2 suicide attempts. Perceived parent and family connectedness was protective against suicide attempts for black, Hispanic, and white girls and boys, with odds ratios ranging from 0.06 to 0.32. For girls, emotional well-being was also protective for all of the racial/ethnic groups studied, while a high grade point average was an additional protective factor for all of the boys. Cross-cutting risk factors included previous suicide attempt, violence victimization, violence perpetration, alcohol use, marijuana use, and school problems. Additionally, somatic symptoms, friend suicide attempt or completion, other illicit drug use, and a history of mental health treatment predicted suicide attempts among black, Hispanic, and white females. Weapon-carrying at school and same-sex romantic attraction were predictive for all groups of boys. Calculating the estimated probabilities of attempting suicide for adolescents with increasing numbers of risk and protective factors revealed that the presence of 3 protective factors reduced the risk of a suicide attempt by 70% to 85% for each of the gender and racial/ethnic groups, including those with and without identified risk factors. In these national samples of black, Hispanic, and white youth, unique and cross-cutting factors derived from a resiliency framework predicted or protected against attempting suicide. In addition to risk reduction, promotion of protective factors may offer an effective approach to primary as well as secondary prevention of adolescent suicidal behavior.


Structural studies of a potent insect maturation inhibitor bound to the juvenile hormone esterase of Manduca sexta.
Juvenile hormone (JH) is an insect hormone containing an alpha,beta-unsaturated ester consisting of a small alcohol and long, hydrophobic acid. JH degradation is required for proper insect development. One pathway of this degradation is through juvenile hormone esterase (JHE), which cleaves the JH ester bond to produce methanol and JH acid. JHE is a member of the functionally divergent alpha/beta-hydrolase family of enzymes and is a highly efficient enzyme that cleaves JH at very low in vivo concentrations. We present here a 2.7 A crystal structure of JHE from the tobacco hornworm Manduca sexta (MsJHE) in complex with the transition state analogue inhibitor 3-octylthio-1,1,1-trifluoropropan-2-one (OTFP) covalently bound to the active site. This crystal structure, the first JHE structure reported, contains a long, hydrophobic binding pocket with the solvent-inaccessible catalytic triad located at the end. The structure explains many of the interactions observed between JHE and its substrates and inhibitors, such as the preference for small alcohol groups and long hydrophobic backbones. The most potent JHE inhibitors identified to date contain a trifluoromethyl ketone (TFK) moiety and have a sulfur atom beta to the ketone. In this study, sulfur-aromatic interactions were observed between the sulfur atom of OTFP and a conserved aromatic residue in the crystal structure. Mutational analysis supported the hypothesis that these interactions contribute to the potency of sulfur-containing TFK inhibitors. Together, these results clarify the binding mechanism of JHE inhibitors and provide useful observations for the development of additional enzyme inhibitors for a variety of enzymes.


Maitotoxin-induced membrane blebbing and cell death in bovine aortic endothelial cells.
Maitotoxin, a potent cytolytic agent, causes an increase in cytosolic free Ca2+ concentration ([Ca2+]i) via activation of Ca2+-permeable, non-selective cation channels (CaNSC). Channel activation is followed by formation of large endogenous pores that allow ethidium and propidium-based vital dyes to enter the cell. Although activation of these cytolytic/oncotic pores, or COP, precedes release of lactate dehydrogenase, an indication of oncotic cell death, the relationship between CaNSC, COP, membrane lysis, and the associated changes in cell morphology has not been clearly defined. In the present study, the effect maitotoxin on [Ca2+]i, vital dye uptake, lactate dehydrogenase release, and membrane blebbing was examined in bovine aortic endothelial cells. Maitotoxin produced a concentration-dependent increase in [Ca2+]i followed by a biphasic uptake of ethidium. Comparison of ethidium (Mw 314 Da), YO-PRO-1 (Mw 375 Da), and POPO-3 (Mw 715 Da) showed that the rate of dye uptake during the first phase was inversely proportional to molecular weight, whereas the second phase appeared to be all-or-nothing. The second phase of dye uptake correlated in time with the release of lactate dehydrogenase. Uptake of vital dyes at the single cell level, determined by time-lapse videomicroscopy, was also biphasic. The first phase was associated with formation of small membrane blebs, whereas the second phase was associated with dramatic bleb dilation. These results suggest that maitotoxin-induced Ca2+ influx in bovine aortic endothelial cells is followed by activation of COP. COP formation is associated with controlled membrane blebbing which ultimately gives rise to uncontrolled bleb dilation, lactate dehydrogenase release, and oncotic cell death.


Deleterious effects of minocycline in animal models of Parkinson's disease and Huntington's disease.
Minocycline has been shown to exert anti-inflammatory effects underlying its putative neuroprotective properties in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson's disease and in the R6/2 mouse model of Huntington's disease (HD). However, contradictory results have recently been reported. We report deleterious effects of minocycline in two phenotypic (toxic) models of Parkinson's disease and HD in monkey and mouse. Of seven MPTP-intoxicated female cynomolgus monkeys (0.2 mg/kg, i.v. until day 15), three received minocycline (200 mg b.i.d.). While placebo-MPTP-treated animals displayed mild parkinsonism at day 15, the minocycline/MPTP-treated animals tended to be more affected (P = 0.057) and showed a greater loss of putaminal dopaminergic nerve endings (P < 0.0001). In the 3-nitropropionic acid (3-NP) mouse model of HD, minocycline (45 mg/kg i.p.) was administered 30 min before each i.p. injection of 3-NP (b.i.d., cumulated dose, 360 mg/kg in 5 days). Mice receiving minocycline exhibited a worsening of the mean motor score with a slower recovery slope, more impaired general activity and significantly deteriorated performances on the rotarod, pole test and beam-traversing tasks. The histopathological outcome demonstrated that minocycline-treated mice presented significantly more severe neuronal cell loss in the dorsal striatum. The effect of minocycline vs. 3-NP was also investigated on hippocampal and cortical cell cultures. minocycline blocked 3-NP-induced neurotoxicity at certain doses (1 mm cortical neurons) but not at higher doses (10 mm). Thus, minocycline may have variable and even deleterious effects in different species and models according to the mode of administration and dose.


Differences in motor unit recruitment patterns and low frequency oscillation of discharge rates between unilateral and bilateral isometric muscle contractions.
Distinct cortical activities contribute to unilateral and bilateral motor control. However, it remains largely unknown whether the behavior of motor neurons differs between unilateral and bilateral isometric force generation. Here, we first investigated motor units (MUs) recruitment patterns during unilateral and bilateral force generation. Considering that the force control is primarily regulated by low-frequency synaptic inputs to motor neurons, we also examined the relation between MU discharge rate and force output during unilateral and bilateral muscle contractions. Using advanced electromyography (EMG) sensor arrays and spike-triggered averaging techniques, we examined a large population of MUs in the right first dorsal interosseous (FDI) muscle during unilateral and bilateral force tracking tasks. Using the principal component analysis, we analyzed the first common component (FCC) of MU discharge rate to describe the force fluctuations during unilateral and bilateral contractions. We found that MU discharge rate decreased during bilateral compared with unilateral contractions. MU recruitment threshold increased, while the amplitude and duration of MU action potential (MUAP) remained unchanged during bilateral compared with unilateral contractions. We found that the coefficients of variation (CV) for the force and FCC signal increased during bilateral compared with unilateral contractions. Notably, the FCC signal captured a great amount of MU discharge variability, and its CV correlated with the CV of the force signal. Our findings suggest that MU recruitment patterns are altered during bilateral compared with unilateral isometric force generation, likely related to changes at the low-frequency portion of the synaptic drive.


[Empyema due to Actinomyces: a diagnostic challenge].
a diagnostic challenge. Isolated pleural effusion due to actinomycosis is rare and poses a diagnostic challenge to clinicians not only because it is uncommon and often forgotten, but also because culture of the causative microorganism is technically difficult. The classic al microscopic appearance of this Gram-positive bacillus on cultures often forms the basis of diagnosis. This is the report of a case of massive left sided pleural effusion due to Actinomyces israelii that improved clinically and radiologically after surgery and prolonged antibiotic treatment.


Improved Spatial Resolution of Metabolites in Tissue Biopsies Using High-Resolution Magic-Angle-Spinning Slice Localization NMR Spectroscopy.
High-resolution magic-angle-spinning 1H NMR spectroscopy (HR-MAS NMR) is a well-established technique for assessing the biochemical composition of intact tissue samples. In this study, we utilized a method based on HR-MAS NMR spectroscopy with slice localization (SLS) to achieve spatial resolution of metabolites. The obtained 7 slice spectra from each of the model samples (i.e., chicken thigh muscle with skin and murine renal biopsy including medulla (M) and cortex (C)) showed distinct metabolite compositions. Furthermore, we analyzed previously acquired 1H HR-MAS NMR spectra of separated cortex and medulla samples using multivariate statistical methods. Concentrations of glycerophosphocholine (GPC) were found to be significantly higher in the renal medulla compared to the cortex. Using GPC as a biomarker, we identified the tissue slices that were predominantly the cortex or medulla. This study demonstrates that HR-MAS SLS combined with multivariate statistics has the potential for identifying tissue heterogeneity and detailed biochemical characterization of complex tissue samples.


Stiffness of the human foot and evolution of the transverse arch.
The stiff human foot enables an efficient push-off when walking or running, and was critical for the evolution of bipedalism1-6. The uniquely arched morphology of the human midfoot is thought to stiffen it5-9, whereas other primates have flat feet that bend severely in the midfoot7,10,11. However, the relationship between midfoot geometry and stiffness remains debated in foot biomechanics12,13, podiatry14,15 and palaeontology4-6. These debates centre on the medial longitudinal arch5,6 and have not considered whether stiffness is affected by the second, transverse tarsal arch of the human foot16. Here we show that the transverse tarsal arch, acting through the inter-metatarsal tissues, is responsible for more than 40% of the longitudinal stiffness of the foot. The underlying principle resembles a floppy currency note that stiffens considerably when it curls transversally. We derive a dimensionless curvature parameter that governs the stiffness contribution of the transverse tarsal arch, demonstrate its predictive power using mechanical models of the foot and find its skeletal correlate in hominin feet. In the foot, the material properties of the inter-metatarsal tissues and the mobility of the metatarsals may additionally influence the longitudinal stiffness of the foot and thus the curvature-stiffness relationship of the transverse tarsal arch. By analysing fossils, we track the evolution of the curvature parameter among extinct hominins and show that a human-like transverse arch was a key step in the evolution of human bipedalism that predates the genus Homo by at least 1.5 million years. This renewed understanding of the foot may improve the clinical treatment of flatfoot disorders, the design of robotic feet and the study of foot function in locomotion.


Surgery for degenerative lumbar spondylosis.
Surgical investigations and interventions account for as much as one third of the health care costs for spinal disorders, but the scientific evidence for most procedures is still unclear. Degenerative conditions affecting the lumbar spine are variously described as lumbar spondylosis or degenerative disc disease (which we regarded as one entity) and are associated with back pain, instability, spinal stenosis and degenerative spondylolisthesis. The objective of this review was to assess the effects of surgical interventions for the treatment of degenerative lumbar spondylosis. We searched the Cochrane Controlled Trials Register, Medline, Embase, Biosis, Dissertation Abstracts, Index to UK Thesis, and reference lists of the retrieved articles and we corresponded with experts. All data found up to 31/12/99 are included. Randomised or quasi-randomised trials of surgical treatment of lumbar spondylosis Two reviewers assessed trial quality and extracted data from published papers. Additional information was sought from the authors if necessary. Sixteen published trials of all forms of surgical treatment for degenerative lumbar spondylosis have been identified. There were many serious weaknesses of trial design, including poor methods of randomisation, lack of blinding and lack of independent assessment of outcome which at times gave considerable potential for bias. Most of the published results were reporting on technical surgical outcomes with some crude ratings of clinical outcome, but few patient-centred outcomes of pain, disability or capacity for work. There was a particular lack of long-term outcomes. This review found no published trials comparing any form of surgery for degenerative lumbar spondylosis compared with natural history, placebo, or any form of conservative treatment. Ten trials randomly compared instrumented and non-instrumented fusion. Instrumented fusion produced a higher fusion rate (though that needs to be qualified by the difficulty of assessing fusion in the presence of metal-work) but did not improve clinical outcomes and there is evidence that it may be associated with higher complication rates. The few and heterogeneous trials on spondylolisthesis, spinal stenosis and nerve compression permitted very limited conclusions. There is no scientific evidence about the effectiveness of any form of surgical decompression or fusion for degenerative lumbar spondylosis compared with natural history, placebo, or conservative treatment.


Understanding primate brain evolution.
We present a detailed reanalysis of the comparative brain data for primates, and develop a model using path analysis that seeks to present the coevolution of primate brain (neocortex) and sociality within a broader ecological and life-history framework. We show that body size, basal metabolic rate and life history act as constraints on brain evolution and through this influence the coevolution of neocortex size and group size. However, they do not determine either of these variables, which appear to be locked in a tight coevolutionary system. We show that, within primates, this relationship is specific to the neocortex. Nonetheless, there are important constraints on brain evolution; we use path analysis to show that, in order to evolve a large neocortex, a species must first evolve a large brain to support that neocortex and this in turn requires adjustments in diet (to provide the energy needed) and life history (to allow sufficient time both for brain growth and for 'software' programming). We review a wider literature demonstrating a tight coevolutionary relationship between brain size and sociality in a range of mammalian taxa, but emphasize that the social brain hypothesis is not about the relationship between brain/neocortex size and group size per se; rather, it is about social complexity and we adduce evidence to support this. Finally, we consider the wider issue of how mammalian (and primate) brains evolve in order to localize the social effects.


Interventions to improve linear growth during complementary feeding period for children aged 6-24 months living in low- and middle-income countries: a systematic review and network meta-analysis.
Background: Optimizing linear growth in children during complementary feeding period (CFP) (6-24 months) are critical for their development. Several interventions, such as micronutrient and food supplements, deworming, maternal education, and water, sanitation and hygiene (WASH), could potentially be provided to prevent stunting, but their comparative effectiveness is currently unclear. In this study, we evaluated comparative effectiveness of interventions under these domains on child linear growth outcomes of height-for-age z-score (HAZ) and stunting (HAZ <-2SD) Methods: For this study, we searched for low- and middle-income country (LMIC)-based randomized clinical trials (RCTs) of aforementioned interventions provided to children during CFP. We searched for reports published until September 17, 2019 and hand-searched bibliographies of existing reviews. We performed random-effects network meta-analysis (NMA) for HAZ and stunting. Results: The evidence base for our NMA was based on 79 RCTs (96 papers) involving 81,786 children. Among the micronutrients, compared to standard-of-care, iron + folic acid (IFA) (mean difference =0.08; 95% credible interval [CrI]: 0.01, 0.15) and multiple micronutrients (MMN) (mean difference =0.06; 95%CrI: 0.01, 0.11) showed improvements for HAZ; MMN also reduced the risks for stunting (RR=0.86; 95%Crl: 0.73, 0.98), whereas IFA did not (RR=0.92; 95%Crl: 0.64, 1.23). For food supplements, flour in the caloric range of 270-340 kcal (RR=0.73; 95%Crl: 0.51, 1.00) and fortified lipid-based nutrient supplements (LNS) containing 220-285 kcal (RR=0.80; 95%Crl: 0.66, 0.97) decreased the risk of stunting compared to standard-of-care, but these interventions and other food supplements did not show improvements for HAZ. Deworming, maternal education, and WASH interventions did not show improvements for HAZ nor stunting. Conclusion: While we found micronutrient and food supplements to be effective for HAZ and/or stunting, the evidence base for other domains in this life stage was limited, highlighting the need for more investigation. Registration: PROSPERO CRD42018110449; registered on 17 October 2018.


Tick box for child? The ethical positioning of children as vulnerable, researchers as Barbarians and reviewers as overly cautious.
Children have historically been marginalized in research with proxies used as appropriate sources of information about children's experiences, perceptions and understandings. In the context of research governance and ethical review, the default setting in many countries reflects a long history of framing children within a discourse of vulnerability and seeing research with (or on) children as inherently risky. This perspective inevitably positions researchers as '(potentially) dangerous' and requires reviewers to adopt a (super-) cautious approach to any research proposal involving children's participation. Halse and Honey [Halse, C., Honey, A., 2007. Rethinking ethics review as institutional discourse. Qualitative Inquiry 13 (3), 336-352] suggest that this forces the researcher into the position of barbarian and the reviewers into the role of the research subject's champion. However, the discourse of child vulnerability competes with the discourse of child participation and involvement. Research with children is further complicated since children also have tended to be viewed as a homogenous group although this clearly misrepresents the diversity, individuality and multiplicity of their geographies. The tensions that arise from children being protected from the predations of the 'barbarian researchers' have meant that researchers have tended to undertake research on the least vulnerable/most adult-like children. This has resulted in some interesting and important research although the findings are likely to have much less utility than if researchers made greater efforts to engage or been given the opportunities to engage with the more marginalized children. Researchers and reviewers are a heterogeneous group each sharing some barbarian and champion tendencies. Both groups need to acknowledge their own 'adultist' assumptions and discursive practices in relation to involving children within research and consider the benefits (as well as the risks) that can accrue from researching children's own perspectives. Currently, the 'tick box' approach automatically identifies a research study as inherently more risky, forcing researchers into a defensive position and framing children as vulnerable even when the risks may be neglible and the risks of not doing the research are higher for children. Considering ways in which we could stretch our current understanding of vulnerability through the use of child-led and oriented ethics review could accommodate the strengths, expertise and capacities of children.


Bypassing the Blood-Brain Barrier: Direct Intracranial Drug Delivery in Epilepsies.
Epilepsies are common chronic neurological diseases characterized by recurrent unprovoked seizures of central origin. The mainstay of treatment involves symptomatic suppression of seizures with systemically applied antiseizure drugs (ASDs). Systemic pharmacotherapies for epilepsies are facing two main challenges. First, adverse effects from (often life-long) systemic drug treatment are common, and second, about one-third of patients with epilepsy have seizures refractory to systemic pharmacotherapy. Especially the drug resistance in epilepsies remains an unmet clinical need despite the recent introduction of new ASDs. Apart from other hypotheses, epilepsy-induced alterations of the blood-brain barrier (BBB) are thought to prevent ASDs from entering the brain parenchyma in necessary amounts, thereby being involved in causing drug-resistant epilepsy. Although an invasive procedure, bypassing the BBB by targeted intracranial drug delivery is an attractive approach to circumvent BBB-associated drug resistance mechanisms and to lower the risk of systemic and neurologic adverse effects. Additionally, it offers the possibility of reaching higher local drug concentrations in appropriate target regions while minimizing them in other brain or peripheral areas, as well as using otherwise toxic drugs not suitable for systemic administration. In our review, we give an overview of experimental and clinical studies conducted on direct intracranial drug delivery in epilepsies. We also discuss challenges associated with intracranial pharmacotherapy for epilepsies.


The ACE-2/Ang1-7/Mas cascade enhances bone structure and metabolism following angiotensin-II type 1 receptor blockade.
The renin angiotensin system (RAS) regulates numerous systemic functions and is expressed locally in skeletal tissues. Angiotensin1-7 (Ang1-7) is a beneficial member of the RAS, and the therapeutic effects of a large number of angiotensin receptors blockers (ARBs) are mediated by an Ang1-7-dependent cascade. This study examines whether the reported osteo-preservative effects of losartan are mediated through the angiotensin converting enzyme2 (ACE-2)/Ang1-7/Mas pathway in ovariectomized (OVX) rats. Sham and OVX animals received losartan (10mg/kg/d p.o.) for 6 weeks. A specific Mas receptor blocker (A-779) was delivered via mini-osmotic pumps during the losartan treatment period. Serum and urine bone metabolism biomarker levels were measured. Bone trabecular and cortical morphometry were quantified in distal femurs, whereas mineral contents were estimated in ashed bones, serum and urine. Finally, the expression of RAS components, the receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) was determined. Losartan significantly improved the elevated bone metabolism marker levels and altered trabecular and cortical structures in OVX animals, and restored normal urinary and skeletal mineral levels. Mas receptor inhibition significantly abolished all osteo-protective effects of losartan and enhanced the deleterious effects of OVX. Losartan enhanced OVX-induced up-regulation of ACE-1, AngII, angiotensin type 1 (AT1) receptor and RANKL expression, and increased ACE-2, Ang1-7, Mas and OPG expression in OVX animals. However, A-779 significantly eradicated the effects of losartan on RAS components and RANKL/OPG expression. Thus, Ang1-7 are involved in the osteo-preservative effects of losartan via Mas receptor, which may add therapeutic value to this well-known antihypertensive agent.


Mechanical plasticity of collagen directs branch elongation in human mammary gland organoids.
Epithelial branch elongation is a central developmental process during branching morphogenesis in diverse organs. This fundamental growth process into large arborized epithelial networks is accompanied by structural reorganization of the surrounding extracellular matrix (ECM), well beyond its mechanical linear response regime. Here, we report that epithelial ductal elongation within human mammary organoid branches relies on the non-linear and plastic mechanical response of the surrounding collagen. Specifically, we demonstrate that collective back-and-forth motion of cells within the branches generates tension that is strong enough to induce a plastic reorganization of the surrounding collagen network which results in the formation of mechanically stable collagen cages. Such matrix encasing in turn directs further tension generation, branch outgrowth and plastic deformation of the matrix. The identified mechanical tension equilibrium sets a framework to understand how mechanical cues can direct ductal branch elongation.


Human microvascular endothelial cells use beta 1 and beta 3 integrin receptor complexes to attach to laminin.
Microvascular endothelial cells (MEC) use a set of surface receptors to adhere not only to the vascular basement membrane but, during angiogenic stimulation, to the interstitium. We examined how cultured human MEC interact with laminin-rich basement membranes. By using a panel of monoclonal antibodies, we found that MEC cells express a number of integrin-related receptor complexes, including alpha 1 beta 1, alpha 2 beta 1, alpha 3 beta 1, alpha 5 beta 1, alpha 6 beta 1, alpha V beta 3. Attachment to laminin, a major adhesive protein in basement membranes, was studied in detail. Blocking monoclonal antibodies specific to different integrin receptor complexes showed that the alpha 6 beta 1 complex was important for MEC adhesion to laminin. In addition, blocking antibody also implicated the vitronectin receptor (alpha V beta 3) in laminin adhesion. We used ligand affinity chromatography of detergent-solubilized receptor complexes to further define receptor specificity. On laminin-Sepharose columns, we identified several integrin receptor complexes whose affinity for the ligand was dependent on the type of divalent cation present. Several beta 1 complexes, including alpha 1 beta 1, alpha 2 beta 1, and alpha 6 beta 1 bound strongly to laminin. In agreement with the antibody blocking experiments, alpha V beta 3 was found to bind well to laminin. However, unlike binding to its other ligands (e.g., vitronectin, fibrinogen, von Willebrand factor), alpha V beta 3 interaction with laminin did not appear to be Arg-Gly-Asp (RGD) sensitive. Finally, immunofluorescent staining demonstrated both beta 1 and beta 3 complexes in vinculin-positive focal adhesion plaques on the basal surface of MEC adhering to laminin-coated substrates. The results indicate that both these subfamilies of integrin heterodimers are involved in promoting MEC adhesion to laminin and the vascular basement membrane.


Selectivity of intracellular proteolysis: protein substrates activate the ATP-dependent protease (La).
A critical enzyme in protein breakdown in Escherichia coli is protease La (the lon gene product), which hydrolyzes proteins and adenosine triphosphate (ATP) in a coupled process. The mechanism of this process was studied with fluorogenic tripeptides. Although proteins and peptides are degraded at the same active site, protein substrates enhance the ability of the enzyme to degrade these peptides two- to tenfold. Proteins that are not substrates had little or no effect. Thus, protein substrates must bind to protease La at two sites, the active site and an allosteric site whose occupancy enhances proteolytic activity. This effect did not require that the proteins themselves be degraded. Proteins could induce peptide breakdown even in the absence of ATP, and proteins and ATP had additive effects in stimulating peptidase activity. A multistep cyclical mechanism is proposed in which the binding of the substrate and ATP activates the protease. The enzyme can then cleave a peptide bond, but is inactivated through ATP hydrolysis. Such a mechanism may help account for the selectivity of protein breakdown and prevent inappropriate or excessive proteolysis in vivo.


Differential Impact of Anastomotic Leak in Patients With Stage IV Colonic or Rectal Cancer: A Nationwide Cohort Study.
Anastomotic leak has a negative impact on the prognosis of patients who undergo colorectal cancer resection. However, data on anastomotic leak are limited for stage IV colorectal cancers. The purpose of this study was to investigate the impact of anastomotic leak on survival and the decision to administer chemotherapy and/or metastasectomy after elective surgery for stage IV colorectal cancer. This was a nationwide, retrospective cohort study. Data were obtained from the Danish Colorectal Cancer Group, the Danish Pathology Registry, and the National Patient Registry. Patients who were diagnosed with stage IV colorectal cancer between 2009 and 2013 and underwent elective resection of their primary tumors were included. The primary outcome was all-cause mortality depending on the occurrence of anastomotic leak. Secondary outcomes were the administration of and time to adjuvant chemotherapy, metastasectomy rate, and risk factors for leak. Of the 774 patients with stage IV colorectal cancer who were included, 71 (9.2%) developed anastomotic leaks. Anastomotic leak had a significant impact on the long-term survival of patients with colon cancer (p = 0.04) but not on those with rectal cancer (p = 0.91). Anastomotic leak was followed by the decreased administration of adjuvant chemotherapy in patients with colon cancer (p = 0.007) but not in patients with rectal cancer (p = 0.47). Finally, anastomotic leak had a detrimental impact on metastasectomy rates after colon cancer but not on resection rates of rectal cancer. Retrospective data on the selection criteria for primary tumor resection and metastatic tumor load were unavailable. The impact of anastomotic leak on patients differed between stage IV colon and rectal cancers. Survival and eligibility to receive chemotherapy and metastasectomy differed between patients with colon and rectal cancers. When planning for primary tumor resection, these factors should be considered.


Anaerobic degradation of linear alkylbenzene sulfonate.
Linear alkylbenzene sulfonate (LAS) found in wastewater is removed in the wastewater treatment facilities by sorption and aerobic biodegradation. The anaerobic digestion of sewage sludge has not been shown to contribute to the removal. The concentration of LAS based on dry matter typically increases during anaerobic stabilization due to transformation of easily degradable organic matter. Hence, LAS is regarded as resistant to biodegradation under anaerobic conditions. We present data from a lab-scale semi-continuously stirred tank reactor (CSTR) spiked with linear dodecylbenzene sulfonate (C12 LAS), which show that C12 LAS was biodegradable under methanogenic conditions. Sorption of C12 LAS on sewage sludge was described with a Freundlich isotherm. The C12 LAS sorption was determined with different concentrations of total solids (TS). In the semi-continuously stirred tank reactor, 18% of the added C12 LAS was bioavailable and 20% was biotransformed when spiking with 100 mg/L of C12 LAS and a TS concentration of 14.2 mg/L. Enhanced bioavailability of C12 LAS was obtained in an upflow anaerobic sludge blanket (UASB) reactor inoculated with granular sludge and sewage sludge. Biodegradation under thermophilic conditions was 37% with LAS as sole carbon source. Benzaldehyde was produced in the UASB reactor during LAS transformation.


Clearance of triamcinolone from vitreous.
The clearance of intravitreally injected triamcinolone acetonide was monitored by both indirect ophthalmoscopy and high-performance liquid chromatography (HPLC) and found to be more rapid than previously reported by others. Twenty-four rabbits were intravitreally injected in each eye with 0.4 mg of triamcinolone acetonide. Three rabbits each were sacrificed at intervals ranging from one hour to 46 days. The vitreous was then harvested and processed for HPLC analysis. Triamcinolone and the internal standard prednisolone were identified and quantitated by the use of HPLC, which was found to be both sensitive and specific for the steroids. The half-life as determined by HPLC was 1.6 days, the level at 13 days postinjection was 66 +/- 19 micrograms, and no drug was detectable by HPLC analysis at 21 days in five of six eyes. The intravitreal masses thought to be triamcinolone were clinically observable to an average of 23.3 days. The chromatographically determined clearance rate did not correlate well with clinical impressions based on indirect ophthalmoscopy.


[Implication of stem cells in the biology and therapy of head and neck cancer].
Stem cells play a central role in re- and generation of tissues. Special importance has been attributed to them in cancer biology. 2 entities can be discriminated: cancer infiltrating stem cells and cancer initiating stem cells. Infiltrating stem cells will be attracted to the tumor in order to be remodelled for tumor expansion, e. g. endothelial cells or other cancerous tissue components, yet these cells are per se benign. Malignant cancer stem cells are capable to generate a new tumor, histologically identical with the cancer they originate from. Many steps in cancer stem cell biology are not understood to date. It is still believed that CSC are only a minor cell fraction in tumor but capable to differentiate in hierarchical manner into any other tissue type. These stem cells are undergoing many steps of differentiation and dedifferentiation in a steady state. The factors of the micromilieu contributing to this are largely not understood. Still these steps are regarded as the key to tumor initiation, metastases and resistance to therapy. The biological functions and associated signal transduction pathways, e. g. self renewal pathways will be the key to future therapeutical strategies.


Capsular sialic acid of Streptococcus suis serotype 2 binds to swine influenza virus and enhances bacterial interactions with virus-infected tracheal epithelial cells.
Streptococcus suis serotype 2 is an important swine bacterial pathogen, and it is also an emerging zoonotic agent. It is unknown how S. suis virulent strains, which are usually found in low quantities in pig tonsils, manage to cross the first host defense lines to initiate systemic disease. Influenza virus produces a contagious infection in pigs which is frequently complicated by bacterial coinfections, leading to significant economic impacts. In this study, the effect of a preceding swine influenza H1N1 virus (swH1N1) infection of swine tracheal epithelial cells (NTPr) on the ability of S. suis serotype 2 to adhere to, invade, and activate these cells was evaluated. Cells preinfected with swH1N1 showed bacterial adhesion and invasion levels that were increased more than 100-fold compared to those of normal cells. Inhibition studies confirmed that the capsular sialic acid moiety is responsible for the binding to virus-infected cell surfaces. Also, preincubation of S. suis with swH1N1 significantly increased bacterial adhesion to/invasion of epithelial cells, suggesting that S. suis also uses swH1N1 as a vehicle to invade epithelial cells when the two infections occur simultaneously. Influenza virus infection may facilitate the transient passage of S. suis at the respiratory tract to reach the bloodstream and cause bacteremia and septicemia. S. suis may also increase the local inflammation at the respiratory tract during influenza infection, as suggested by an exacerbated expression of proinflammatory mediators in coinfected cells. These results give new insight into the complex interactions between influenza virus and S. suis in a coinfection model.


Microscopic colitis syndrome: lymphocytic colitis and collagenous colitis.
Microscopic colitis is a syndrome consisting of chronic watery diarrhea, a normal or near-normal gross appearance of the colonic lining, and a specific histological picture described as either lymphocytic colitis or collagenous colitis. Since its initial descriptions a quarter of a century ago, microscopic colitis has become a frequent diagnosis in patients with chronic diarrhea. Understanding of the cause and pathogenesis of microscopic colitis remain incomplete, but potentially important clues have been discovered that shed light on predisposing factors. In particular, specific HLA-DQ genotypes may be permissive for the development of microscopic colitis, and suggest a linkage to the pathogenesis of celiac sprue. Although the differential diagnosis of chronic watery diarrhea is broad, the diagnosis of microscopic colitis is straightforward, involving endoscopic inspection of the colonic mucosa and proper pathologic interpretation of biopsy specimens. As the limitations of drugs ordinarily used for other forms of inflammatory bowel disease are being recognized, new approaches, such as the use of bismuth subsalicylate, are being evaluated. The prognosis of patients with microscopic colitis syndrome remains good, and symptomatic improvement can be expected in most patients.


The evolution of immunosuppressive cell populations in experimental mycobacterial infection.
Immunosuppressor activity of considerable potency and complexity was generated during the course of chronic, progressive infection of C3H/Anf mice by Mycobacterium lepraemurium. From the 5th through 10th week after inoculation, spleen cells from infected mice mildly but reproducibly suppressed the direct plaque-forming cell response of normal spleen cell cultures to sheep erythrocytes. Suppression at this stage of infection was mediated by cells with macrophage-like characteristics. A marked increase in splenic suppressor activity at 10 to 11 weeks was associated with the appearance of a second suppressor cell subpopulation composed of T lymphocytes. The appearance of these cells was closely related in time to the onset of rapid splenic enlargement and a loss of cutaneous delayed type hypersensitivity to antigens of M. lepraemurium in mice at 10 to 11 weeks of infection. Suppressor cells were not present in peripheral lymph nodes until terminal infection at 22 to 25 weeks. Suppressor spleen cells depressed the T-dependent antibody response most severely, but there was also a direct effect upon B cells as shown by moderate suppression of responses to TNP-LPS and DNP-Ficoll. Spleen cells from 14-week-infected mice generated a soluble suppressor factor(s) that induces depression of moderate severity, however, the immunosuppression by intact cells was far greater.


Partial characterization of biliary metabolites of pulegone by tandem mass spectrometry. Detection of glucuronide, glutathione, and glutathionyl glucuronide conjugates.
The hepatotoxic monoterpene pulegone is a major constituent of the herbal abortifacient pennyroyal oil. An approximately equimolar mixture of 2H3- and 14C-labeled pulegone was administered to rats to study its phase II metabolism. Radioactive conjugates that were excreted into the bile were isolated by selective derivatization and HPLC separation, and subsequently characterized from the daughter ion mass spectra of protio- and deutero-analogs of each metabolite. The biliary metabolites characterized were glucuronide and glutathione (GSH) conjugates, accounting for approximately 3% of the radioactivity excreted in bile. The glucuronides, which were 2-fold more abundant than GSH conjugates, were mainly of hydroxylated pulegone and hydroxylated, reduced pulegone. The three GSH conjugates contained xenobiotic moieties that varied in their oxidation state; one of these was tentatively identified as the GSH conjugate of the proximate oxygenated metabolite, menthofuran. The two other GSH conjugates apparently underwent subsequent glucuronidation since novel glutathionyl glucuronide conjugates were identified that contained nonhydroxylated xenobiotic moieties. The results indicate that pulegone is bioactivated via at least three distinct pathways, each marked by a different GSH conjugate. Characterization of these conjugates represents a first step in the identification of the reactive metabolites from which they are derived.


Pain and discomfort associated with common hospital procedures and experiences.
To determine whether reliable and valid rankings of pain and discomfort resulting from hospital procedures encountered by advanced dementia patients could be developed from interviews with cognitively intact adults, rankings of pain and discomfort resulting from 16 common procedures were obtained from two samples of hospitalized, nondemented adults using ten- (N = 100) and five- (N = 35) point numeric rating scales (NRS). Reliability was assessed by having 30 additional subjects complete ten-point NRS representing the ten most frequent procedures in a re-arranged order. By repeated measure analysis of variance, the scales discriminated between procedures (F = 35.1, P < 0.001). Subjects could discriminate between pain and discomfort (F = 21.6, P < 0.001). The five-point NRS exhibited better subject discrimination between experiences. Reliability was also acceptable. A five-point NRS produced reliable and valid pain and discomfort rankings for 16 common hospital procedures and experiences. These rankings should prove useful in reducing suffering and can serve as surrogates for quantifying pain and discomfort in dementia patients.


Effects of the 5-HT(1A) agonist (+/-)-8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) on cocaine choice in cynomolgus monkeys.
Drugs that alter brain serotonin (5-HT) function can modulate the behavioral effects of cocaine, but the underlying receptor mechanisms are poorly understood. The present study examined the effects of the selective 5-HT1A receptor agonist (+/-)-8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT, 0.01-0.1 mg/kg, i.v.) on cocaine self-administration in the context of a choice procedure. Five adult male cynomolgus monkeys self-administered cocaine (saline, 0.003-0.03 mg/kg per injection) under a concurrent fixed-ratio 50 schedule of food (1-g banana-flavored pellets) and cocaine presentation. Allocation of responses to the cocaine-associated lever (cocaine choice) increased in a dose-related manner from < or =20% of total responses when saline or 0.003 mg/kg per injection cocaine was the alternative to food to > or =75% when 0.03 mg/kg per injection cocaine was available. In four of five monkeys, when choice was between a low cocaine dose and food, 0.01 mg/kg 8-OH-DPAT increased injection-lever responding. At cocaine doses which occasioned > or =75% cocaine choice, 8-OH-DPAT did not alter response allocation. In the fifth monkey, 8-OH-DPAT only decreased injection-lever responding. When choice was between saline and food, 8-OH-DPAT did not reliably shift responding to the injection lever, except at doses that disrupted operant performance. These results suggest that a 5-HT1A receptor agonist can increase the reinforcing strength of a low cocaine dose relative to a concurrently available non-drug reinforcer.


Chronic ethanol feeding and folate deficiency activate hepatic endoplasmic reticulum stress pathway in micropigs.
Previously, we showed that feeding micropigs ethanol with a folate-deficient diet promoted the development of hepatic injury while increasing hepatic levels of homocysteine and S-adenosylhomocysteine (SAH) and reducing the level of S-adenosylmethionine (SAM) and the SAM-to-SAH ratio. Our present goals were to evaluate mechanisms for hepatic injury using liver specimens from the same micropigs. The effects of ethanol feeding or folate-deficient diets, singly or in combination, on cytochrome P-450 2E1 (CYP2E1) and signal pathways for apoptosis and steatosis were analyzed using microarray, real-time PCR, and immunoblotting techniques. Apoptosis was increased maximally by the combination of ethanol feeding and folate deficiency and was correlated positively to liver homocysteine and SAH. Liver CYP2E1 and the endoplasmic reticulum stress signals glucose-regulated protein 78 (GRP78), caspase 12, and sterol regulatory element binding protein-1c (SREBP-1c) were each activated in pigs fed folate-deficient or ethanol diets singly or in combination. Liver mRNA levels of CYP2E1, GRP78, and SREBP-1c, and protein levels of CYP2E1, GRP78, nuclear SREBP, and activated caspase 12 each correlated positively to liver levels of SAH and/or homocysteine and negatively to the SAM-to-SAH ratio. The transcripts of the lipogenic enzymes fatty acid synthase, acetyl-CoA carboxylase, and stearoyl-CoA desaturase were elevated in the ethanol-fed groups, and each was positively correlated to liver homocysteine levels. The induction of abnormal hepatic methionine metabolism through the combination of ethanol feeding with folate deficiency is associated with the activation of CYP2E1 and enhances endoplasmic reticulum stress signals that promote steatosis and apoptosis.


The practical analysis of food: the development of Sakalar quantification table of DNA (SQT-DNA).
Practical and highly sensitive Sakalar quantification table of DNA (SQT-DNA) has been developed for the detection% of species-specific DNA amount in food products. Cycle threshold (Ct) data were obtained from multiple curves of real-time qPCR. The statistical analysis was done to estimate the concentration of standard dilutions. Amplicon concentrations versus each Ct value were assessed by the predictions of targets at known concentrations. SQT-DNA was prepared by using the percentage versus each Ct values. The applicability of SQT-DNA to commercial foods was proved by using sausages containing varying ratios of beef, chicken, and soybean. The results showed that SQT-DNA can be used to directly quantify food DNA by a single PCR without the need to construct a standart curve in parallel with the samples every time the experiment is performed, and also quantification by SQT-DNA is as reliable as standard curve quantification for a wide range of DNA concentrations.


Genome-wide association analysis reveals new targets for carotenoid biofortification in maize.
Genome-wide association analysis in CIMMYT's association panel revealed new favorable native genomic variations in/nearby important genes such as hydroxylases and CCD1 that have potential for carotenoid biofortification in maize. Genome-wide association studies (GWAS) have been used extensively to identify allelic variation for genes controlling important agronomic and nutritional traits in plants. Provitamin A (proVA) enhancing alleles of lycopene epsilon cyclase (LCYE) and β-carotene hydroxylase 1 (CRTRB1), previously identified through candidate-gene based GWAS, are currently used in CIMMYT's maize breeding program. The objective of this study was to identify genes or genomic regions controlling variation for carotenoid concentrations in grain for CIMMYT's carotenoid association mapping panel of 380 inbred maize lines, using high-density genome-wide platforms with ~476,000 SNP markers. Population structure effects were minimized by adjustments using principal components and kinship matrix with mixed models. Genome-wide linkage disequilibrium (LD) analysis indicated faster LD decay (3.9 kb; r (2) = 0.1) than commonly reported for temperate germplasm, and therefore the possibility of achieving higher mapping resolution with our mostly tropical diversity panel. GWAS for various carotenoids identified CRTRB1, LCYE and other key genes or genomic regions that govern rate-critical steps in the upstream pathway, such as DXS1, GGPS1, and GGPS2 that are known to play important roles in the accumulation of precursor isoprenoids as well as downstream genes HYD5, CCD1, and ZEP1, which are involved in hydroxylation and carotenoid degradation. SNPs at or near all of these regions were identified and may be useful target regions for carotenoid biofortification breeding efforts in maize; for example a genomic region on chromosome 2 explained ~16% of the phenotypic variance for β-carotene independently of CRTRB1, and a variant of CCD1 that resulted in reduced β-cryptoxanthin degradation was found in lines that have previously been observed to have low proVA degradation rates.


Heparin-associated thrombocytopenia: antibody binding specificity to platelet antigens.
Sera from four patients with heparin-associated thrombocytopenia (HAT) were evaluated by a quantitative enzyme-linked immunosorbent assay (ELISA) to detect heparin-dependent serum platelet-bindable immunoglobulin (S-PBIg) and by Western blotting and immunoprecipitation to investigate the specificity of the antibody binding. All HAT sera showed mildly increased S-PBIg (mean, 7.8 fg per platelet; normal, less than 6.0 fg per platelet) to intact target platelets in the ELISA, which was markedly increased in the presence of heparin (mean, 20.9 fg per platelet). This increase was 20-fold greater than normal control sera, which showed a mean differential increase of only 0.5 fg per platelet. Immunoglobulin binding specificity to platelet antigens was investigated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis of platelet lysate with transfer of the platelet fractions onto nitrocellulose strips (Western blotting) and subsequent immunoassay using HAT and normal sera. In the presence of heparin, the four HAT patients demonstrated increased binding of immunoglobulin to platelet antigens of apparent molecular weights of 180, 124, and 82 kd. Radiolabeled heparin when incubated with HAT sera, normal sera, or albumin blanks bound to platelet proteins of the same apparent molecular weights. These observations are consistent with current hypotheses suggesting that HAT antibody is directed to heparin-platelet complexes or, alternatively, that heparin induces conformational change of antigenic sites on the platelet membrane.


To Effectively Address Complex Healthcare Problems, Continuing Professional Development Must Evolve.
Continuing professional development aims to provide health professionals with the knowledge, skills, and competencies needed to improve care. Physicians and other clinicians increasingly practice within complex health care delivery organizations aiming to improve the care of populations of patients with multiple problems and differing needs. These organizations are composed of local units in different departments and venues; these teams and the patients they care for change over time. Improving outcomes within constantly changing complex organizations delivering population care takes time and persistence. It takes time to equip critical masses of clinicians and other personnel with knowledge and skills to effect change. Although some changes might be simple, those involving new workflows require implementation support. Not all change will be smooth; individuals need opportunities to learn from and adjust their early intervention efforts, measure effectiveness of change, and sustain successful practices. Longitudinal support is necessary to affect change over complex organizations. This essay proposes that to be more supportive and valuable to health care delivery organizations, continuing professional development needs to intentionally participate in longitudinal, collaborative, context-specific, team-based interventions. An expanded menu of evaluation approaches will better describe the role of continuing professional development in helping health care professionals and organizations address increasingly complex health care delivery problems and improve patient and population outcomes. Selected concepts to achieve these ends are introduced at a high level in this article. Readers are invited to explore concepts that resonate with their current situation in further detail.