Heterologous expression, purification, and properties of human cytochrome P450 27C1.
Cytochrome P450 (P450) 27C1 is one of the "orphan" P450 enzymes without a known biological function. A human P450 27C1 cDNA with a nucleotide sequence modified for Escherichia coli usage was prepared and modified at the N-terminus, based on the expected mitochondrial localization. A derivative with residues 3-60 deleted was expressed at a level of 1350nmol/L E. coli culture and had the characteristic P450 spectra. The identity of the expressed protein was confirmed by mass spectrometry of proteolytic fragments. The purified P450 was in the low-spin iron state, and the spin equilibrium was not perturbed by any of the potential substrates vitamin D(3), 1alpha- or 25-hydroxy vitamin D(3), or cholesterol. P450s 27A1 and 27B1 are known to catalyze the 25-hydroxylation of vitamin D(3) and the 1alpha-hydroxylation of 25-hydroxy vitamin D(3), respectively. In the presence of recombinant human adrenodoxin and adrenodoxin reductase, recombinant P450 27C1 did not catalyze the oxidation of vitamin D(3), 1alpha- or 25-hydroxy vitamin D(3), or cholesterol at detectable rates. P450 27C1 mRNA was determined to be expressed in liver, kidney, pancreas, and several other human tissues.


Emotional intelligence: an integrative meta-analysis and cascading model.
Research and valid practice in emotional intelligence (EI) have been impeded by lack of theoretical clarity regarding (a) the relative roles of emotion perception, emotion understanding, and emotion regulation facets in explaining job performance; (b) conceptual redundancy of EI with cognitive intelligence and Big Five personality; and (c) application of the EI label to 2 distinct sets of constructs (i.e., ability-based EI and mixed-based EI). In the current article, the authors propose and then test a theoretical model that integrates these factors. They specify a progressive (cascading) pattern among ability-based EI facets, in which emotion perception must causally precede emotion understanding, which in turn precedes conscious emotion regulation and job performance. The sequential elements in this progressive model are believed to selectively reflect Conscientiousness, cognitive ability, and Neuroticism, respectively. "Mixed-based" measures of EI are expected to explain variance in job performance beyond cognitive ability and personality. The cascading model of EI is empirically confirmed via meta-analytic data, although relationships between ability-based EI and job performance are shown to be inconsistent (i.e., EI positively predicts performance for high emotional labor jobs and negatively predicts performance for low emotional labor jobs). Gender and race differences in EI are also meta-analyzed. Implications for linking the EI fad in personnel selection to established psychological theory are discussed.


The role of hyphenated chromatography-mass spectrometry techniques in exploratory drug metabolism and pharmacokinetics.
The advances in high-speed synthesis technologies have produced a large number of biologically active new chemical entities (NCEs) for developability assessment. Current drug discovery efforts have been focused on identifying drug metabolism and pharmacokinetic (DMPK) issues at the earliest possible stage in order to reduce the attrition rate of drug candidates during the development phase. Mass spectrometry (MS) has proven a powerful tool in providing rapid qualitative and quantitative measurements of drug molecules for DMPK studies in both drug discovery and development. Although mass spectrometers can serve as separation devices, for most pharmaceutical applications, some form of chromatography is combined with MS. For most MS-based methods, tandem mass spectrometry (MS/MS) utilizes atmospheric pressure ionization (API) and chromatographic techniques. This review describes the major hyphenated chromatography-mass spectrometry techniques and their applications in supporting exploratory DMPK studies including various in vitro and in vivo PK and metabolite identification profiles.


Direct quantification in bioanalytical LC-MS/MS using internal calibration via analyte/stable isotope ratio.
The possibility to rationalize and simplify bioanalysis, without compromising the analytical quality, by omitting the calibration curves was studied. Using mass spectrometry (MS) and a stable isotope labeled internal standard it was possible to get equally good results by calculating the results directly from the analyte/internal standard area ratio and a predetermined response factor as by the traditional way, using a calibration curve run at the same occasion. To be able to use this simplified quantification method, that we call internal calibration, in its most simple form there are some prerequisites that must be considered: (1) The relative response should not be concentration dependent. (2) The relative response should be constant between batches/days. (3) The level of analyte in the internal standard should not be detectable. (4) There should be no influence from naturally occurring isotopes of the analyte on the internal standard peak area. A bioanalytical LC-MS/MS method for a research compound was validated both with and without calibration curves and no significant differences were found regarding precision and accuracy. It was shown that all four prerequisites above were fulfilled. Validation data were very good for the whole concentration range, 0.010-30 micromol/L. Long-term data for QC samples showed excellent precision and accuracy.


Network tuned multiple rank aggregation and applications to gene ranking.
With the development of various high throughput technologies and analysis methods, researchers can study different aspects of a biological phenomenon simultaneously or one aspect repeatedly with different experimental techniques and analysis methods. The output from each study is a rank list of components of interest. Aggregation of the rank lists of components, such as proteins, genes and single nucleotide variants (SNV), produced by these experiments has been proven to be helpful in both filtering the noise and bringing forth a more complete understanding of the biological problems. Current available rank aggregation methods do not consider the network information that has been observed to provide vital contributions in many data integration studies. We developed network tuned rank aggregation methods incorporating network information and demonstrated its superior performance over aggregation methods without network information.


Pseudomonas cepacia typing systems: collaborative study to assess their potential in epidemiologic investigations.
To determine the utility of available Pseudomonas cepacia typing systems for confirming the relatedness of isolates, we applied these methods to isolates associated with previously investigated nosocomial outbreaks. We compared chromosome analysis, serologic reactions, biochemical tests, bacteriocin production and susceptibility, and antimicrobial susceptibility in their ability to determine outbreak relatedness. Chromosome analysis, serologic reactions, and biochemical tests were each demonstrated to be epidemiologically useful methods for typing isolates. Determination of the sensitivity and specificity of these typing techniques will facilitate their application in the epidemiologic study of this increasingly important nosocomial pathogen.


Identification of conserved structural features at sequentially degenerate locations in transcription factor binding sites.
Many locations within transcription factor binding sites are not sequentially conserved and appear to be degenerate. We hypothesize that some of these positions contain essential structural codes that are recognized by the transcription factors that bind to them. The structural codes can be defined by base-pair step parameters that describe the relative displacement and orientation of two adjacent base pairs in a nucleic acid structure. We have developed a method, Identification of Conserved Structural Features (ICSF), which uses base-pair step parameters obtained from a collection of high-resolution DNA crystal structures to discover structural conservation that exists in the sequentially degenerate areas within a binding site and produce profiles of the structural features along the entire site. We have focused our study on the transcription factor binding sites in the JASPAR database and have found that one-third (P-value > or = 0.05) of the binding sites contain sequentially degenerate locations with highly conserved structural features as described by the base-pair step parameters. These results will help us to gain a better understanding of the process by which transcription factors recognize their binding sites and possibly lead to an improvement in our ability to find these sites in genomic sequences. ICSF is freely available to academic users at http://zlab.bu.edu/ICSF . http://zlab.bu.edu/ICSF .


The hydrogen games and other adventures in chemistry.
I seem to have started off on the wrong foot in life, but I am extremely fortunate that I soon found my footing in the company of physical chemists. I consider myself to be very lucky to be doing something that constantly brings me in contact with bright minds, stimulating conversations, and exciting experiments. My work has allowed me to learn astounding facts about the molecules and atoms that make up our surroundings and ourselves. For this article, I focus on one aspect of my research, understanding the fundamental principles of the simple reaction between a hydrogen atom and a hydrogen molecule. Although my group and others have been studying this seemingly simple reaction for well over 30 years, it continues to provoke questions about the properties of matter.


Thyroid status regulates particulate but not soluble TRH-degrading pyroglutamate aminopeptidase activity in the rat liver.
Rat liver contains two topologically different TRH-degrading pyroglutamate aminopeptidases. The particulate pyroglutamate aminopeptidase, unlike the soluble one, was highly specific for TRH and shared many physico-chemical properties with serum thyroliberinase, which is controlled by thyroid hormones. Both enzymes convert pGlu His Pro NH2 into His Pro NH2; the latter may be cyclized to cyclo His Pro known to possess several biological activities and specific binding sites in liver. The aim of the present study was to determine the effects of thyroid status on the particulate and soluble enzymes activity and gain more insight into their biological role. The regulatory pathway for the particulate pyroglutamate aminopeptidase was found similar to that of serum thyroliberinase: their specific activities decrease in hypothyroid rats and increase in hyperthyroid rats, whereas that of soluble enzyme remains unchanged. We postulate that the particulate pyroglutamate aminopeptidase may be a determining factor in the concentrations of TRH and/or cyclo His Pro reaching liver cells and a possible source for serum thyroliberinase. Taken together, these data suggest that this "converting enzyme" acts as a physiological regulator.


A new variant Cu/Zn superoxide dismutase (Val7-->Glu) deduced from lymphocyte mRNA sequences from Japanese patients with familial amyotrophic lateral sclerosis.
We have identified a new mutant Cu/Zn superoxide dismutase (SOD1) deduced from the nucleotide sequences of peripheral blood lymphocyte mRNA from Japanese patients with familial amyotrophic lateral sclerosis (FALS). Sequence analysis of reverse transcriptase-initiated PCR amplified mRNA revealed a heterozygosity indicative of one normal allele and one variant allele with a T-->A transversion. This base change led to replacement of valine by glutamic acid at position 7 of 153-residue SOD1 molecule, and produced a new restriction site for Alu I in the exon 1. Restriction fragment length polymorphism analysis confirmed the linkage of this mutation with this type of FALS. Both enzymatic activity and protein of the SOD1 were reduced in red blood cells from the patient.


Multimodality imaging, single center, cross-sectional study in adolescents or young adults with repaired tetralogy of Fallout.
Proper integration of multiple imaging modalities in the routine follow-up of patients with repaired tetralogy of Fallout (TOF) is poorly supported by data. We report our single center comparative study between cardiac magnetic resonance (CMR) and echocardiography to assess equipoise in the clinical utility of these two imaging methods in an unselected consecutive cohort of TOF patients referred to our outpatient clinic. In this cross-sectional study, repaired TOF patients who underwent CMR and echocardiography within a 4-week period between 2010 and 2011 at our Center were included. Linear regression was used to analyze degree of inter modality correlation. A prediction model tested the association between functional data/probrain natriuretic peptide (Pro-BNP) with CMR. Fifty patients were included in the study (mean age 31 ± 18 years). The best predictors of right ventricle (RV) ejection fraction at CMR were tricuspid anular plane systolic excursion (tricuspid valve anular plane systolic excursion, R 0.37, P < 0.0001) and RV peak S-wave velocity (R 0.40, P < 0.001). Pro-BNP levels did present weak correlation with New York Heart Association functional class (R 0.31, P < 0.002) and QRS duration (R 0.32, P < 0.002) and a moderate correlation with right atrium area at CMR (R 0.46, P < 0.0001). We found limited correlation between the two imaging modalities in the evaluation of RV after intracardiac repair of TOF. Pro-BNP level presents moderate correlation with right atrium area measured with echocardiography. Serial CMR evaluations are needed in this patient population, but they may be interchanged by routine echocardiography in particular in patients with normal or stable echocardiographic parameters.


Self-organization at the interface and in aqueous solution of a cationic gemini surfactant from the dioctyl ester of cystine.
The cationic surfactant, dioctyl ester of cystine hydrochloride (DOEC), was characterized for interfacial adsorption and aggregation behavior in water. The cmc of DOEC was measured as 1.42±0.27×10(-5) mol dm(-3) using the techniques of tensiometry, conductivity and fluorimetry. From specific conductivity measurements, the degree of dissociation (α) of the amine hydrochloride was measured as 0.612. The standard free energy change of micellization (ΔG(m)(°)) and adsorption (ΔG(a)(°)) were calculated to be -25.07 and -44.37 kJ mol(-1), respectively. The aggregated structures provide non-polar microdomains as inferred from the I(3)/I(1) emission intensity ratio of 1.05 of pyrene fluoroprobe and also a blue shift of fluorescence emission wave length (λ(emi.)) maximum down to 470 nm with enhanced intensity of ANS probe in micellar solutions. From Langmuir film balance experiments, it is shown that DOEC forms stable viscoelastic films at the interface with A(0) at 0.69 nm(2)molecule(-1) that agree with the result from surface tension measurements. Molecular modeling suggests the tilted orientation of DOEC at the interface. A large packing parameter (P) of 0.58 and the fibril structures as observed from microscopy studies demonstrate that DOEC favors one-dimensional growth to form elongated micelles.


Vascular endothelial growth factor from retinal pigment epithelium is essential in choriocapillaris and axial length maintenance.
Myopia, which prevalence is rapidly increasing, causes visual impairment; however, the onset mechanism of pathological axial length (AL) elongation remains unclear. A highly vascularized choroid between the retinal pigment epithelium (RPE) and sclera not only maintains physiological activities, but also contributes to ocular development and growth regulation. Vascular endothelial growth factor (VEGF) secreted from the RPE to the choroid is essential for retinal function and maintenance of the choriocapillaris. Herein, we demonstrated that the loss of VEGF secreted from the RPE caused abnormal choriocapillaris development and AL elongation, with features similar to those of the lens-induced myopia (LIM) mouse model, whereas VEGF overexpression by knocking-out von Hippel-Lindau (VHL) specific to the RPE expands the choriocapillaris and shortens the AL. Additionally, LDL Receptor Related Protein 2 (LRP2) deletion in the RPE downregulated VEGF expression and leads to pathological AL elongation. Furthermore, high-myopia patients without choriocapillaris demonstrated longer ALs than did those with preserved choriocapillaris. These results suggest that physiological secretion of VEGF from the RPE is required for proper AL development by maintaining the choriocapillaris. The pinpoint application of VEGF to the choriocapillaris may become a potential intervention for the prevention and treatment of axial myopia progression.


Interferon lambda as a potential treatment for COVID-19.
Pegylated interferon lambda substantially reduced the risk of COVID-19-related hospitalizations or emergency room visits in a recent phase 3, multi-center, randomized, double-blind, placebo-controlled study of high-risk, non-hospitalized adult patients with SARS-CoV-2 infection compared to treatment with placebo. Interferons are a family of signaling molecules produced as part of the innate immune response to viral infections. The administration of exogenous interferon may limit disease progression in patients with COVID-19. Interferons have been used to treat viral infections, including hepatitis B and hepatitis C, and malignancies such as non-Hodgkin's lymphoma, as well as the autoimmune condition multiple sclerosis. This manuscript examines what is known about the role of interferon lambda in the treatment of COVID-19, including potential limitations, and explores how this approach may be used in the future.


Description of the novel perchlorate-reducing bacteria Dechlorobacter hydrogenophilus gen. nov., sp. nov.and Propionivibrio militaris, sp. nov.
Novel dissimilatory perchlorate-reducing bacteria (DPRB) were isolated from enrichments conducted under conditions different from those of all previously described DPRB. Strain LT-1(T) was enriched using medium buffered at pH 6.6 with 2-(N-morpholino)ethanesulfonic acid (MES) and had only 95% 16S rRNA gene identity with its closest relative, Azonexus caeni. Strain MP(T) was enriched in the cathodic chamber of a perchlorate-reducing bioelectrical reactor (BER) and together with an additional strain, CR (99% 16S rRNA gene identity), had 97% 16S rRNA gene identity with Propionivibrio limicola. The use of perchlorate and other electron acceptors distinguished strains MP(T) and CR from P. limicola physiologically. Strain LT-1(T) had differences in electron donor utilization and optimum growth temperatures from A. caeni. Strains LT-1(T) and MP(T) are the first DPRB to be described in the Betaproteobacteria outside of the Dechloromonas and Azospira genera. On the basis of phylogenetic and physiological features, strain LT-1(T) represents a novel genus in the Rhodocyclaceae; strain MP(T) represents a novel species within the genus Propionivibrio. The names Dechlorobacter hydrogenophilus gen. nov., sp. nov and Propionivibrio militaris sp. nov. are proposed.


Improved in vitro blood compatibility of polycaprolactone nanowire surfaces.
There are a multitude of polymeric materials currently utilized to prepare a variety of blood-contacting implantable medical devices. These devices include tissue grafts, coronary artery and vascular stents, and orthopedic implants. The thrombogenic nature of such materials can cause serious complications in patients, and ultimately lead to functional failure. To date, there is no truly hemocompatible biomaterial surface. Nanostructured surfaces improve cellular interactions but there is a limited amount of information regarding their blood compatibility. In this study, the in vitro blood compatibility of four different surfaces (control, PCL; nanowire, NW; collagen immobilized control, cPCL; collagen immobilized nanowire, cNW) were investigated for their use as interfaces for blood-contacting implants. The results presented here indicate enhanced in vitro blood compatibility of nanowire surfaces compared control surfaces. Although there were no significant differences in leukocyte adhesion, there was a decrease in platelet adhesion on NW surfaces. Scanning electron microscopy images showed a decrease in platelet/leukocyte complexes on cNW surfaces and no apparent complexes were formed on NW surfaces compared to PCL and cPCL surfaces. The increase in these complexes likely contributed to a higher expression of specific markers for platelet and leukocyte activation on PCL and cPCL surfaces. No significant differences were found in contact and complement activation on any surface. Further, thrombin antithrombin complexes were significantly reduced on NW surfaces. A significant increase in hemolysis and fibrinogen adsorption was identified on PCL surfaces likely caused by its hydrophobic surface. This work shows the improved blood-compatibility of nanostructured surfaces, identifying this specific nanoarchitecture as a potential interface for promoting the long-term success of blood-contacting biomaterials.


Appendectomy, cholecystectomy and diagnostic laparoscopy conducted before pregnancy and risk of adverse birth outcomes: a nationwide registry-based prevalence study 1996-2015.
Non-obstetric surgery conducted during pregnancy may increase the risk of adverse birth outcomes like small for gestational age, preterm birth, and miscarriage. Mechanisms are unclear but possibly longer lasting. We examined whether appendectomy, cholecystectomy and diagnostic laparoscopy conducted before pregnancy affect these outcomes. This nationwide Danish prevalence study included all pregnancies during 1996-2015 that had an appendectomy, cholecystectomy or diagnostic laparoscopy registered before last menstrual period in the years 1992-2015. We excluded pregnancies with surgery and categorized pre-pregnancy surgery according to timing (0-11, 12-23, and 24+ months before last menstrual period). Outcomes were small for gestational age, late preterm birth (32-37 weeks), early preterm birth (22-31 weeks) and miscarriage (7-21 weeks). We computed absolute risks and used logistic regression comparing pregnancies with surgery 0-11 or 12-23 to 24+ months before last menstrual period, computing odds ratios for each outcome, adjusting for maternal age and smoking. We identified 15,939 pregnancies with appendectomy, 12,869 pregnancies with cholecystectomy and 19,330 pregnancies with diagnostic laparoscopy. The absolute risk of small for gestational age was 2.2% for patients with appendectomy 0-11 months before last menstrual period, 3.2% 12-23 months before compared with 2.2% when appendectomy was conducted more than 24 months before (adjusted OR 0.95 (95% CI; 0.65 to 1.31) and 1.37(95% CI;1.00 to 1.86). For early preterm birth, the absolute risks were 0.7, 0.5 and 0.8%, for late preterm birth 4.8, 4.4 and 4.7% and for miscarriage 5.7, 6.2 and 5.4%.We observed similar results for cholecystectomy. For diagnostic laparoscopy 0-11 months before pregnancy we found increased risks of small for gestational age (4.0, 2.8 and 2.6%) and late preterm birth (5.9, 5.0 and 4.8%). We found no increased risk of adverse birth outcomes among pregnancies with appendectomy or cholecystectomy conducted within 2 years before pregnancy compared to more than 2 years before pregnancy. The increased risks 0-11 months after diagnostic laparoscopy are likely explained by confounding by underlying indication. It appears safe to become pregnant any time following appendectomy and cholecystectomy, but, probably depending on indication, attention should be payed 0-11 months after diagnostic laparoscopy.


Chronic consumption of a hypercaloric diet increases neuroinflammation and brain senescence, promoting cognitive decline in middle-aged female Wistar rats.
Being overweight and obesity are world health problems, with a higher prevalence in women, defined as abnormal or excessive fat accumulation that increases the risk of chronic diseases. Excess energy leads to adipose expansion, generating hypertrophic adipocytes that produce various pro-inflammatory molecules. These molecules cause chronic low-intensity inflammation, affecting the organism's functioning and the central nervous system (CNS), inducing neuroinflammation. The neuroinflammatory response during obesity occurs in different structures of the CNS involved in memory and learning, such as the cortex and the hippocampus. Here we analyzed how obesity-related peripheral inflammation can affect CNS physiology, generating neuroinflammation and promoting cellular senescence establishment. Since some studies have shown an increase in senescent cells during aging, obesity, and neurodegenerative diseases, we proposed that cellular senescence participation may contribute to the cognitive decline in an obesity model of middle-aged female Wistar rats. The inflammatory state of 6 and 13 months-old female Wistar rats fed with a hypercaloric diet was measured in serum and CNS (cortex and hippocampus). Memory was evaluated using the novel object recognition (NOR) test; the presence of senescent markers was also determined. Our data suggest that the systemic inflammation generated by obesity induces a neuroinflammatory state in regions involved in learning and memory, with an increase in senescent markers, thus proposing senescence as a potential participant in the negative consequences of obesity in cognition.


Early Intravenous Magnesium Sulfate Administration in the Emergency Department for Severe Asthma Exacerbations.
Severe asthma exacerbations in pediatric patients occur frequently and can require pediatric intensive care unit (PICU) admission. To determine if early administration of intravenous magnesium sulfate (IVMg) to pediatric patients experiencing severe asthma exacerbations, defined as a respiratory clinical score (RCS) of 9 to 12, resulted in fewer PICU admissions. Retrospective chart review of pediatric patients aged from 2 to 17 years presenting with a severe asthma exacerbation to a single tertiary care pediatric emergency department. Univariable and multivariable logistic regression analyses were used to determine if admission to the PICU was associated with early IVMg treatment, within 60 minutes of registration. A total of 1911 patients were included in the study, of which 1541 received IVMg. The average time to IVMg was 79 minutes, with 35% of the patients receiving it within 60 minutes of arrival. Two hundred forty-eight (13%) were admitted to the PICU, 641 (34%) were admitted to the general inpatient floor, and 1022 (53%) were discharged home. Factors associated with increased odds ratio (OR) of PICU admission were: early IVMg (OR, 1.63; 95% CI: 1.16-2.28), arrival mode to the emergency department via ambulance (OR, 2.23; 95% CI: 1.45-3.43), history of PICU admission for asthma (OR, 1.73; 95% CI: 1.22-2.44), and diagnosis of status asthmaticus (OR, 8.88; 95% CI: 3.49-30.07). Calculated OR of PICU admission subcategorized by RCS for early IVMg patients, after controlling for PICU risk factors, are as follows: RCS 9 (reference), RCS 10 (OR, 2.52; 95% CI: 0.89-2.23), RCS 11 (OR, 2.19; 95% CI: 1.3-3.70), and RCS 12 (OR, 4.12; 95% CI: 2.13-7.95). Early administration of IVMg to pediatric patients experiencing severe asthma exacerbations does not result in fewer PICU admissions.


Highly biocompatible nanofibrous microspheres self-assembled from chitin in NaOH/urea aqueous solution as cell carriers.
In this work, chitin microspheres (NCM) having a nanofibrous architecture were constructed using a "bottom-up" fabrication pathway. The chitin chains rapidly self-assembled into nanofibers in NaOH/urea aqueous solution by a thermally induced method and subsequently formed weaved microspheres. The diameter of the chitin nanofibers and the size of the NCM were tunable by controlling the temperature and the processing parameters to be in the range from 26 to 55 nm and 3 to 130 μm, respectively. As a result of the nanofibrous surface and the inherent biocompatibility of chitin, cells could adhere to the chitin microspheres and showed a high attachment efficiency, indicating the great potential of the NCM for 3D cell microcarriers.


Atypical alpha-conotoxin LtIA from Conus litteratus targets a novel microsite of the alpha3beta2 nicotinic receptor.
Different nicotinic acetylcholine receptor (nAChR) subtypes are implicated in learning, pain sensation, and disease states, including Parkinson disease and nicotine addiction. alpha-Conotoxins are among the most selective nAChR ligands. Mechanistic insights into the structure, function, and receptor interaction of alpha-conotoxins may serve as a platform for development of new therapies. Previously characterized alpha-conotoxins have a highly conserved Ser-Xaa-Pro motif that is crucial for potent nAChR interaction. This study characterized the novel alpha-conotoxin LtIA, which lacks this highly conserved motif but potently blocked alpha3beta2 nAChRs with a 9.8 nm IC(50) value. The off-rate of LtIA was rapid relative to Ser-Xaa-Pro-containing alpha-conotoxin MII. Nevertheless, pre-block of alpha3beta2 nAChRs with LtIA prevented the slowly reversible block associated with MII, suggesting overlap in their binding sites. nAChR beta subunit ligand-binding interface mutations were used to examine the >1000-fold selectivity difference of LtIA for alpha3beta2 versus alpha3beta4 nAChRs. Unlike MII, LtIA had a >900-fold increased IC(50) value on alpha3beta2(F119Q) versus wild type nAChRs, whereas T59K and V111I beta2 mutants had little effect. Molecular docking simulations suggested that LtIA had a surprisingly shallow binding site on the alpha3beta2 nAChR that includes beta2 Lys-79. The K79A mutant disrupted LtIA binding but was without effect on an LtIA analog where the Ser-Xaa-Pro motif is present, consistent with distinct binding modes.


Synthesis, optical properties, and electronic structures of fully core-modified porphyrin dications and isophlorins.
The synthesis, structures, optical properties, and electronic structures of the tetraphenyltetrathiaporphyrin dication (S(4)TPP(2+), 6) and tetrakis(pentafluorophenyl)tetrathiaisophlorin (S(4)F(20)TPP, 7) are reported. S(4)TPP(2+) (6) and S(4)F(20)TPP (7) were synthesized by acid-catalyzed condensation of the corresponding hydroxylmethylthiophene, followed by oxidation. The electronic structures of S(4)TPP(2+) (6) and S(4)F(20)TPP (7) were analyzed by using UV/Vis-absorption spectroscopy and by magnetic circular dichroism (MCD) spectroscopy and the bands were assigned by using time-dependent density functional theory (TD-DFT) and ZINDO/s calculations. A red-shift of the Q bands of S(4)TPP(2+) (6) is observed relative to the spectra of tetraphenylporphyrins because a destabilization of the HOMO leads to a narrower HOMO-LUMO band-gap. Michl's perimeter model was used to assign the absorption bands and MCD spectra of S(4)F(20)TPP (7). Current-density maps and nucleus-independent chemical-shift (NICS) calculations of S(4)TPP(2+) (6) and of a model compound predict marked modification to the diamagnetic ring current, whilst nonaromatic character is predicted for S(4)F(20)TPP (7).


Climate, immigration and speciation shape terrestrial and aquatic biodiversity in the European Alps.
Quaternary climate fluctuations can affect speciation in regional biodiversity assembly in two non-mutually exclusive ways: a glacial species pump, where isolation in glacial refugia accelerates allopatric speciation, and adaptive radiation in underused adaptive zones during ice-free periods. We detected biogeographic and genetic signatures associated with both mechanisms in the assembly of the biota of the European Alps. Age distributions of endemic and widespread species within aquatic and terrestrial taxa (amphipods, fishes, amphibians, butterflies and flowering plants) revealed that endemic fish evolved only in lakes, are highly sympatric, and mainly of Holocene age, consistent with adaptive radiation. Endemic amphipods are ancient, suggesting preglacial radiation with limited range expansion and local Pleistocene survival, perhaps facilitated by a groundwater-dwelling lifestyle. Terrestrial endemics are mostly of Pleistocene age and are thus more consistent with the glacial species pump. The lack of evidence for Holocene adaptive radiation in the terrestrial biome is consistent with faster recolonization through range expansion of these taxa after glacial retreats. More stable and less seasonal ecological conditions in lakes during the Holocene may also have contributed to Holocene speciation in lakes. The high proportion of young, endemic species makes the Alpine biota vulnerable to climate change, but the mechanisms and consequences of species loss will likely differ between biomes because of their distinct evolutionary histories.


Circulating gonadotropin pattern in anorchia syndrome. Response to treatment.
Two anorchidic brothers, 16 and 25 years of age were studied; three, 60 min interval samples were drawn on the day -1 followed by a daily sample for five days, and after every seven days. 25 mg of testosterone propionate were administered daily during 24 days and after, 100 mg of testosterone enanthate were administered every 14 days (prolonged action). Testosterone, F S H and L H were quantified by R I A. Testosterone concentrations were 0.75 and 0.52 ng/ml; F S H 22.0 and 24.0 ng/ml and L H, 18.0 and 25.0 ng/ml respectively in A. F. M. and A.F.J. With testosterone propinate, F S H basal levels were decreased in 53 and 55 per cent, and L H, 80 and 86 per cent in A.F.M. and A.F.J. respectively. With prolonged action testosterone, F S H and L H levels were within normal limits after a 7 day administration course. Due to the decrease in F S H and L H initial levels as a result of treatment we concluded that in the anorchia syndrome, receptors in the hypothalamus - pituitary axis maintain their normal ability of feedback mechanism response with testosterone administration and thus prolonged action testosterone is the best substitute.


[Study on effect of Xianzhong Injection on morphology of knee and insulin-like growth factor-1 in rabbits with early stage osteoarthritis].
To investigate the effect of Xianzhong Injection (XI) on morphology of knee and insulin-like growth factor-1 (IGF-1) in rabbits with early stage osteoarthritis (OA). One hundred and twenty rabbits were randomly divided into 5 groups, the blank group, the model group treated with 0.5 mL normal saline once a week, the positive control group treated with 0.5 mL hyaluronate once a week, and the XI 1 group and XI 2 group with 0.5 mL XI once and twice a week, respectively, all the medicines were medicated via intra-articular injection. Knee joint function was examined 8 weeks after modeling, IGF-1 content in serum and synovia was detected, and the histologic examination of articular cartilage was observed by Mallory staining as well. The improvement of knee joint function was better in the XI 1 and 2 groups than that in the model group (P < 0.01), and that in the XI 1 group was equivalent to that in the positive control group (P > 0.05). Histologic examination showed there was no calcification in all the layers of cartilage in the positive control group and the XI 1 group with complete cartilaginous bluish-green collagen preserved. The content of IGF-1 increased in synovia and serum in the positive control group and the two XI groups as compared with that in the model group (P < 0.05). XI can improve knee joint function to certain extent in early OA, and repair the degenerative cartilage.


[Astragaloside IV regulates STAT1/IκB/NF-κB signaling pathway to inhibit activation of BV-2 cells].
The study was aimed to investigate the inhibitory effect and mechanism of astragaloside IV (ASI) on the activation of microglial cells. After pre-incubated with ASI for 2 h, microglial cells BV-2 were stimulated with interferon-γ (IFN-γ) for 1. 5 h and 24 h, respectively. Secretion of nitric oxide (NO) in the medium was measured by Griess method. Production of tumor necrosis factor alpha (TNF-α) was detected by ELISA approach. Cellular gene expressions of CD11b, TNF-α, interleukin 1β (IL-1β) and induced nitric oxide synthase (iNOS) were examined by quantitative-PCR analysis. Total and phosphorylation of STAT1, IκB and NF-κB was analyzed by Western blot method. ASI could significantly inhibit the increased secretion of TNF-α and NO from BV-2 cells upon IFN-γ stimulation (P < 0.001). Further study showed that ASI significantly down-regulated gene expression of IL-1β and TNF-α (P < 0.01, P < 0.05) and exhibited a trend to reduce that of iNOS. IFN-γ and ASI have no obvious effect on gene expression of CD11b. Moreover, ASI inhibited the phosphorylation of STAT1, IκB and NF-κB elicited by IFN-γ stimulation. ASI could restrain microglial activation through interfering STAT1/IκB/NF-κB signaling pathway, reducing gene expres- sion of IL-1β and TNF-α, and thus inhibiting the production of proinflammatory mediators such as NO and TNF-α.


[Diabetes and osteoporosis].
Over the last decades a considerable amount of data has accumulated to indicate that metabolic and endocrine alterations of diabetes affect bone quantity and quality. These skeletal changes may increase the risk of bone fracture. There is strong evidence that in type 1 diabetes the decreased bone mass, lack of insulin and insulin-like growth factor-1, dysregulation of adipokines, and increased levels of proinflammatory cytokines are in the background of fragility fractures. In type 2 diabetes hyperinsulinemia, insulin resistance and increased body weight may result in an increase of bone mass; however, accumulation of advanced glycation end products within the bone collagen driven by glucotoxicity may increase the cortical porosity. There is a higher incidence of falls resulting from diabetes-related co-morbidities such as diabetic retinopathy, peripheral neuropathy, hypoglycemic episodes and sometimes from the medications. Vitamin D deficiency has special impact on glucose metabolism and the prevalence of diabetes. Vitamin D supplementation in childhood can decrease incidence of type 1 diabetes by 80%. The effect of thiazolidinediones, glucagon-like peptide-1 agonists and metformin, agents for treatment of diabetes open a new connection between bone, carbohydrate and fat metabolism.


Recombinant Protein Expression Chassis Library of Vibrio natriegens by Fine-Tuning the Expression of T7 RNA Polymerase.
Vibrio natriegens is the fastest-growing bacteria, and its doubling time is less than 10 min. At present, the T7 expression system has been introduced into V. natriegens for heterologous protein expression, including the commercial strain Vmax1 and the variant VnDX,2 which is a backup expression chassis of Escherichia coli BL21(DE3). However, the strength of the existing T7 expression system is not optimal for every recombinant protein. The different expression strengths of T7 RNA polymerase (T7 RNAP) can be obtained by changing the promoter and ribosome binding site (RBS) sequences of T7 RNAP at different transcription and translation levels. In this work, we obtained a robust VnDX variant library with the fine-tuning T7 RNAP using the industrially used enzyme glucose dehydrogenase (GDH) as the reporter protein. Among this library, the variant VnDX-tet, whose promoter of T7 RNAP was changed from PlacUV5 to Ptet, showed that the reporter enzyme GDH activity was increased by 109% by the T7 expression system. Similarly, variants with different T7 RNAP translation levels were obtained by changing RBS sequences upstream of T7 RNAP, and the results showed that the variant VnDX-RBS12/pGDH had the highest GDH activity, which increased by 12.6%. The VnDX variant library constructed in this study with different T7 expression strengths provides a choice for expressing various recombinant proteins, greatly expanding the application of V. natriegens.


Contribution of the intensive care unit environment to sleep disruption in mechanically ventilated patients and healthy subjects.
Recent studies have challenged the traditional hypothesis that excessive environmental noise is central to the etiology of sleep disruption in the intensive care unit (ICU). We characterized potentially disruptive ICU noise stimuli and patient-care activities and determined their relative contributions to sleep disruption. Furthermore, we studied the effect of noise in isolation by placing healthy subjects in the ICU in both normal and noise-reduced locations. Seven mechanically ventilated patients and six healthy subjects were studied by continuous 24-hour polysomnography with time-synchronized environmental monitoring. Sound elevations occurred 36.5 +/- 20.1 times per hour of sleep and were responsible for 20.9 +/- 11.3% of total arousals and awakenings. Patient-care activities occurred 7.8 +/- 4.2 times per hour of sleep and were responsible for 7.1 +/- 4.4% of total arousals and awakenings. Healthy subjects slept relatively well in the typically loud ICU environment and experienced a quantitative, but not qualitative, improvement in sleep in a noise-reduced, single-patient ICU room. Our data indicate that noise and patient-care activities account for less than 30% of arousals and awakenings and suggest that other elements of the critically ill patient's environment or treatment should be investigated in the pathogenesis of ICU sleep disruption.


Heavy-chain amyloidosis in TGFBI-negative and gelsolin-negative atypical lattice corneal dystrophy.
An atypical case of late-onset lattice corneal dystrophy is described in a 61-year-old man without a family history of eye disease. Mutational analysis of the TGFBI gene excluded any pathogenic sequence variants. However, 2 years later, renal impairment and nephrotic syndrome were diagnosed, resulting in a diagnosis of systemic heavy-chain amyloidosis. Slit-lamp examination, corneal photography, and in vivo confocal microscopy were performed. General systemic evaluation included blood and urine assessment, bone marrow and renal biopsies, and cardiologic evaluation. A DNA sample underwent initial mutational analysis of TGFBI and, subsequently, gelsolin. The renal biopsy sample was subject to direct protein sequencing by mass spectrometry. A bilateral, atypical, fine, midperipheral lattice corneal dystrophy with minor central subepithelial scarring was clinically characterized. Subsequently, abnormal renal functions with proteinuria, IgG lambda paraproteinemia, extensive deposition of amyloid in renal glomeruli, and increased plasma cells in bone marrow were identified. No pathogenic sequence mutations were identified in TGFBI or the gelsolin genes. Direct protein sequencing by mass spectrometry showed amyloid to be heavy-chain deposition rather than the more usual light-chain deposition. Atypical midperipheral lattice corneal dystrophy presenting with adult onset and negative family history should arouse suspicion for an association with paraproteinemias or amyloidosis. Exclusion of TGFBI mutations should alert the clinician to the possibility of potentially life-threatening conditions, with referral for careful systemic evaluation.


[Medical crisis, clinical engagement].
Medicine is changing, and so are the causes of dissatisfaction among physicians. In a broader context characterized by the dominance of an economic rationality, in which medicine is shaped by the phenomenon of acceleration typical of modernity, we argue, in this brief article, that clinical practice is based on an engagement involving a form of suspension of the flow of time. Engagement is thereby an essential dimension of medicine, and is proposed as a lasting foundation of clinical practice.


Emerging strategies for antibody-mediated rejection.
Antibody-mediated rejection (AMR) is the leading cause of kidney graft loss. Very few treatment options are available to the clinician to counter this disease process. In this review we describe the available therapeutics and the novel approaches that are being currently developed. AMR treatment requires a multidrug approach. Imlifidase, a new immunoglobulin G cleaving agent, may prove to be the perfect replacement of apheresis. New complement blockers other than eculizumab are in development in order to block acute kidney damage in the delicate phase following antibody removal. Plasma cell depletion is being explored in chronic AMR: studies are in progress with daratumumab and felzartamab. Interleukin 6 inhibition is generating enthusiasm in the chronic setting with preliminary encouraging results. In acute AMR, the clinicians will have to remove the antibodies, avoid rebound and block specific damage effectors. In chronic AMR they will need to reduce the inflammatory response induced by donor specific antibodies. New drugs are available and transplant physicians are starting to develop effective multidrug strategies to counter the complex disease mechanisms. Safety of these drugs needs to be further explored especially when used together with other potent immunosuppressive drugs.


v-sis oncogene-induced transformation of human fibroblasts into cells capable of forming benign tumors.
Normal human fibroblasts do not synthesize platelet-derived growth factor (PDGF), but they respond to its mitogenic action. In contrast, cells from several human fibrosarcomas have been shown to synthesize PDGF at significant levels. To investigate the possible role of PDGF expression in the development of tumors of mesenchymal origin in humans, we transfected a plasmid carrying the v-sis oncogene and a selectable marker into an infinite lifespan, non-tumorigenic human fibroblast cell strain, MSU-1.1. The v-sis gene codes for a protein homolog of PDGF-B. Of the six independent drug-resistant transfectants clonally isolated, three expressed a relatively low level of v-sis mRNA and protein, grew to a saturation density only 2-2.5 times higher than MSU-1.1 cells, did not form colonies in agar, were not growth factor independent and did not form tumors. The other three expressed v-sis mRNA and protein at a high level, grew to a very high saturation density (> 750,000 cells/cm2), replicated in medium lacking exogenous growth factors almost as rapidly as with 10% serum, formed large colonies in 0.33% agarose and, when injected into athymic mice, formed tumors that grew rapidly. Tumors that were removed after 6 weeks were classified as benign (fibromas). However, several of the tumors that were left in the animals for 6 months developed focal areas of cells showing features of poorly differentiated spindle cell or round cell sarcomas. Similarly, cells isolated from a very large sized agarose colony formed by one of the other v-sis strains expressed very high levels of v-sis mRNA and protein and formed large, very fast growing benign tumors. Re-injection of cells from the tumors yielded tumors composed of two distinct areas: spindle cell fibromas and high grade sarcomas. These results suggest that if mesenchymal cells in the body were induced to express their PDGF-B gene, this could lead to the formation of benign tumors of mesenchymal origin. The system described can serve as a model for studying the mechanisms of formation of such tumors in humans and their progression to the malignant state.


Neighborhood Deprivation and Rurality Associated With Patient-Reported Outcomes and Survival in Men With Prostate Cancer in NRG Oncology RTOG 0415.
Rurality and neighborhood deprivation can contribute to poor patient-reported outcomes, which have not been systematically evaluated in patients with specific cancers in national trials. Our objective was to examine the effect of rurality and neighborhood socioeconomic and environmental deprivation on patient-reported outcomes and survival in men with prostate cancer in NRG Oncology RTOG 0415. Data from men with prostate cancer in trial NRG Oncology RTOG 0415 were analyzed; 1,092 men were randomized to receive conventional radiation therapy or hypofractionated radiation therapy. Rurality was categorized as urban or rural. Neighborhood deprivation was assessed using the area deprivation index and air pollution indicators (nitrogen dioxide and particulate matter with a diameter less than 2.5 micrometers) via patient ZIP codes. Expanded Prostate Cancer Index Composite measured cancer-specific quality of life. The Hopkins symptom checklist measured anxiety and depression. EuroQoL-5 Dimension assessed general health. We analyzed 751 patients in trial NRG Oncology RTOG 0415. At baseline, patients from the most deprived neighborhoods had worse bowel (P = .011), worse sexual (P = .042), and worse hormonal (P = .015) scores; patients from the most deprived areas had worse self-care (P = .04) and more pain (P = .047); and patients from rural areas had worse urinary (P = .03) and sexual (P = .003) scores versus patients from urban areas. Longitudinal analyses showed that the 25% most deprived areas (P = .004) and rural areas (P = .002) were associated with worse EuroQoL-5 Dimension visual analog scale score. Patients from urban areas (hazard ratio, 1.81; P = .033) and the 75% less-deprived neighborhoods (hazard ratio, 0.68; P = .053) showed relative decrease in risk of recurrence or death (disease-free survival). Patients with prostate cancer from the most deprived neighborhoods and rural areas had low quality of life at baseline, poor general health longitudinally, and worse disease-free survival. Interventions should screen populations from deprived neighborhoods and rural areas to improve patient access to supportive care services.


Multidrug-resistant nontyphoidal Salmonella in New York state's foodborne diseases active surveillance network counties.
With the emergence of multidrug-resistant nontyphoidal (NT) Salmonella, knowledge of resistance patterns is critical for appropriate presumptive treatment. This report describes the prevalence and trends of NT Salmonella antimicrobial susceptibility within the New York State (NYS) Foodborne Diseases Active Surveillance Network (FoodNet). The NYS Department of Health, Wadsworth Center Public Health Laboratory tested all Salmonella isolates from the NYS FoodNet catchment area between May 2003 and December 2007 for antimicrobial susceptibility to ampicillin, chloramphenicol, streptomycin, sulfisoxazole, tetracycline, nalidixic acid, and ciprofloxacin. Isolate susceptibility results were linked to their corresponding demographic and clinical data and analyzed. Multidrug-resistant isolates were defined as resistant to ampicillin, chloramphenicol, streptomycin, sulfisoxazole, and tetracycline (R-type ACSSuT). Antimicrobial susceptibility for 2189 FoodNet cases (98.5% of total cases) showed 79.6% pansusceptible, 6.9% R-type ACSSuT, and 13.5% resistant to at least one antimicrobial agent but not R-type ACSSuT. Four (0.2%) isolates were resistant to ciprofloxacin. From 2004 to 2007, cases with R-type ACSSuT significantly decreased from 8.7% (37/424) to 4.8% (24/499) (p < 0.01). Serotypes with the highest proportion of R-type ACSSuT included Salmonella Typhimurium 17.9% (79/444), and Salmonella Newport 29.1% (51/175). Among Salmonella Typhimurium isolates, over 40% of the African-American cases (19/46) had R-type ACSSuT isolates, compared with 15.7% of the Caucasian cases (58/369) (p < 0.01). R-type ACSSuT Salmonella Typhimurium cases were hospitalized (41.8%) more frequently than pansusceptible Salmonella Typhimurium cases (24.9%), after controlling for age (p < 0.05). Length of hospitalization was not significantly different. Although R-type ACSSuT NT Salmonella has decreased since 2003 within the NYS FoodNet catchment area, monitoring resistance patterns remains important in identifying emerging resistant strains, vulnerable populations, and determining appropriate presumptive treatment regimens. The higher rate of R-type ACSSuT among the African-American cases requires further study.


The hemodynamic response of calves to tiletamine-zolazepam anesthesia.
Six isoflurane-anesthetized calves were instrumented for hemodynamic studies and allowed to recover from anesthesia. When the mean arterial blood pressure rose to 100 mmHg or when vigorous movement occurred, a 1:1 tiletamine-zolazepam mixture (4 mg/kg) was administered intravenously (IV). Values for cardiac output, cardiac index, stroke index, central venous pressure, and right ventricular stroke work index did not change significantly. Systolic, mean, and diastolic arterial blood pressures and systemic vascular resistance were significantly decreased below baseline at 5 minutes; they were significantly increased above baseline at 20 minutes and remained so throughout the 60 minute study. Changes in left ventricular stroke work index and rate pressure product were similar to those of arterial blood pressure and systemic vascular resistance, although they were not significant. Heart rate and pulmonary capillary wedge pressure decreased significantly but gradually returned to baseline at 40 minutes and then increased significantly above baseline by the end of the study. Minor venous-arterial shunting or perhaps mismatching of ventilation and perfusion appeared to have developed in the later stages of the study. This was reflected in a minor increase in the arterial partial pressure of carbon dioxide (PaCO2) and a decrease in the arterial partial pressure of oxygen (PaO2) and arterial pH. At the dose administered, the hemodynamic changes induced by tiletamine-zolazepam were minimal and were compatible with safe anesthesia in calves.


Cellulitis of the penis: a case report.
Cellulitis of the penis is an uncommon clinical condition, most often seen in young men, and presents with local and systemic signs that progress rapidly in the absence of treatment. It needs to be differentiated from sexually transmitted infections and dermatological conditions. The present report concerns a case of penile cellulitis in a young, heterosexual man, following sexual intercourse. The clinical presentation, aetiology and management are discussed. Penile swelling in any age group should be viewed with high index of suspicion and sexually transmitted infections should be excluded in young men. Group B haemolytic streptococci are the usual causative organisms, although less virulent organisms should be considered in patients who are immunocompromised. Administration of appropriate antibiotics resolves the local and systemic symptoms and avoids complications.


Enhanced interleukin-1 beta release and longevity of glioma-associated peripheral blood monocytes in vitro.
Interleukin-1 (IL-1) plays a controversial role in the immune response. Besides its activation of immune cells and juvenile central nervous system cells, monocyte-derived IL-1 may be able to stimulate the malignant transformation and proliferation of glial brain tumor cells expressing IL-1 receptors. The aim of this study was to determine the growth pattern and the IL-1 beta release of long-term cultured peripheral blood monocytes of glioma patients. At 6- to 7-day intervals, the vital monocytes, characterized by CD14 immunophenotyping, were counted. By the use of a specific IL-1 beta enzyme-linked immunosorbent assay, the IL-1 beta content of monocyte culture supernatants derived from 13 subjects with glioma and from 12 controls were compared at Days 7, 21, and 100 of culture. Cell clusters of monocytes derived from glioblastoma patients survived more than 250 days in culture, whereas control monocytes survived only up to 114 days. The IL-1 beta release of glioma-associated peripheral blood monocyte cultures was about 50 times higher as compared with control monocyte cultures. Dexamethasone treatment at the time of blood sampling and recurrences of the gliomas did not influence the increase in the IL-1 beta expression of glioma monocytes. It seemed that at least subsets of glioma-associated blood monocytes, although they had been removed from the circulation, remained activated for a long period of time. We conclude that increased IL-1 beta production of glioma-associated peripheral blood monocytes and their longevity in vitro may be features of aberrant immune cell subsets. In future studies, the exact phenotyping of monocyte subsets will be mandatory.


Prevalence and clinical characteristics of fragile X syndrome at child development clinic, Ramathibodi Hospital.
Fragile X syndrome, the most common cause of inherited mental retardation, is an X-linked genetic disorder caused by an expanded CGG repeat in the fragile X mental retardation 1 gene. It is characterized by mental retardation, behavioral features, and physical features, such as a long face with large protruding ears and macro-orchidism. A screening for the syndrome was conducted in a representative sample of pediatric patients, who had developmental delay or mental retardation with unknown cause, at the Child Development Clinic, Ramathibodi Hospital. The DNA test was performed on all patients using PCR and southern blot techniques. Five positive cases were detected from 114 screened subjects, and more four cases confirmed among other family members. Two of five positive families initially denied a family history of mental retardation. Among 9 cases of fragile X syndrome, four had hyperactivity and two had autistic like behavior. More than half had rather a long face or prominent ears. Three boys had macro-orchidism.


Circulating tumor cells: a window into cancer biology and metastasis.
The presence of circulating tumor cells (CTCs) accompanies tumor invasion into the bloodstream. Detection, monitoring, and molecular analysis of these rare cancer cells shed into blood will provide a powerful and noninvasive approach for the detection of early disease, assessing prognosis and therapeutic response in established cancers, and targeting metastatic precursor cells. We review current and emerging technologies for CTC isolation, with a focus on capture efficiency, purity, and viability of CTCs, and their potential clinical applications.


Surgical observation of the agenesis of the foramen of Magendie.
We report a young girl who presented with headache and back pain. Dynamic MRI revealed no cerebrospinal egress from the median aperture (Foramen of Magendie) of the fourth ventricle and syringomyelia. A posterior cranial fossa exploration was performed and agenesis of the median aperture was observed. Following surgical penetration of the posterior aspect of the fourth ventricle and at the most recent follow-up examination, this patient's syringomyelia had resolved, as had her symptoms. Agenesis of the foramen of Magendie may be a rare cause of inhibition of normal cerebrospinal egress from the fourth ventricle with resultant syringomyelia.


Differentiated localizations of phosphorylated focal adhesion kinase in endothelial cells of rat splenic sinus.
The splenic sinus endothelium adhering via adherens junctions and tight junctions regulates the passage of blood cells through the splenic cord. Focal adhesion kinase (FAK) regulates the focal adhesion complex in the basal part of endothelial cells and is an integrated component of cell-cell adhesion, depending on its phosphorylation status. The objectives of this study are to assess the localization of FAK phosphorylated at tyrosine residues and the related proteins of integrin β5, talin, paxillin, p130Cas, vinculin, RhoA, Rac1, Rac2, Cdc42 and VE-cadherin, in the sinus endothelial cells of rat spleen and to examine the roles of FAK in regulating endothelial adhesion and the passage of blood cells. Immunofluorescence microscopy of tissue cryosections revealed that FAK was localized in the entire circumference of sinus endothelial cells and FAK phosphorylated at Try397 residue (pFAKy397) and pFAKy576 were precisely localized in the adherens junctions of the endothelial cells, whereas pFAKy925 was localized in the basal part of the endothelial cells. Paxillin and vinculin were prominently localized in the basal part of the endothelial cells. Integrin β5, talin and p130Cas were colocalized with FAK in the entire circumference of sinus endothelial cells. RhoA, Rac2 and Cdc42 were localized in the entire circumference of sinus endothelial cells close to FAK, stress fibers and cortical actin filaments. Immunogold electron microscopy revealed that pFAKy397 and pFAKy576 were colocalized with VE-cadherin, RhoA, Rac2 and Cdc42 in the adherens junctions of the endothelial cells. Possible functional roles of FAK in splenic sinus endothelial cells are also discussed.


Protein-binding partners of the tobacco syntaxin NtSyr1.
Syntaxins and other SNARE (soluble NSF-attachment protein receptor) complex proteins play a key role in the cellular processes of vesicle trafficking, vesicle fusion and secretion. Intriguingly, the SNARE NtSyr1 (=NtSyp121) from Nicotiana tabacum also appears to have a role in signalling evoked by the plant stress hormone abscisic acid. However, partner proteins contributing to its function(s) remain unknown. We used an affinity chromatography approach to identify proteins from tobacco leaf microsomes that directly interact with the hydrophilic (cytosolic) domains of NtSyr1 and report several interacting proteins with sensitivities to the endopeptidase activity of Clostridium botulinum neurotoxins, including one protein that was recognised by alphaAtSNAP33 antiserum, raised against the Arabidopsis SNAP25 homologue. Treatment of microsomal membrane fractions indicated a protein near 55 kDa was sensitive to proteolysis by BotN/A and BotN/E, yielding degradation products of approximately 34 and 23 kDa. Expressed and purified AtSNAP33 also bound directly to the cytosolic domain of NtSyr1 and was sensitive to proteolysis by these toxins, suggesting that NtSyr1, a tobacco homologue of AtSNAP33, and coordinate SNAREs are likely to associate as partners for function in vivo.


[Cardiobacterium hominis pericarditis: an unusual case].
The case of a male patient under hemodialytic therapy, who developed right heart failure is presented. Echocardiography revealed pericardial effusion, constrictive pattem in the right cavities, septation, without valvular damage and preserved systolic and diastolic function. Pericardial drainage and extensive pericardiectomy was performed obtaining cultures of pericardial tissue positive for an HACEK group organism, Cardiobacterium hominis, with repeatedly negative blood cultures. This is a rare clinical presentation of isolated bacterial pericarditis by an atypical microorganism, without associated endocarditis. The infection mechanisms are presented and the scarce available scientific literature is discussed in this study.


Initial whole-genome sequencing and analysis of the host genetic contribution to COVID-19 severity and susceptibility.
The COVID-19 pandemic has accounted for millions of infections and hundreds of thousand deaths worldwide in a short-time period. The patients demonstrate a great diversity in clinical and laboratory manifestations and disease severity. Nonetheless, little is known about the host genetic contribution to the observed interindividual phenotypic variability. Here, we report the first host genetic study in the Chinese population by deeply sequencing and analyzing 332 COVID-19 patients categorized by varying levels of severity from the Shenzhen Third People's Hospital. Upon a total of 22.2 million genetic variants, we conducted both single-variant and gene-based association tests among five severity groups including asymptomatic, mild, moderate, severe, and critical ill patients after the correction of potential confounding factors. Pedigree analysis suggested a potential monogenic effect of loss of function variants in GOLGA3 and DPP7 for critically ill and asymptomatic disease demonstration. Genome-wide association study suggests the most significant gene locus associated with severity were located in TMEM189-UBE2V1 that involved in the IL-1 signaling pathway. The p.Val197Met missense variant that affects the stability of the TMPRSS2 protein displays a decreasing allele frequency among the severe patients compared to the mild and the general population. We identified that the HLA-A*11:01, B*51:01, and C*14:02 alleles significantly predispose the worst outcome of the patients. This initial genomic study of Chinese patients provides genetic insights into the phenotypic difference among the COVID-19 patient groups and highlighted genes and variants that may help guide targeted efforts in containing the outbreak. Limitations and advantages of the study were also reviewed to guide future international efforts on elucidating the genetic architecture of host-pathogen interaction for COVID-19 and other infectious and complex diseases.


Transcultural validation of the Maastricht Clinical Teaching Questionnaire.
The evaluation of the clinical teacher's performance provides feedback to motivate them to improve their teaching skills. To perform the cross-cultural adaptation of the Maastricht Clinical Teaching Questionnaire, the International Guide for the Adaptation of the Questionnaires was followed. The validity of content, response process, construct and reliability were investigated. After cross-cultural adaption, residents of two University hospitals evaluated 187 clinical teachers. Content and answering process were validated. In the confirmatory factor analysis, all indexes and criteria for a good fit suited the 5 factors and 14 items model. The Cronbach's alpha coefficient was 0.80. The G coefficient was > 0.70, with low variance of the absolute error. Every clinical teacher should receive at least 6 evaluations to achieve a reliable evaluation of every domain and of their global performance. The Spanish version of the questionnaire is a valid, reliable instrument for medical residents to evaluate teachers.


An unexpected base-induced [1,4]-phospho-Fries rearrangement.
An unexpected [1,4]-phospho-Fries rearrangement that gives rise to the formation of a O,O,O,O-tetraethyl methylenebis(thiophosphonate) derivative is reported. The regioselectivity of the metallation with n-BuLi or t-BuLi is the key factor that explains either the [1,4] or [1,3] rearrangement observed.


Tin oxide nanowire sensor with integrated temperature and gate control for multi-gas recognition.
The selectivity of a chemiresistive gas sensor comprising an array of single-crystalline tin oxide nanowires (NWs) is shown to be greatly enhanced by combined temperature and gate voltage modulation. This dual modulation was effected by a novel microsensor platform that consisted of a suspended nitride membrane embedded with independently addressable platinum heater and back-gate structures. The sensor was evaluated in a chemical vapor exposure test consisting of three volatile organic compound (VOC) analytes in an air background; VOC concentrations ranged from 20 μmol/mol to 80 μmol/mol. During the exposure test, the temperature and gating conditions of the NW sensor were modulated in order to induce variations in the sensor's analyte response behavior. By treating these temperature- and gate-dependent analyte response variations as an identifying "fingerprint," analyte identification was achieved using a statistical pattern recognition procedure, linear discriminant analysis (LDA). Through optimization of this pattern recognition procedure, a VOC recognition rate of 98% was obtained. An analysis of the recognition results revealed that this high recognition rate could only be achieved through the combined modulation of temperature and gate bias as compared to either parameter alone. Overall, the highly accurate VOC analyte discrimination that was achieved here confirms the selectivity benefits provided by the utilized dual modulation approach and demonstrates the suitability of miniature nanowire sensors in real-world, multi-chemical detection problems.


Rupture of the atherosclerotic plaque: does a good animal model exist?
By its very nature, rupture of the atherosclerotic plaque is difficult to study directly in humans. A good animal model would help us not only to understand how rupture occurs but also to design and test treatments to prevent it from happening. However, several difficulties surround existing models of plaque rupture, including the need for radical interventions to produce the rupture, lack of direct evidence of rupture per se, and absence of convincing evidence of platelet- and fibrin-rich thrombus at the rupture site. At the present time, attention should therefore focus on the processes of plaque breakdown and thrombus formation in humans, whereas the use of animal models should probably be reserved for studying the function of particular genes and for investigating isolated features of plaques, such as the relationship between cap thickness and plaque stability.


[Effects of 7 d head down tilt on cardiopulmonary circulation during orthostasis].
Objective. To accumulate data for further studies on the mechanisms of the decrease of cardiovascular function after weightlessness. Method. Cardiopulmonary circulatory function under supine position and head up tilting (HUT) for 20 min before and after exposure to 7 d 6 degrees head down tilt (HDT) in 6 healthy adults who aged 19-21 were measured with a XXH-2000 lesser (pulmonary) circulation and cardiac function instrument. Result. The 7 d HDT resulted in orthostatic intolerance of the subjects; the reduction of RVET (j-z) and the elevation of RPEP [RVET(Q-j)/(j - z)] indicated a decrease of the right heart function. An obvious decrease of the right heart reserves was also observed under HUT after HDT. Conclusion. hc and hc/hz were demonstrated to be useful parameters to evaluate orthostatic tolerance or to forecast orthostatic syncope. The lesser (pulmonary) circulation and cardiac function testing methods are valuable in evaluating the cardiopulmonary circulatory function during HUT.


Radiological characteristics of primary intestinal lymphoma of the "Mediterranean" type: observations on twelve cases.
Radiological findings in 12 cases of "Mediterranean" lymphoma are analyzed, 4 of which are associated with gammapathy. These findings have not permitted differentiation of this type of lymphoma from those classically described, although there are two signs that strongly suggest this diagnosis: extension of the lesions along wide areas of the small intestine, almost always including the duodenum, and spiculation of the mucosal fold edges, representing massive plasmocyte infiltration of the lamina propria. The principal clinical, immunological, and pathological aspects are described, as well as the radiological differential diagnosis.


[Effect of cations on the quantum yield and the lifetime of the chloroplast fluorescence].
The interrelationship between the cation-induced fluorescence changes and the state of the photosystem 2 (PS-2) reaction centers for pea chloroplasts and their osmotic fragments was studied. The effects of K+ and Mg2+ on the fluorescence quantum yield (phi f1) under varying light intensities as well as on the fluorescence lifetime (tau f1) in the saturating light were demonstrated. K+ induces the decrease in tau f1; Mg2+ exerts an opposite effect. The effects were more pronounced when the reaction centers of PS-2 were converted into an inactive state by illuminating the sample with a saturating light or by adding DCMU. Under these conditions the cations' effect on tau f1 was accompanied by proportional changes in tau f1. It was concluded that in Mg-deficient chloroplasts an efficient channel of the excitation quenching appears in antenna chlorophyll of PS-2 with the rate constant of 7 . 10(8) s-1. The simultaneous measurements of tau f1 by phase and modulation type techniques allowed to reveal the emission heterogeneity within the nanosecond time interval and the DCMU-sensitive delayed fluorescence with the lifetime exceeding 10(-7) s and the overall quantum yield approximately equal to 2 . 10(-3).


An Improved Kinect Recognition Method for Identifying Unsafe Behaviors of Metro Passengers.
In order to solve the problem of the low action recognition accuracy of passengers' unsafe behaviors caused by redundant joints, this study proposes an efficient recognition method based on a Kinect sensor. The method uses the pelvis as the starting point of the vector and high-frequency bone joints as the end point to construct the recognition feature vector. The joint angle difference between actions is obtained by using the cosine law, and the initial test result is converted into action similarity combined with the DTW similarity algorithm. Considering the combination of 3 angle features and 4 joint feature selection methods, 12 combined recognition models are formed. A comparative experiment was carried out to identify five types of unsafe behaviors of metro passengers-punch, one-armed call for help, arms for help, trip forward and trip backwards. The results show that the overall selection of joints has a poor similarity effect and cannot achieve the purpose of recognition. The overall recognition model effect of the local "pelvis divergence method" is higher than that of the local "adjacent joint method", and the local "pelvis divergence method" has the highest recognition result of the maximum angle difference model, and the recognition results of the five unsafe behaviors are 86.9%, 89.2%, 85.5%, 86.7%, and 88.3%, respectively, and the recognition accuracy of this method is 95.7%, indicating the feasibility of the model. The recognition results are more concentrated and more stable, which significantly improves the recognition rate of metro passengers' unsafe behavior.


Ostreibacterium oceani gen. nov., sp. nov., isolated from oyster, and description of Ostreibacteriaceae fam. nov.
A novel Gram-stain-negative, short rod-shaped, facultatively anaerobic, non-motile, non-gliding, oxidase-positive and catalase-negative bacterium, designated ML27T, was isolated from oyster homogenate in Rushan, Weihai, PR China. Growth occurred at 20-33 °C (optimum, 30 °C), at pH 7.0-9.0 (optimum, pH 7.5-8.0) and in the presence of 1-6 % (w/v) NaCl (optimum, 3 %). Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain ML27T was 90.7 % similar to Suttonella ornithocola DSM 18249T, 89.2 % to Suttonella indologenes JCM 1478T and 88.2 % to Cardiobacterium hominis DSM 8339T; similarities to other species were less than 90 %. The average amino acid identity between strain ML27T, S. indologenes JCM 1478T, S. ornithocola DSM 18249T, C. hominis DSM 8339T and Dichelobacter nodosus ATCC 25549T were 46.23, 45.86, 45.54 and 45.84 %, respectively. Phylogenomic tree and phylogenetic analyses based on 16S rRNA gene sequences showed that the isolate formed a novel family-level clade in the order Cardiobacteriales. The sole respiratory quinone was ubiquinone-7 (Q-7). The dominant cellular fatty acids were summed feature 8 (C18 : 1 ω7c/C18 : 1 ω6c; 46.3 %), C16 : 0 (17.8 %) and summed feature 3 (C16 : 1 ω7c/C16 : 1 ω6c; 13.5 %). The DNA G+C content of strain ML27T was 45.6 mol%. Polar lipids included phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and one unidentified lipid. Comparative analyses of 16S rRNA gene sequences, genomic distinctiveness and characterization indicated that strain ML27T represents a novel species of a new genus within a novel family of the order Cardiobacteriales, for which the name Ostreibacterium oceani gen. nov., sp. nov. is proposed. The type strain of Ostreibacterium oceani is ML27T (=MCCC 1H00372T=KCTC 72155T). In addition, a novel family, Ostreibacteriaceae fam. nov., is proposed to accommodate the genus Ostreibacterium.


Identification of pyroptosis-related subtypes, development of a prognostic model, and characterization of tumour microenvironment infiltration in gastric cancer.
As a new programmed death mode, pyroptosis plays an indispensable role in gastric cancer (GC) and has strong immunotherapy potential, but the specific pathogenic mechanism and antitumor function remain unclear. We comprehensively analysed the overall changes of pyroptosis-related genes (PRGs) at the genomic and epigenetic levels in 886 GC patients. We identified two molecular subtypes by consensus unsupervised clustering analysis. Then, we calculated the risk score and constructed the risk model for predicting prognostic and selected nine PRGs related genes (IL18RAP, CTLA4, SLC2A3, IL1A, KRT7,PEG10, IGFBP2, GPA33, and DES) through LASSO and COX regression analyses in the training cohorts and were verified in the test cohorts. Consequently, a highly accurate nomogram for improving the clinical applicability of the risk score was constructed. Besides, we found that multi-layer PRGs alterations were correlated with patient clinicopathological features, prognosis, immune infiltration and TME characteristics. The low risk group mainly characterized by increased microsatellite hyperinstability, tumour mutational burden and immune infiltration. The group had lower stromal cell content, higher immune cell content and lower tumour purity. Moreover, risk score was positively correlated with T regulatory cells, M1 and M2 macrophages. In addition, the risk score was significantly associated with the cancer stem cell index and chemotherapeutic drug sensitivity. This study revealed the genomic, transcriptional and TME multiomics features of PRGs and deeply explored the potential role of pyroptosis in the TME, clinicopathological features and prognosis in GC. This study provides a new immune strategy and prediction model for clinical treatment and prognosis evaluation.


Modulatory role of BV6 and chloroquine on the regulation of apoptosis and autophagy in non-small cell lung cancer cells.
Non-small cell lung cancer (NSCLC) is one of the aggressive tumors mostly diagnosed in the advanced stage. Therapeutic failure and drug resistance pose a major problem in NSCLC treatment primarily due to alterations in autophagy and loss of apoptosis. Therefore, the present study aimed to investigate the importance of the second mitochondria-derived activator of caspase mimetic BV6 and autophagy inhibitor chloroquine (CQ) on the regulation of apoptosis and autophagy, respectively. Study was conducted on NCI-H23 and NCI-H522 cell lines to evaluate the effect of BV6 and CQ on the transcription and translation level of LC3-II, caspase-3, and caspase-9 genes by quantitative real-time-polymerase chain reaction and western blotting techniques. In NCI-H23 cell line, BV6 and CQ treatments showed increased mRNA and protein expression of caspase-3, and caspase-9 compared to its untreated counterpart. BV6 and CQ treatments also caused downregulation of LC3-II protein expression compared to its counterpart. In NCI-H522 cell line, BV6 treatment showed a significantly increased expression of caspase-3 and caspase-9 mRNA and protein expression levels whereas BV6 treatment downregulated the expression level of LC3-II protein. A similar pattern was also observed in CQ treatment when compared with the respective controls. Both BV6 and CQ modulated in vitro expression of caspases and LC3-II which have critical regulatory roles in apoptosis and autophagy, respectively. Our findings suggest that BV6 and CQ could be promising candidates in NSCLC treatment and there is a need to explore them in vivo and in clinical applications.


Exploring the service and knowledge gap between rehabilitation professionals and employers in the return-to-work process of people with mental health problems in Sweden.
Research is needed on how to optimize the return-to-work process for persons with mental health problems. To develop a model that explains how employer and rehabilitation professional mental health literacy may influence strategies used in the return-to-work of persons with mental health problems. A grounded theory study design was used to collect and analyse interview data from 23 employers and 22 rehabilitation professionals. The analysis resulted in four categories, illustrated in a conceptual process model: 1) seeing mental health problems through past experiences; 2) separating understandable and incomprehensible mental health problems; 3) balancing safeguarding one's personal interest with providing adequate support; and 4) facing conflicts and uncertainty in employee/service user return-to-work. A positional map of employer/rehabilitation types was created based on level of negative attitudes and uncertainty in supporting employees/service users and the employer in relation to the level of comprehensive understanding of mental health problems. Professional mental health literacy needs to be increased. Interventions need to be developed that provide employers with targeted support and knowledge in mental health literacy. Education on how to prevent and detect common mental health problems in the workplace is needed.


Plaque formation assay for human parainfluenza virus type 1.
Human parainfluenza virus type 1 (hPIV1) generally does not show visible plaques in common cell lines, including Lewis lung carcinoma-monkey kidney (LLC-MK(2)) cells, by plaque formation assays for human parainfluenza virus type 3 (hPIV3) and Sendai virus. In several conditions of the plaque formation assay, complete elimination of serum proteins in the overlay medium was necessary for visualization of hPIV1-induced plaque formation in LLC-MK(2) cells. We developed a plaque formation assay for hPIV1 isolation and titration in LLC-MK(2) cells using an initial overlay medium of bovine serum albumin-free Eagle's minimum essential medium containing agarose and acetylated trypsin for 4-6 d followed by a second overlay staining medium containing agarose and neutral red. The assay allowed both laboratory and clinical hPIV1 strains to form large plaques. The plaque reduction assay was also performed with rabbit anti-hPIV1 antibody as a general evaluation model of viral inhibitors to decrease both the plaque number and size. The results indicate that the plaque formation assay is useful for hPIV1 isolation, titration, evaluation of antiviral reagents and epidemiologic research.


Building antimicrobial stewardship through massive open online courses: a pilot study in Macedonia.
The global struggle against antibiotic resistance requires antimicrobial stewardship (AMS). Massive open online courses (MOOCs) offer health professionals unprecedented access to high-quality instructional material on AMS; the question is how apprehensible it is to non-native English speakers. Furthermore, to better understand how education interventions promote change towards rational antibiotic prescribing, leading institutions call for studies integrating behavioural science. Research from lower- and middle-income countries is particularly needed. To measure the knowledge improvement from an AMS MOOC, the influence of language, course satisfaction and subsequent effect on intention to change antibiotic prescribing behaviour. Fifty-five physicians from Macedonia completed the MOOC. Pre- and post-course knowledge test scores were compared using a one-sample t-test. The effect of a language barrier was assessed using self-reported English level. Scores were compared with participants' intention to change behaviour in clinical practice. Scores significantly improved from 77.8% to 82.2%. Participants with a higher English level improved most, while the low-level group showed no significant improvement. Physicians reported a high or very high intention to change behaviour. This was independent of knowledge improvements. First, lower self-reported English proficiency hindered knowledge acquisition from a MOOC platform. AMS programmes should commit to bridge this barrier so as to enable a global spread of education in AMS. Second, factors underlying the physicians' intentions to engage in AMS appear to be more complex than simple knowledge improvements. This suggests that less time-consuming interventions could be as effective.


[A comparative study on acellular small intestinal submucosa and acellular amnion as dressings for traumatic skin defects].
To compare the reparative effects between the acellular small intestinal submucosa and the acellular amnion as dressings for traumatic skin defects. Three full-thickness skin defects, which were close to the vertebral column of the pig, were created on both sides of the dorsum. The skin defects were randomly divided into three groups. In each group, the following different materials were used to cover the skin defects: the acellular amnion in Group A, the acellular small intestinal submucosa (SIS) in Group B, and the physiological saline gauze in Group C (the control group). The specimens from the skin defects were harvested for a histological evaluation and for determination of the hydroxyproline content at 10 (2 pigs), 20 (2 pigs), and 30 days (3 pigs). We observed the healing process of the wound and its healing rate, counted the inflammatory cells, vascular endothelial cells, and proliferating cells, and determined the hydroxyproline content. The acellular amnion in Group A and the acellular SIS in Group B adhered to the wound tightly, but they did not adhere to the dressing; when the dressing was changed, the wound did not bleed. The saline gauze in Group C adhered to the wound tightly, but when the dressing was changed, the wound bled until 22 days after operation. Under the microscope, the collagen in the tissue below the epithelium was arranged more regularly and there were fewer cells concerned with inflammation in Groups A and B than in Group C at 10, 20, and 30 days after operation. At 10, 20, and 30 days after operation, the wound healing rate was greater in Groups A and B than in Group C. The number of the inflammatory cells and the proliferating cells were greater in Group C than in Groups A and B. There was a statistically significant difference (P < 0.05), At 20 and 30 days after operation, the content of hydroxyproline was greater in Group C than in Group A and B. There was a statistically significant difference (P < 0.05). However, there was no statistically significant difference between Group A and Group B in the wound healing rate, the numbers of the inflammatory cells, vascular endothelial cells and proliferating cells, and the content of hydroxyproline (P > 0.05). There was no statistically significant difference among the three groups in the number of the vascular endothelial cells. Compared with Group C, the wound healing rate is improved, the collagen in the tissue below the epithelium is arranged more regularly, and the inflammatory cells, bleeding, and effusion are reduced in Groups A and B. The reparative effects of the acellular amnion and the acellular small intestinal submucosa as dressings on the skin defects are almost the same.


Rodent carcinogenicity bioassay: past, present, and future.
Toxicity/carcinogenicity studies in rodents have played a pivotal role in identifying chemicals that are potentially hazardous to humans. In fact, nearly all of the known human carcinogens are also carcinogenic in 1 or more rodent species. During the past 20 yr the quality and consistency of rodent studies has improved considerably, and much has been learned about mechanisms whereby chemicals initiate or promote the carcinogenic process in rats and mice. The process of identifying chemicals that cause toxicity or carcinogenicity in rodents is quite well established, but the procedures for extrapolating this data for risk management decisions in the protection of human health have lagged far behind. While many would accept the assumptions that genotoxic chemicals that cause cancer in animals pose a cancer risk to humans and that genotoxic chemicals causing cancer at high doses pose a risk at lower doses, there is much less certainty with respect to nongenotoxic chemicals. The confusion about risk extrapolation for nongenotoxic chemicals has often lead to criticism of the hazard identification process for chemicals in general. There is increasing awareness of the complexity of the carcinogenic process that has made species extrapolation and dose extrapolation from rodent studies to humans more complex. Although newer molecular biological techniques and cell kinetic measurements offer exciting possibilities for better risk assessment, it is the combination of well-designed rodent studies with appropriate mechanistic studies that offers the best hope for regulatory decisions based on sound scientific principles.


The use of polyion complex micelles to enhance the oral delivery of salmon calcitonin and transport mechanism across the intestinal epithelial barrier.
The objective of the present study was to demonstrate the effect of polyanionic copolymer mPEG-grafted-alginic acid (mPEG-g-AA)-based polyion complex (PIC) micelles on enhancing the oral absorption of salmon calcitonin (sCT) in vivo and in vitro and identify the transepithelial transport mechanism of PIC micelles across the intestinal barrier. mPEG-g-AA was first successfully synthesized and characterized in cytotoxicity. The PIC micelles were approximately of 72 nm in diameter with a narrow distribution. The extremely significant enhancement of hypocalcemia efficacy of sCT-loaded PIC micelles in rats was evidenced by intraduodenal administration in comparison with sCT solution. The presence of mPEG-grafted-chitosan in PIC micelles had no favorable effect on this action in the referred content. In the Caco-2 transport studies, PIC micelles could significantly increase the permeability of sCT across Caco-2 monolayers without significantly affecting transepithelial electrical resistance values during the transport study. No evident alterations in the F-actin cytoskeleton were detected by confocal microscope observation following treatment of the cell monolayers with PIC micelles, which further certified the incapacity of PIC micelles to open the intercellular tight junctions. In addition, TEM observations showed that the intact PIC micelles were transported across the everted gut sac. These suggested that the transport of PIC micelles across Caco-2 cell monolayers involve a predominant transcytosis mechanism via endocytosis rather than paracellular pathway. Furthermore, PIC micelles were localized in both the cytoplasm and the nuclei observed by CLSM. Therefore, PIC micelles might be a potentially applicable tool for enhancing the oral absorption of cationic peptide and protein drugs.


Risk factors for surgical wound infection and bacteraemia following coronary artery bypass surgery.
There has been no consensus from previous studies of risk factors for surgical wound infections (SWI) and postoperative bacteraemia for patients undergoing coronary artery bypass graft (CABG) surgery. Data on 15 potential risk factors were prospectively collected on all patients undergoing CABG surgery during a 12-month period. Of 693 patients, 62 developed 65 SWI using the Centres for Disease Control definition: 23 were sternal wound infections and 42 were arm or leg wound infections at the site of conduit harvest. There were 19 episodes of postoperative bacteraemia. Multivariate analysis revealed that: (i) diabetes, obesity and previous cardiovascular procedure were independent predictors of SWI; and (ii) obesity was an independent risk factor for postoperative bacteraemia. These findings suggest that improved diabetic control and pre-operative weight reduction may result in a decrease in the incidence of SWI. But further prospective studies need to be undertaken to examine (i) whether the increased SWI risk in diabetes occurs with both insulin- and non-insulin-requiring diabetes, and whether improved peri-operative diabetes control decreases SWI; and (ii) what degree of obesity confers a risk of SWI and postoperative bacteraemia, and whether pre-operative weight reduction, if a realistic strategy in this patient group, results in a decrease in SWI.


[Clinical and surgical therapy of phlegmasia coerulea dolens].
Venous thrombectomy was performed on 123 patients with phlegmasia coerulea dolens (blue phlebitis). Best results are obtained if treatment is initiated at the earliest stage. Arterial involvement must be closely monitored. A fasciotomy, which in some cases may involve the fascia lata, must be performed in case of compartment compression. Palma's bypass procedure and Av-fistula are indicated in some cases.


Prognostic Impact of Hybrid Comprehensive Telerehabilitation Regarding Diastolic Dysfunction in Patients with Heart Failure with Reduced Ejection Fraction-Subanalysis of the TELEREH-HF Randomized Clinical Trial.
The objective of the study was to evaluate the effects of individually prescribed hybrid comprehensive telerehabilitation (HCTR) implemented at patients' homes on left ventricular (LV) diastolic function in heart failure (HF) patients. The Telerehabilitation in Heart Failure Patients trial (TELEREH-HF) is a multicenter, prospective, randomized (1:1), open-label, parallel-group, controlled trial involving HF patients assigned either to HCTR involving a remotely monitored home training program in conjunction with usual care (HCTR group) or usual care only (UC group). The patient in the HCTR group underwent a 9-week HCTR program consisting of two stages: an initial stage (1 week) conducted in hospital and the subsequent stage (eight weeks) of home-based HCTR five times weekly. Due to difficulties of proper assessment and differences in the evaluation of diastolic function in patients with atrial fibrillation, we included in our subanalysis only patients with sinus rhythm. Depending on the grade of diastolic dysfunction, patients were assigned to subgroups with mild diastolic (MDD) or severe diastolic dysfunction (SDD), both in HCTR (HCTR-MDD and HCTR-SDD) and UC groups (UC-MDD and UC-SDD). Changes from baseline to 9 weeks in echocardiographic parameters were seen only in A velocities in HCTR-MDD vs. UC-MDD; no significant shifts between groups of different diastolic dysfunction grades were observed after HCTR. All-cause mortality was higher in UC-SDD vs. UC-MDD with no difference between HCTR-SDD and HCTR-MDD. Higher probability of HF hospitalization was observed in HCTR-SDD than HCTR-MDD and in UC-SDD than UC-MDD. No differences in the probability of cardiovascular mortality and hospitalization were found. HCTR did not influence diastolic function in HF patients in a significant manner. The grade of diastolic dysfunction had an impact on mortality only in the UC group and HF hospitalization over a 12-24-month follow-up in HCTR and UC groups.


A series of site-specific fluorescently labeled BPTI derivatives prepared by nonselective acylation and chromatographic separations.
Investigation of conformational transitions and, in particular, the folding/unfolding transitions of globular proteins by means of excitation energy transfer measurements depends on the availability of protein derivatives carrying donor and acceptor probes at well-defined pairs of sites. A series of bovine pancreatic trypsin inhibitor (BPTI) derivatives, each labeled at one of the epsilon-amino groups, was prepared. This was achieved by a nonselective acylation reaction using 7-dimethyl-amino-coumarin-4-acetyl-N-hydroxysuccinimide ester (DACA-NHSIE) as a reagent yielding a mixture of products. The mixture was resolved by affinity chromatography and reversed phase high performance liquid chromatography (HPLC). Four derivatives were obtained, each carrying the probe at one of the four amino groups. Identification of site of labeling and determination of the purity of the products was achieved by HPLC-tryptic peptide mapping. The labeled derivatives are active and can undergo a reversible denaturation/renaturation cycle. The spectral characteristics of the probe make it a suitable acceptor in energy-transfer measurements. The advantage of the approach described here, namely nonselective reaction combined with efficient fractionation procedures for the preparation of site specifically labeled derivatives, is that each of the amino groups can be labeled in a simple procedure, thus allowing for a maximal number of labeling sites which cannot be achieved when site-directed reagents (e.g. specific particular protection) are used. The present method yields derivatives which are useful in energy transfer measurements for determination of intramolecular distances between labeled sites. The derivatives should be useful in the analysis of the mechanism of protein folding and the intermediate structures involved.


Biallelic intragenic tandem duplication of CPLANE1 in Joubert syndrome: A case report.
Joubert syndrome (JS) is a clinically and genetically heterogeneous genetic disorder. To date, 40 JS-causing genes have been reported and CPLANE1 is one of the most frequently mutated, with biallelic pathogenic missense and truncating variants explaining up to 14% of JS cases. We present a case of JS diagnosed after the identification of a novel biallelic intragenic duplication of exons 20-46 of CPLANE1. The quadruplication was identified by short-read sequencing and copy number variant analysis and confirmed in tandem by long PCR with the breakpoints defined by a nanopore-based long-read sequencing approach. Based on the genetic findings and the clinical presentation of the patient, a brain MRI was ordered, evidencing the molar tooth sign, which confirmed the diagnosis of JS in the patient. This is, to the best of our knowledge, the first report of an intragenic duplication in this gene as the potential molecular mechanism of JS.


[Firstline Therapy of Intermittent Claudication - Claim and Reality].
This narrative review summarizes the situation of research concerning exercise therapy in patients suffering from intermittent claudication. In depth we present the evidence-based guidelines and their underlying studies. Aspects as angiomorphology, adherence, long-term effect, quality of studies and their comparability are discussed. We focus on the gap between guidelines and therapy of healthcare providers. Therefore, we discuss the relevance of exercise therapy in healthcare policy. Reasons for the missing congruence between guidelines and the implementation in the healthcare system in Germany are analysed. According to this we express recommendations corresponding to international experiences (Netherlands, Denmark).