Molecular evidence that human ocular ciliary epithelium expresses components involved in phototransduction.
Here we report the expression, in the human ocular ciliary epithelium and in a human nonpigmented (NPE) ciliary epithelial cell line, of genes usually restricted to cone and rod photoreceptor cells of the retina. By RT-PCR and DNA sequencing we identified the expression of rhodopsin and components linked to its deactivation, including rhodopsin kinase, recoverin, and visual arrestin. We also detected the expression of transducin (T-alpha), phosphodiesterase (PDE-alpha), and cGMP-gated channel alpha-subunits. Cultured NPE cells responded to treatment with phorbol ester by enhancing the expression of rhodopsin mRNA three- to fourfold. Indirect immunofluorescence of the intact ciliary epithelium with monoclonal antibodies (MAbs) against rhodopsin, rhodopsin kinase, and visual arrestin revealed labeling preferentially restricted to the NPE cells. Furthermore, Western blot analysis of whole lysates from the pars plicata region of the human ciliary epithelium with MAbs demonstrated immunochemical cross-reactivity with proteins of molecular mass similar to rhodopsin (36 kDa), rhodopsin kinase (64 to 66 kDa), and arrestin (48-52 kDa) from the human retina. These results provide the first molecular evidence that components of a non-visual phototransduction pathway are expressed in the human ocular NPE ciliary epithelium, which may be linked to circadian entrainment tasks.


Novel KCNQ2/Q3 agonists as potential therapeutics for epilepsy and neuropathic pain.
Current drugs for the treatment of seizure disorders, although effective in many patients, still suffer from a number of failures and are not effective in some forms of resistant epilepsies. Historically, many of these drugs have multiple mechanisms of action including calcium and sodium channel blockade as well as GABAergic activity and thus a number of associated side effects. Modulation of the M-current through opening of KCNQ channels has been proposed as a way to attenuate neuroexcitability and have a therapeutic benefit for the treatment of seizure disorders. Therefore, as part of our program to identify new treatments for epilepsy, we set out to identify agonists of KCNQ channels. High throughput screening of our corporate collection led to the identification of 1, adamantane-1-carboxylic acid (3-methyl-3H-benzothiazol-2-ylidine) hydrazide, a potent KCNQ2/Q3 agonist. Herein, we describe the syntheses and structure-activity relationships of analogues of 1 as well as their in vivo activity in animal models of epilepsy and neuropathic pain.


Transdermal Buprenorphine for Pain Management Following a Neck of Femur Fracture.
Pain management in patients with hip fractures can be challenging. Poor pain control is associated with reduced mobility and increased morbidity. Inadequate analgesia in patients with dementia is a concern. After using several different alternatives, transdermal buprenorphine was chosen as a standardised approach for analgesia in patients with fragility fracture in our hospital. There is limited evidence on the use of buprenorphine in this population. Our aim was to investigate the safety and effectiveness of transdermal buprenorphine in patients with hip fractures. A review of consecutive patients presenting with a hip fracture from June 2018 to December 2018 was conducted using medical records. Our primary outcome was the incidence of complications as a consequence of transdermal buprenorphine. Our secondary outcome was adequate analgesia measured by reviewing the requirement for analgesia during the first week following the patient's admission. Analgesia demands were considered adequate if patients required less than 20 mg of oral morphine in total during the first week following injury. In total, 148 patients presented with a hip fracture during the study period. 128 patients had documented evidence of buprenorphine patch application. Complete data was available for the primary outcome of complications in all cases. Data was available for the secondary outcome in 124 patients. Buprenorphine was discontinued in 24 patients (19%), most commonly due to due to concerns about contribution to hypoactive delirium (9%), and when strong analgesia was no longer required (4%). There were no severe complications. Adequate analgesia was achieved using this regime in 68% patients. 38 patients (32%) required more than 20 mg of oral morphine sulphate solution in the first week post-admission. This series suggests that transdermal buprenorphine is safe and effective in the management of pain following a neck of femur fragility fracture.


High Expression of the Component 3a Receptor 1 (C3AR1) Gene in Stomach Adenocarcinomas Infers a Poor Prognosis and High Immune-Infiltration Levels.
BACKGROUND This study was designed to explore the incompletely investigated role of the complement component 3a receptor 1 (C3AR1) in the prognosis of stomach adenocarcinomas (STAD). MATERIAL AND METHODS Using bioinformatic methods, we systematically determined the expression and prognosis value of C3AR1 in various cancers by using the TIMER (Tumor Immune Estimation Resource) database, UALCAN platform, GEPIA (Gene Expression Profiling Interactive Analysis) server, and the OncoLnc tool. The biological processes influenced by C3AR1 were determined using the GSEA (Gene Set Enrichment Analysis) software (Copyright 2004-2020 Broad Institute, Inc., Massachusetts Institute of Technology, and Regents of the University of California). The correlation between C3AR1 expression and the immune-infiltrating cells as well as the correlation analysis between C3AR1 expression and the corresponding immune-marker sets were conducted using the TIMER and GEPIA databases. RESULTS The expression of C3AR1 was significantly (P<0.001) differentially expressed on several tumor types, while its prognosis value could only be determined on STAD, with a high expression of C3AR1 closely correlated with a poor prognosis. The GSEA analysis revealed that the differential expression of C3AR1 profoundly affected the immune-related biological processes. The expression of C3AR1 was strongly and positively correlated with the infiltration of monocytes, tumor-associated macrophages, M2 macrophages, dendritic cells, and exhausted T cells. CONCLUSIONS Our results have revealed that a high expression of C3AR1 is positively correlated with a poor prognosis and increased tumor-immune infiltration. C3AR1 can promote the polarization of M2 macrophages and T cell exhaustion, leading to the immune escape of STAD. These findings suggest that C3AR1 could be used as a prognostic and immune-infiltration marker in the pathogenesis of STAD.


Single-armed phenylsulfonated pyridine derivative of DOTA is rigid and stable paramagnetic tag in protein analysis.
Single-armed DOTA-like phenylsulfonated pyridine derivatives are rigid and stable paramagnetic tags for site-specific labeling of proteins. Their reactions with a solvent-exposed protein thiol group generate a stable C-S bond and produce one single paramagnetic species in solution NMR. The generated large paramagnetic effects yield valuable long-range structural restraints for proteins.


Selection and regeneration of Vitis vinifera Chardonnay hydroxyproline-resistant calli.
Proline (Pro) accumulation protects plant cell under abiotic stress. Hydroxyproline (Hyp) as selection agent is a toxic analog of proline and promotes Pro overaccumulation. In this study, Chardonnay calli were firstly irradiated with different dosages of 60Co and then cultured on a Hyp-added medium. Finally, some stable hydroxyproline-resistant (HR) calli were obtained. When calli were cultured on 4 mM Hyp medium for 7 days, intracellular Pro content of the HR calli was five times higher than that detected in the normal calli. The regeneration of HR calli into plantlets was much slower than that of normal ones. When cultured on woody plant medium (WPM) containing 10 mM NaCl for 14 days, HR plantlets still grew well with lower Pro than withered normal plantlets. qRT-PCR results of Pro biosynthesis-related genes in HR plantlets showed that three genes VvP5CS, VvOAT, and VvP5CDH were conducive for Pro accumulation. These results confirmed that HR plantlets acquired salt tolerance ability. We prospect that this procedure to obtain salt-tolerant plants may be valuable to breed programs and improve grapevine genotypes with increased tolerance to salt and other abiotic stresses.


Antibiotic Expenditures by Medication, Class, and Healthcare Setting in the United States, 2010-2015.
Improving antibiotic use has the potential to decrease healthcare costs by reducing the incidence of antibiotic-resistant infections, antibiotic-associated adverse events, and expenditures due to unnecessary prescriptions. Antibiotic expenditures in 2009 totaled $10.7 billion in the United States. Since then, national and local antibiotic stewardship initiatives have grown. The purpose of this study was to assess trends in antibiotic expenditures by healthcare setting in the United States between 2010 and 2015. Systemic (nontopical) antibiotic expenditures from January 2010 to December 2015 were extracted from the QuintilesIMS National Sales Perspectives database. These data represent a statistically valid projection of US medication purchases. Regression analyses evaluated trends in expenditures over the study period. Antibiotic expenditures totaled $56.0 billion over the 6-year period; the majority (59.1%) of expenditures were associated with the outpatient setting. Overall antibiotic expenditures in 2015 ($8.8 billion) were 16.6% lower than in 2010 ($10.6 billion). Antibiotic expenditures similarly decreased in the community by 25.5% (P = .05), but outpatient clinics and mail service pharmacy expenditures experienced significant growth (148% and 67% increase, respectively; P < .01 for both). In 2015, 16.5% of antibiotic expenditures in the community were for parenteral formulations, an increase of 25%. From 2010 to 2015, antibiotic expenditures decreased. The majority of antibiotic expenditures were in the outpatient setting, specifically community pharmacies. Expenditures for intravenous agents in the community are increasing and may represent increased use. These results reinforce the importance of antibiotic stewardship efforts across the spectrum of healthcare.


Angiogenesis in pre-malignant conditions.
Angiogenesis is an essential process involved in the normal growth and differentiation. In its defective and excessive form, angiogenesis is a crucial event in the progression of many human diseases. Excessive angiogenesis was largely investigated in psoriasis, arthritis, diabetic retinopathy and malignant tumours. Soon after the discovery of angiogenic factors and their inhibitors, the angiogenesis jumped from the experimental studies to clinical application. Tumour-associated angiogenesis is nowadays considered as a priority in oncology based on numerous evidences that showed a significant reduction in tumour growth following anti-angiogenic therapy. However, few data are available on pre-malignant conditions. First evidences on angiogenesis in pre-malignant lesions came from the evaluation of microvessel density (MVD). MVD was found to be significantly increased in a relatively large spectrum of pre-malignant squamous cell lesions, such as in the oral mucosa, skin, uterine cervix, vulva and anal canal. For many of them, a correlation was found between MVD and the expression of vascular endothelial growth factor (VEGF). Based on these data, it was suggested that tumour angiogenesis is not necessarily a characteristic of invasive tumour, but may be an early event during tumourigenesis. Additional evidences came from pre-malignant lesions of glandular epithelia, in which the angiogenic switch was demonstrated by the immunohistochemical expression of VEGF in gastric metaplasia and dysplasia, in atypical adenoma of the colon, atypical hyperplasia and carcinoma in situ of the breast and others. Actually, there are convincing evidences for an active angiogenesis in many cases with pre-malignant conditions, and this supports a more accurate evaluation of different chemopreventive agents.


Effect of chitosan on lingual hemostasis in rabbits with platelet dysfunction induced by epoprostenol.
Chitosan, a complex carbohydrate derivative of shellfish exoskeleton, is shown to enhance lingual hemostasis in rabbits treated with a known antagonist of platelet function, epoprostenol (prostacyclin or PGI2). Bleeding times were measured for bilateral (15 mm x 2 mm) tongue incisions in 10 New Zealand white rabbits. Using a randomized, blinded experimental design, one incision in each animal was treated with chitosan and the other was treated with control vehicle without chitosan. Extraoral bleeding and coagulation times were measured for each animal before, during, and after infusion of epoprostenol. Continuous infusion of epoprostenol increased mean systemic bleeding time 95%. In this platelet dysfunction animal model, lingual incisions receiving the experimental substance showed a 56% improvement in bleeding time in comparison with lingual incisions receiving control solution (P = .003).


Ultrafast exciton delocalization and localization dynamics of a perylene bisimide quadruple π-stack: a nonadiabatic dynamics simulation.
Unraveling the photogenerated exciton dynamics of π-stacked molecular aggregates is of great importance for both fundamental studies and industrial applications. Among various π-stacked molecular aggregates, perylene tetracarboxylic acid bisimide (PBI) based aggregates are regarded as one of the prototypes due to their inherent high fluorescence quantum yield and excellent photostability and flexibility in controlling intermolecular forces via chemical modifications. However, the exciton dynamics of these PBI based aggregates remain elusive up to now. In this work, we have first employed LR-TDDFT-based nonadiabatic dynamics simulations and static electronic structure calculations to investigate the ultrafast exciton dynamics of a newly synthesized perylene bisimide quadruple (PBQ) π-stack. Upon photoexcitation, the S6 to S10 states are the most likely populated excited states, which can be regarded as a combination of local excited (LE) excitons and charge transfer (CT) excitons of those four PBI chromophores. Then, the excited PBQ π-stack relaxes ultrafast to the lowest lying excited S1 state within 500 fs, which is accompanied by the complicated exciton conversion as well as exciton localization/delocalization dynamics. In short, the initially populated hybrid LE and CT excitons convert to the LE excitons of B/C and A/D, in which the LE excitons of B/C contribute the most (∼0.44) while the LE excitons of A/D also have minor contributions (0.21), indicating the formation of the localized excimer state. We use the notations A/B/C/D here to represent the four PBI fragments of PBQ π-stacks along the direction perpendicular to the PBI molecular plane. Additionally, using a recently defined root mean square deviation (RMSD) of electron and hole spatial distributions along three Cartesian coordinates, we could investigate the exciton localization/delocalization dynamics in a quantitative way. Our simulation results indicate that the photoinduced electrons and holes of the PBQ π-stack exhibit an ultrafast localization(∼10 fs)-delocalization(∼60 fs)-localization(∼200 fs) dynamics, during which both LE and CT excitons play crucial roles. Our present work is not only consistent with previous experimental studies, but also provides more detailed insights into the relevant processes, which might be useful for the future design of PBI based optoelectronic devices with improved performances.


Comparison of failure characteristics of a range of cancellous bone-bone cement composites.
Over the past decade, orthopedic surgery has embraced an increase in the depth of cement penetration into the adjacent cancellous bone structure. The resultant interdigitation transforms this zone into a thick layer of continuous interpenetrating composite material. The failure behavior of the composite formed with a number of potential bone cements with different bonding ability was investigated. The cancellous bone-cement composites exhibit considerable resistance to crack extension, and in situ optical observation indicates that the contribution of the cancellous bone is analogous to that of a typical fiber bridging process. The critical stress intensity factor and the work of fracture have been used to quantify the failure characteristics of the cancellous bone-cement composites. The nature of the crack propagation through these cement-bone composites was also captured via optical microscopy, and scanning electron microscopic images were taken of the failure surfaces. The R-curve behavior, or crack extension characteristic, of the cancellous bone-cement composite was also determined. The interesting outcome is that the cancellous bone-PMMA (poly-methylmethacrylate) composite, despite the absence of chemical bonding with bone, required the highest energy to fracture. In addition, the dimensional stability of the cement has a great effect on the interface.


Optimising experimental design for high-throughput phenotyping in mice: a case study.
To further the functional annotation of the mammalian genome, the Sanger Mouse Genetics Programme aims to generate and characterise knockout mice in a high-throughput manner. Annually, approximately 200 lines of knockout mice will be characterised using a standardised battery of phenotyping tests covering key disease indications ranging from obesity to sensory acuity. From these findings secondary centres will select putative mutants of interest for more in-depth, confirmatory experiments. Optimising experimental design and data analysis is essential to maximise output using the resources with greatest efficiency, thereby attaining our biological objective of understanding the role of genes in normal development and disease. This study uses the example of the noninvasive blood pressure test to demonstrate how statistical investigation is important for generating meaningful, reliable results and assessing the design for the defined research objectives. The analysis adjusts for the multiple-testing problem by applying the false discovery rate, which controls the number of false calls within those highlighted as significant. A variance analysis finds that the variation between mice dominates this assay. These variance measures were used to examine the interplay between days, readings, and number of mice on power, the ability to detect change. If an experiment is underpowered, we cannot conclude whether failure to detect a biological difference arises from low power or lack of a distinct phenotype, hence the mice are subjected to testing without gain. Consequently, in confirmatory studies, a power analysis along with the 3Rs can provide justification to increase the number of mice used.


The investigational Cystic Fibrosis drug Trimethylangelicin directly modulates CFTR by stabilizing the first membrane-spanning domain.
Cystic Fibrosis (CF) is caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. The most common mutation, deletion of phenylalanine 508 (F508del), disrupts tertiary assembly, causing protein misprocessing and loss of CFTR function in epithelial tissues. Lumacaftor (VX-809) is a Class 1 corrector molecule shown to partially rescue misprocessing of F508del and together with the potentiator of channel activity: ivacaftor (VX-770) has been approved for treatment of CF patients homozygous for the F508del mutation. The specificity of these modulators for CFTR is thought to be conferred through direct binding. Trimethylangelicin (TMA) is a distinct small molecule modulator, previously shown to exhibit both corrector and potentiator activities. We were prompted to determine if TMA also mediates these activities by direct binding. Interestingly, we found that like VX-770, TMA was effective in enhancing anion efflux mediated by purified WT-CFTR reconstituted in phospholipid liposomes. Furthermore, like VX-809, TMA was effective in stabilizing the functional expression of CFTR lacking the regulatory "R" domain or second nucleotide-binding domain (NBD2). The smallest domain that was stabilized by TMA binding was the first membrane-spanning domain (MSD1) as previously observed for VX-809. Together, our findings support the claim that TMA binds directly to CFTR, and despite its distinct chemical structure, shares similar mechanisms as VX-770 and VX-809 to potentiate and stabilize CFTR, respectively.


[Actin is located in the nucleus and nuclear matrix of HeLa cells].
HeLa cell nuclei were isolated and the nuclear matrix specimens were prepared. After labelled with an anti-actin antibody and FITC-conjugated secondary antibody, both the nuclei and nuclear matrix specimens were observed to emanate specific yellow-green fluorescence. The fluorescent signals in the nuclear matrix were much stronger than that in the nuclei, and the signals in former were widespread throughout the whole structure while that in the latter were mainly defined to their peripheral regions. A 43 kD band was revealed in nuclei and nuclear matrix specimens by SDS-PAGE, and was then proved to be actin by Western blot, confirming the immuno-fluorescence observations. When stained with TRICT-conjugated phalloidin, both the nuclei and nuclear matrix specimens were found to give off specific, red fluorescent signals which represent the location of F-actin (filamentous actin), and the pattern of TRITC signals distribution in the nuclei and nuclear matrix showed similarity with that of FITC signals. The existence and significance of actin and F-actin in the nuclei and nuclear matrix were discussed.


Spatial Stability of Functional Networks: A Measure to Assess the Robustness of Graph-Theoretical Metrics to Spatial Errors Related to Brain Parcellation.
There is growing interest in studying human brain connectivity and in modelling the brain functional structure as a network. Brain network creation requires parcellation of the cerebral cortex to define nodes. Parcellation might be affected by possible errors due to inter- and intra-subject variability as a consequence of brain structural and physiological characteristics and shape variations related to ageing and diseases, acquisition noise, and misregistration. These errors could induce a knock-on effect on network measure variability. The aim of this study was to investigate spatial stability, a measure of functional connectivity variations induced by parcellation errors. We simulated parcellation variability with random small spatial changes and evaluated its effects on twenty-seven graph-theoretical measures. The study included subjects from three public online datasets. Two brain parcellations were performed using FreeSurfer with geometric atlases. Starting from these, 100 new parcellations were created by increasing the area of 30% of parcels, reducing the area of neighbour parcels, with a rearrangement of vertices. fMRI data were filtered with linear regression, CompCor, and motion correction. Adjacency matrices were constructed with 0.1, 0.2, 0.3, and 0.4 thresholds. Differences in spatial stability between datasets, atlases, and threshold were evaluated. The higher spatial stability resulted for Characteristic-path-length, Density, Transitivity, and Closeness-centrality, and the lower spatial stability resulted for Bonacich and Katz. Multivariate analysis showed a significant effect of atlas, datasets, and thresholds. Katz and Bonacich centrality, which was subject to larger variations, can be considered an unconventional graph measure, poorly implemented in the clinical field and not yet investigated for reliability assessment. Spatial stability (SS) is affected by threshold, and it decreases with increasing threshold for several measures. Moreover, SS seems to depend on atlas choice and scanning parameters. Our study highlights the importance of paying close attention to possible parcellation-related spatial errors, which may affect the reliability of functional connectivity measures.


Studies on O-glycans of Plasmodium-falciparum-infected human erythrocytes. Evidence for O-GlcNAc and O-GlcNAc-transferase in malaria parasites.
O-Glycosylation is the major form of protein glycosylation in human erythrocytes infected with the asexual intraerythrocytic stage of the malaria parasite. Plasmodium falciparum. This study compares aspects of O-glycosylation in P. falciparum-infected and uninfected erythrocytes. Non-labeled and metabolically glucosamine-labeled O-glycans were obtained from the protein fraction of infected or uninfected erythrocytes by beta elimination. Additional label was introduced by reduction with sodium borohydride, or by the attachment of radioactive Gal to peripheral GlcNAc using galactosyltransferase. 2-4-times more labeled O-glycans were obtained from infected erythrocytes compared to the same number of uninfected ones, consistent with additional biosynthesis by the parasite. Our analysis of these O-glycans showed no significant qualitative divergence between the O-glycans of the infected and those of the uninfected red cell. According to preliminary alditol analyses, the O-glycans of P. falciparum-infected red cells do not contain GalNAc at their reducing terminus. Moreover, GalNAc was not synthesized by P. falciparum from either Glc, Gal, GlcN or GalN. At least one O-glycan found in P. falciparum-infected erythrocytes contains GlcNAc at its reducing terminus. Gel-filtration results had suggested the presence of O-GlcNAc on proteins in the infected erythrocyte. Probing with a synthetic pentapeptide, we could show that P. falciparum expresses its own O-GlcNAc transferase during intraerythrocytic development. Using this peptide, the enzyme was characterized to some degree. The localization and function of O-GlcNAc in P. falciparum remains to be elucidated.


Discovery of surfactants for metal/semiconductor separation of single-wall carbon nanotubes via high-throughput screening.
We report novel surfactants that can be used for the separation of metallic (M) and semiconducting (S) single-wall carbon nanotubes (SWCNTs). Among the M/S separation methods using surfactants in an aqueous solution, sodium dodecyl sulfate plays a key role in density gradient ultracentrifugation (DGU) and agarose gel separations. In this study, we screened 100 surfactants for M/S separation using a high-throughput screening system. We identified five surfactants, which could be used for both DGU and agarose gel separations, suggesting that the basic principle of these separations is common. These surfactants have relatively low dispersibilities, which is likely due to their common structural features, i.e., straight alkyl tails and charged head groups, and appeared to enable M- and S-SWCNTs to be distinguished and separated. These surfactants should stimulate research in this field and extend the application of electrically homogeneous SWCNTs not only for electronics but also for biology and medicine.


Solution conformation and mutagenic specificity of 1,N6-ethenoadenine.
Site-specific studies in several laboratories established that each of the three etheno adducts, 1,N6-ethenoadenine (epsilon A), 3,N4-ethenocytosine (epsilon C) and N2,3-ethenoguanine (N2,3-epsilon G), is mutagenic. In Escherichia coli, epsilon A is only weakly mutagenic in single-stranded DNA (mutation frequency, 0.1%), and epsilon C is at least 20 times more mutagenic than epsilon A. Prior treatment of host cells with ultraviolet irradiation enhances the mutagenic frequency of epsilon C by 30-60%, even when the E. coli is recA. Likewise, enhanced mutagenicity was observed when the host cells lacked 3'-->5' exonuclease activity of DNA polymerase III. epsilon A induces all three base substitutions, but A-->G predominates. epsilon C induces epsilon C-->T and epsilon C-->A substitutions, but only the latter was enhanced after ultraviolet irradiation of host cells. In contrast to the results in bacteria, both epsilon A and epsilon C are potent mutagenic lesions in simian kidney cells, inducing 70 and 81% base substitutions, respectively. In simian kidney cells, epsilon A exclusively induces epsilon A-->G transitions, whereas epsilon C-->A transversions are the major type of mutation induced by epsilon C. Nuclear magnetic resonance (NMR) spectrometry of the four possible pairs containing epsilon C indicated that both epsilon C:G and epsilon C:T pairs are stabilized by hydrogen bonds. Even though the latter forms the most stable pair containing epsilon C, the etheno adduct is in syn alignment. DNA polymerase appears to continue DNA synthesis with a syn-orientated base only in the absence of proofreading exonuclease activity or when ultraviolet irradiation-inducible proteins are present. For epsilon A, only epsilon A:T and epsilon A:G pairs have been studied by NMR, which showed that the former has no hydrogen bond whereas the latter maintains two hydrogen bonds with the etheno base in syn orientation. Determination of the relationship between a particular conformation of epsilon A and its mutagenic activity must await further studies. In a site-specific study of epsilon A with human cell extracts, an 11-mer oligonuclotide with a single epsilon A was inserted into an M13 bacteriophage containing an SV40 origin of replication. This vector was replicated in vitro with human fibroblast cell extracts, and the replicated products were analysed. In this experiment, epsilon A induced predominantly epsilon A-->G transitions but at a mutation frequency of 0.14%.


The effect of T cell-derived cytokines on B cell motility in vitro.
We have investigated the ability of interleukin-1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, and interferon-gamma (IFN-gamma) to induce motility in murine, splenic B lymphocytes. Two parameters of cellular locomotion were studied, namely acquisition of motile morphology (polarization) and in vitro migration through polycarbonate filters. Of the tested cytokines, only IL-4 gave a strong motile response among B cells. At the optimal concentration of 3 ng/ml, IL-4 induced polarization in 10-20% of the B cells within 30 min and in up to 40% after overnight exposure. The same amount IL-4 present in a microchemotaxis chamber stimulated migration through polycarbonate filters after overnight incubation in up to 15% of the input B cells. IFN-gamma could also induce some degree of polarization and migration, but only after 19 hr of exposure and to a much lesser extent than IL-4.


The Fitting Optimization Path Analysis on Scale Missing Data: Based on the 507 Patients of Poststroke Depression Measured by SDS.
To explore the optimal fitting path of missing data of the Scale to make the fitting data close to the real situation of patients' data. Based on the complete data set of the SDS of 507 patients with stroke, the data simulation sets of Missing Completely at Random (MCAR), Missing at Random (MAR), and Missing Not at Random (MNAR) were constructed by R software, respectively, with missing rates of 5%, 10%, 15%, 20%, 25%, 30%, 35%, and 40% under three missing mechanisms. Mean substitution (MS), random forest regression (RFR), and predictive mean matching (PMM) were used to fit the data. Root mean square error (RMSE), the width of 95% confidence intervals (95% CI), and Spearman correlation coefficient (SCC) were used to evaluate the fitting effect and determine the optimal fitting path. when dealing with the problem of missing data in scales, the optimal fitting path is ① under the MCAR deletion mechanism, when the deletion proportion is less than 20%, the MS method is the most convenient; when the missing ratio is greater than 20%, RFR algorithm is the best fitting method. ② Under the Mar mechanism, when the deletion ratio is less than 35%, the MS method is the most convenient. When the deletion ratio is greater than 35%, RFR has a better correlation. ③ Under the mechanism of MNAR, RFR is the best data fitting method, especially when the missing proportion is greater than 30%. In reality, when the deletion ratio is small, the complete case deletion method is the most commonly used, but the RFR algorithm can greatly expand the application scope of samples and save the cost of clinical research when the deletion ratio is less than 30%. The best way to deal with data missing should be based on the missing mechanism and proportion of actual data, and choose the best method between the statistical analysis ability of the research team, the effectiveness of the method, and the understanding of readers.


Differences in Incidence and Biological Characteristics of Breast Cancer between Roma and Non-Roma Patients in Slovakia.
Roma (Gypsies) constitute one of the largest ethnic minorities in Slovakia. Some reports have supported a higher prevalence of communicable diseases in Roma but data on cancer prevalence in Roma is absent. The aim of this study was to compare differences in the incidence and pathological characteristics of breast cancer between Roma and non-Roma in Slovakia. Roma and non-Roma breast cancer patients were identified using the Slovak HER2 Registry. The database from the last Census of Slovakia in 2011 was matched by gender, date of birth, and residency with the HER2 Registry from 2011 to 2013. Based on the match, Roma and non-Roma breast cancer patients were identified. Thirty-two and 5,775 women with breast cancer were identified as Roma and non-Roma, resp. The age-standardized breast cancer incidence rate was 2.12 times higher for non-Roma than for Roma patients (36 vs. 17 per 100,000 people). Roma patients were younger than non-Roma patients (median 49 vs. 61 years; p = 0.00001). Roma patients had more hormone receptor negative (34.4% vs. 18.1%; p = 0.03) and triple negative tumors (28.1% vs. 12.3%; p = 0.01) than non-Roma, and these differences remained statistically significant in multivariate analysis. For the first time, this study has revealed that the incidence and biological characteristics of breast cancer are different between Roma and non-Roma. Our data suggests that Roma patients are younger at diagnosis, have a lower age-standardized breast cancer incidence rate, and have more aggressive tumors than non-Roma.


Mitogen-activated protein kinases mediate the oxidative burst and saponin synthesis induced by chitosan in cell cultures of Panax ginseng.
Chitosan (CHN) specially induced the activities of 39 kD and 42 kD protein kinases in ginseng cells, which could be suppressed by an inhibitor of mitogen-activated protein kinase (MAPK) pathway, PD98059. The immunoprecipitation (IP) using MAPK antibody or kinase assay in vitro also showed that CHN-induced 42 kD and 39 kD protein kinases belonged to the MAPK family. PD98059 suppressed CHN-induced transcriptions of ginseng squalene synthase and ginseng squalene epoxidase genes (gss and gse), CHN-induced accumulation of beta-Amyrin synthase (beta-AS) and synthesis of saponin. These results showed that CHN-induced activities of MAPKs were necessary for the CHN-induced saponin synthesis. EGTA and LaCl3 suppressed CHN-induced 39 kD and 42 kD MAPK activities. Ruthenium red (RR) could suppress CHN-induced 39 kD activity. All of them suppressed CHN-induced saponin synthesis. These results indicated that CHN-induced increment of cytosolic calcium was necessary for CHN-induced saponin synthesis. PD98059 also suppressed CHN-induced oxidative burst (including the increment of activity of plasma membrane NADPH oxidase and production of H2O2), but diphenylene iodonium (DPI), dimethylthiourea (DMTU) and 2,5-dihydroxycinnamic acid methyl ester (DHC) could not suppress CHN-induced MAPK activities, which indicated that MAPK was possibly function upstream of CHN-induced oxidative burst.


Health spending and ability to pay: business, individuals, and government.
Health care spending has grown almost twice as fast as has the gross national product since 1965. Various parties in the health care financing arena have been affected to different degrees by this rising health care spending. As discussed in this article, households, businesses, and government all have had to devote increasing shares of their resources to financing health care. Although businesses have been increasingly burdened, either directly or through higher insurance premiums and Medicare taxes, that burden is less than is popularly believed.


Acute immune response to Mycobacterium massiliense in C57BL/6 and BALB/c mice.
Mycobacterium massiliense is an environmental opportunistic pathogen that has been associated with soft tissue infection after minor surgery. We studied the acute immune response of C57BL/6 and BALB/c mice infected intravenously with 10(6) CFU of an M. massiliense strain isolated from a nosocomial infection in Brazil. The results presented here show that M. massiliense is virulent and pathogenic to both C57BL/6 and BALB/c mice, inducing a granulomatous inflammatory reaction that involves the activation of macrophages, dendritic cells, and natural killer cells induced by gamma interferon and interleukin-17 (IL-17) in C57BL/6 mice and by IL-12 in BALB/c mice.


High-resolution melting analysis of the spa locus reveals significant diversity within sequence type 93 methicillin-resistant Staphylococcus aureus from northern Australia.
High-resolution melting analysis is an inherently robust, easy and inexpensive approach to the examination of genomic regions containing single-nucleotide polymorphisms and hypervariable loci. Staphylococcus aureus sequence type (ST) 93 is a singleton, Panton-Valentine leukocidin-positive clone unique to Australia. A high-resolution melting-based method for the identification of ST93 was developed, and a similar approach was used to reveal diversity within the spa locus of this lineage. Statistical and graphical methods that account for instrumental and operator-dependent variation in high-resolution melting curves were developed, to allow greater confidence and reproducibility in deciding whether another curve is truly different from the baseline curve of an amplicon with known sequence. The data support a very early acquisition, or multiple independent acquisitions, of SCCmec by ST93 methicillin-susceptible S. aureus (MSSA), and the coexistence of MSSA and methicillin-resistant S. aureus versions of the same lineage within northern Australia.