Connective Tissue Growth Factor (CTGF/CCN2) enhances lactogenic differentiation of mammary epithelial cells via integrin-mediated cell adhesion.
Connective Tissue Growth Factor (CTGF/CCN2), a known matrix-associated protein, is required for the lactogenic differentiation of mouse mammary epithelial cells. An HC11 mammary epithelial cell line expressing CTGF/CCN2 was constructed to dissect the cellular responses to CTGF/CCN2 that contribute to this differentiation program. Tetracycline-regulated expression of CTGF/CCN2 in HC11 cells enhanced multiple markers of lactogenic differentiation including beta-casein transcription and mammosphere formation. In a separate measure of mammary differentiation the addition of CTGF/CCN2 to cultures of MCF10A cells increased the development of acini in vitro. In HC11 cells the elevated levels of CTGF/CCN2 diminished the requirement for extracellular matrix proteins in the activation of beta-casein transcription, indicating that CTGF/CCN2 contributed to lactogenic differentiation through the regulation of matrix dependent cell adhesion. CTGF/CCN2 expression in HC11 cells increased expression of extracellular matrix proteins and integrins, enhanced the formation of focal adhesion complexes, and increased survival signaling. In addition, HC11 cells adhered to immobilized CTGF/CCN2 and this was inhibited by function-blocking antibodies to the integrins alpha6 and beta1, and to a lesser degree by antibody to beta3 integrin. CTGF/CCN2 expression in HC11 cells led to an increase in multiple markers of lactogenic differentiation. The mechanisms by which CTGF/CCN2 contributed to lactogenic differentiation include direct binding of CTGF/CCN2 to integrin complexes and CTGF/CCN2-induced matrix protein expression resulting in elevated integrin functionality.


Targeted mutagenesis in Atlantic salmon (Salmo salar L.) using the CRISPR/Cas9 system induces complete knockout individuals in the F0 generation.
Understanding the biological function behind key proteins is of great concern in Atlantic salmon, both due to a high commercial importance and an interesting life history. Until recently, functional studies in salmonids appeared to be difficult. However, the recent discovery of targeted mutagenesis using the CRISPR/Cas9 (clustered regularly interspaced palindromic repeats/CRISPR-associated) system enables performing functional studies in Atlantic salmon to a great extent. We used the CRISPR/Cas9 system to target two genes involved in pigmentation, tyrosinase (tyr) and solute carrier family 45, member 2 (slc45a2). Embryos were assayed for mutation rates at the 17 somite stage, where 40 and 22% of all injected embryos showed a high degree of mutation induction for slc45a2 and tyr, respectively. At hatching this mutation frequency was also visible for both targeted genes, displaying a graded phenotype ranging from complete lack of pigmentation to partial loss and normal pigmentation. CRISPRslc45a2/Cas9 injected embryos showing a complete lack of pigmentation or just a few spots of pigments also lacked wild type sequences when assaying more than 80 (slc45a2) sequence clones from whole embryos. This indicates that CRISPR/Cas9 can induce double-allelic knockout in the F0 generation. However, types and frequency of indels might affect the phenotype. Therefore, the variation of indels was assayed in the graded pigmentation phenotypes produced by CRISPR/Cas9-slc45a2. The results show a tendency for fewer types of indels formed in juveniles completely lacking pigmentation compared to juveniles displaying partial pigmentation. Another interesting observation was a high degree of the same indel type in different juveniles. This study shows for the first time successful use of the CRISPR/Cas9 technology in a marine cold water species. Targeted double-allelic mutations were obtained and, though the level of mosaicism has to be considered, we demonstrate that F0 fish can be used for functional studies in Atlantic salmon.


Chemoselective Coatings of GL13K Antimicrobial Peptides for Dental Implants.
Dental implant-associated infection is a clinical challenge which poses a significant healthcare and socio-economic burden. To overcome this issue, developing antimicrobial surfaces, including antimicrobial peptide coatings, has gained great attention. Different physical and chemical routes have been used to obtain these biofunctional coatings, which in turn might have a direct influence on their bioactivity and functionality. In this study, we present a silane-based, fast, and efficient chemoselective conjugation of antimicrobial peptides (Cys-GL13K) to coat titanium implant surfaces. Comprehensive surface analysis was performed to confirm the surface functionalization of as-prepared and mechanically challenged coatings. The antibacterial potency of the evaluated surfaces was confirmed against both Streptococcus gordonii and Streptococcus mutans, the primary colonizers and pathogens of dental surfaces, as demonstrated by reduced bacteria viability. Additionally, human dental pulp stem cells demonstrated long-term viability when cultured on Cys-GL13K-grafted titanium surfaces. Cell functionality and antimicrobial capability against multi-species need to be studied further; however, our results confirmed that the proposed chemistry for chemoselective peptide anchoring is a valid alternative to traditional site-unspecific anchoring methods and offers opportunities to modify varying biomaterial surfaces to form potent bioactive coatings with multiple functionalities to prevent infection.


Validation of a cell-based ELISA as a screening tool identifying anti-alphavirus small-molecule inhibitors.
Venezuelan (VEEV), eastern, and western equine encephalitis viruses, members of the genus Alphavirus, are causative agents of debilitative and sometimes fatal encephalitis. Although human cases are rare, these viruses pose a threat to military personnel, and to public health, due to their potential use as bioweapons. Currently, there are no licensed therapeutics for treating alphavirus infections. To address this need, small-molecules with potential anti-alphavirus activity, provided by collaborators, are tested routinely in live alphavirus assays utilizing time-consuming virus yield-reduction assays. To expedite the screening/hit-confirmation process, a cell-based enzyme-linked immunosorbent assay (ELISA) was developed and validated for the measurement of VEEV infection. A signal-to-background ratio of >900, and a z-factor of >0.8 indicated the robustness of this assay. For validation, the cell-based ELISA was compared directly to results from virus yield reduction assays in a single dose screen of 21 compounds. Using stringent criteria for anti-VEEV activity there was 90% agreement between the two assays (compounds displaying either antiviral activity, or no effect, in both assays). A concurrent compound-induced cell toxicity assay effectively filtered out false-positive hits. The cell-based ELISA also reproduced successfully compound dose-response virus inhibition data observed using the virus yield reduction assay. With available antibodies, this assay can be adapted readily to other viruses of interest to the biodefense community. Additionally, it is cost-effective, rapid, and amenable to automation and scale-up. Therefore, this assay could expedite greatly screening efforts and the identification of effective anti-alphavirus inhibitors.


A comparison of ultrasound treatment on primary and secondary sludges.
Ultrasound treatment of primary and secondary sludges was conducted to improve the qualities of sludges for the anaerobic digestion. The impacts of different sonication times, sonication densities and solids concentrations on ultrasonication efficiency were examined. The experimental results indicated that the significant reduction in particle size and increase in soluble organics could be achieved, implying that ultrasonication could offer a feasible treatment method to efficiently disintegrate sludge. The greater decrease in particle size and increase in soluble organics of sludge indicated that the secondary sludge has a more remarkable improvement after sonication over the primary sludge. With respects to the extent of disintegration and energy consumption, higher sonication density performed more effectively in terms of specific energy. There exists an optimal solids concentration range for both the sludges for optimum sonication. Within the optimal solids concentration range, efficient sonication can be effected and sludge would be disintegrated efficiently. The ultrasound would be attenuated by scattering and absorption if the solids concentration exceeds the optimal range. It appeared from the study that the mechanical shear forces caused by ultrasonic cavitation could be a key factor for sludge disintegration and collapse of cavitation bubbles could significantly alter the sludge characteristics.


The relation between the determinable quantities of volatile N-nitroso compounds and the peroxide number in soya bean oil.
Comparative studies of soya bean oil with and without addition of N-nitroso compounds (NDMA and NDEA) at different hydroperoxide concentrations have shown that the determinable quantities of substances having the retention time of N-nitroso compounds in soya bean oil and of nitrosoamines added to the oil are dependent upon the peroxide number. The determination was carried out by gas chromatography (nitrogen detector, nitrogen-sensitive, flame-ionization detector) prior to and following irradiation with UV light (360 nm). When the peroxide number was above 4, the determinability and the recovery were reduced by more than 60 per cent. In advanced autoxidation and after reduced recoveries, large amounts of NMDA and NDEA may be encountered for a short time. A possible linkage between N-nitroso compounds and peroxide is discussed. Comparative investigations of soya beans, crude oil, intermediates and commercially available steamed oils have revealed that the concentrations of the compounds with NDMA and NDEA properties in crude oil (peroxide number about 3) is higher than in soya beans. The recovery of these compounds is very poor in intermediate products with high peroxide numbers (about 5 to 9). However, N-nitroso compounds can be demonstrated in commercially available oils treated with steam (peroxide number about 0.7), although to a lesser extent than in crude oil.


Estimating the population burden of injuries: a comparison of household surveys and emergency department surveillance.
Injuries represent an important public health problem but their incidence is difficult to estimate. We conducted a population-based household survey in Greece covering 4079 interviewed individuals. The interviewees reported, for themselves and for cohabitating adults (age 15 years and older; n = 7157), injuries that occurred during the preceding year. Major injuries were defined as those requiring contact with a health institution. We compared these survey data with data obtained through a national Emergency Department Injury Surveillance System (EDISS). For the month closest to the survey interview, the incidence reported for the responders was 5.9 per 100 person-year, whereas the incidence for cohabitating adults was 3.7 per 100 person-years. These incidence rates declined for months more remote to the interview. Comparison of survey and EDISS data suggested that survey reporting was less accurate for nontraffic-related injuries. Taking into account possible recall and telescoping biases, the best survey estimate of the national annual number of major injuries is 525,000 (5.9 per 100 person-year), whereas the EDISS data yielded an estimate of 1,150,000 major injuries (12.9 per 100 person-years) Comparison of survey and EDISS data systems provides quantitative assessment of accuracy of the survey data in relation to time of injury before report date, to severity of injury, and to whether the injury is to the interviewee or to a cohabitant. The 2 systems could be used in a complementary way, although EDISS generates information that is medically more accurate and is a more cost-effective data collection system.


Binding-protein-dependent sugar transport by Agrobacterium radiobacter and A. tumefaciens grown in continuous culture.
Binding-protein-dependent sugar transport has been investigated in Agrobacterium radiobacter and A. tumefaciens. A. radiobacter contained two high-affinity glucose-binding proteins (GBP1 and GBP2) that additionally bound D-galactose (KD 0.26 microM) and D-xylose (KD 0.04 microM) respectively and were involved in the transport of these sugars. Partial sequencing of GBP1 and GBP2 showed that GBP2 exhibited significant homology with both the arabinose-binding protein (ABP) and the galactose-binding protein (GalBP) from Escherichia coli, whereas GBP1 exhibited significant homology only with ABP. Antiserum raised against GBP1 cross-reacted with GBP1 but not with GBP2, and vice versa. Anti-GBP1 and anti-GBP2 also cross-reacted with proteins corresponding to GBP1 and GBP2 respectively in A. tumefaciens, but little or no cross-reaction was observed with selected members of the Enterobacteriaceae, Rhizobiaceae and Pseudomonadaceae families grown under glucose limitation. GBP1 was less strongly repressed than GBP2 following batch growth of A. radiobacter on various carbon sources. The growth of A. radiobacter for more than approximately 10 generations in continuous culture under galactose or xylose limitation (D 0.045 h-1) led to the emergence of new strains which exhibited increased rates of glucose/galactose or glucose/xylose uptake, and which respectively hyperproduced GBP1 (strain AR18a) or GBP2 (strain AR9a). Similarly, growth of A. tumefaciens for more than approximately 15 generations under glucose or galactose limitation produced new strains which exhibited increased rates of glucose/xylose or glucose/galactose uptake and which respectively hyperproduced proteins analogous to GBP2 (strain AT9) or GBP1 (strain AT18a). It is concluded that growth of Agrobacterium species under carbon-limited conditions leads to the predictable emergence of new strains which specifically hyperproduce the transport system for the limiting nutrient. The GBP1-dependent system of A. radiobacter is unique amongst these transport systems in that the mutations that lead to its hyperproduction under carbon limitation render it least susceptible to repression by excess glucose during ammonia limitation, with the result that succinoglucan exopolysaccharide is produced from glucose at an enhanced rate.


Histological Analysis of Early Osteolysis in Total Ankle Arthroplasty.
The purpose of this study was to perform a histological comparative analysis of tibiotalar joint samples taken from areas of osteolysis adjacent to total ankle arthroplasties vs control synovial specimens to determine the reaction to and presence of polyethylene (PE) particles. A total of 57 pathology samples were identified in the osteolysis group, while 11 were identified in the control group. For each sample, hematoxylin and eosin, Oil Red O (ORO), and macrophage marker CD163-stained slides were created. Polarized light and ORO stain were used to identify PE particles. The presence of metal particles and giant cell reaction to PE particles were also scored. Macrophages, PE particles, metallosis, and foreign body giant cell reaction scores were significantly higher in the osteolysis group compared with the control group. In the osteolysis group, ORO staining was positive in 93% (53/57), birefringent material was present in 96.5% (55/57), and macrophage infiltrates were present in 96.5% (55/57). Foreign body giant cell reaction with giant cells surrounding PE particles was present in 49.1% (28/57) of osteolytic specimens. The presence of foreign body giant cell reaction was associated with significantly higher macrophage, ORO, and polarizable material scores. The average time to surgery for osteolysis from the index ankle replacement was 6.0 (range, 0-15) years for the 57 patients in the osteolysis group. This study is the largest ankle arthroplasty histological analysis to show that areas of osteolysis consist of abundant polyethylene wear particles, present both intracellularly and extracellularly. Furthermore, these areas were associated with a CD163+ macrophage infiltrate and frequently a foreign body reaction with giant cells engulfing PE particles. It is likely that implant wear particles play a significant role in osteolysis based on the histopathology. Level III, retrospective comparative series.


Injectable PAMAM dendrimer-PEG hydrogels for the treatment of genital infections: formulation and in vitro and in vivo evaluation.
Local intravaginal drug therapy is preferred for treatment of ascending genital infections during pregnancy. In the present study, an in situ forming biodegradable hydrogel for sustained release of amoxicillin in the cervicovaginal region is described. A generation 4 poly(amidoamine) [G4-(NH(2))(64)] dendrimer with peripheral thiopyridyl terminations is cross-linked with 8-arm polyethylene glycol (PEG) bearing thiol terminations. The hydrogels were formulated and tested in vivo in a pregnant guinea pig model for volume, retention times, biodegradation, tolerability and transport across fetal membrane. The physicochemical characterization of the hydrogels was carried out using differential calorimetry, SEM, and confocal imaging. The hydrogels offer antibacterial activity arising from sustained release of amoxicillin from gels. The in vivo studies in guinea pig showed that 100-200 μL of gel sufficiently covered the cervicovaginal region with a residence time of at least 72 h and gel was primarily retained in the maternal tissues without crossing the fetal membranes into the fetus. The dendrimer gels were stable up to 72 h, and the in vivo biodegradation of gel occurred after 72 h; this correlated well with the in vitro degradation pattern. The pH of the vagina was not altered upon application of the gel, and none of the animals aborted up to 72 h after application of gel. The histological evaluation of the cervical tissues showed absence of edema in the epithelial cell layer, no sloughing of the epithelial or superficial mucous layer, and absence of necrosis and infiltration of inflammatory cells in the submucosal layers, confirming that tissues were tolerant to the gel. The immunohistofluorescence images showed the localization of the gel components on the superficial mucified epithelial layer. The cross-linking density and swelling of hydrogels was impacted by the polymer content, and the 10% hydrogels exhibited the highest cross-link density. The in vitro drug release studies carried out using Franz diffusion cells showed that amoxicillin release from 6 and 10% gels was sustained for 240 h as compared to 3% gels. As the polymer concentration increased to 10%, the release pattern from gels approached diffusion controlled mechanism with diffusional exponent n = 0.49. In conclusion, the biodegradable in situ forming hydrogels of the present study offer a therapeutic option to provide sustained localized delivery of amoxicillin intracervically to the pregnant woman for the treatment of ascending genital infections.


FOXP1 negatively regulates intrinsic excitability in D2 striatal projection neurons by promoting inwardly rectifying and leak potassium currents.
Heterozygous loss-of-function mutations in the transcription factor FOXP1 are strongly associated with autism. Dopamine receptor 2 expressing (D2) striatal projection neurons (SPNs) in heterozygous Foxp1 (Foxp1+/-) mice have higher intrinsic excitability. To understand the mechanisms underlying this alteration, we examined SPNs with cell-type specific homozygous Foxp1 deletion to study cell-autonomous regulation by Foxp1. As in Foxp1+/- mice, D2 SPNs had increased intrinsic excitability with homozygous Foxp1 deletion. This effect involved postnatal mechanisms. The hyperexcitability was mainly due to down-regulation of two classes of potassium currents: inwardly rectifying (KIR) and leak (KLeak). Single-cell RNA sequencing data from D2 SPNs with Foxp1 deletion indicated the down-regulation of transcripts of candidate ion channels that may underlie these currents: Kcnj2 and Kcnj4 for KIR and Kcnk2 for KLeak. This Foxp1-dependent regulation was neuron-type specific since these same currents and transcripts were either unchanged, or very little changed, in D1 SPNs with cell-specific Foxp1 deletion. Our data are consistent with a model where FOXP1 negatively regulates the excitability of D2 SPNs through KIR and KLeak by transcriptionally activating their corresponding transcripts. This, in turn, provides a novel example of how a transcription factor may regulate multiple genes to impact neuronal electrophysiological function that depends on the integration of multiple current types - and do this in a cell-specific fashion. Our findings provide initial clues to altered neuronal function and possible therapeutic strategies not only for FOXP1-associated autism but also for other autism forms associated with transcription factor dysfunction.


Interactions between inflammation, oxidative stress, and endothelial dysfunction in end-stage renal disease.
Despite a rapid improvement in dialysis technology during the last 20 years, the mortality rate is still very high in patients with end-stage renal disease (ESRD), and the death rate is comparable with that of many cancer patients with metastases. The main cause of mortality in ESRD is cardiovascular disease (CVD), and cardiac mortality for dialysis patients aged 45 years or younger is more than 100-fold greater than in the general population. The high cardiovascular mortality rate suggests that ESRD patients are subjected to a process of accelerated atherogenesis. Because factors proven to contribute to atherosclerosis in the general population, such as dyslipidemia, smoking, diabetes mellitus, and hypertension are highly prevalent in ESRD patients, it is reasonable to assume that such risk factors also apply to these patients. However, as it has been shown that the high cardiovascular risk in ESRD is incompletely accounted for by traditional risk factors, it may be speculated that nontraditional risk factors, seemingly more difficult to reconcile, also contribute. Among several putative nontraditional risk factors, chronic inflammation has attracted a lot of interest recently because it seems to be associated to both increased vascular oxidative stress and endothelial dysfunction, both of which are important predictors of cardiovascular events in nonrenal patient groups.


CARDIOVASCULAR DISEASE: PREVENTION AND REHABILITATION.
Cardiovascular related diseases are the major cause of death in the United States. The primary goal of the physician should be prevention of these diseases; however, once problems occur, a definitive rehabilitation program must be instituted in addition to the medical and surgical treatment. Although risk factors for the development of cardiovascular disease are numerous, statistical reports suggest that control of these factors might delay or prevent such development. This presentation describes the recommended Physical Medicine and Rehabilitation Program for high-risk patients or those with existing cardiovascular disease. The basis of the physical fitness and rehabilitation program is controlled progression-of-energy-expenditure physical activity and exercise in metabolic equivalents (METS). Education plays a key role in decreasing the incidence of cardiovascular disease, and should start at an early age. Adults at high risk of developing cardiovascular disease should be offered medically supervised physical fitness exercise programs, with monitoring where necessary.


Critical variables in the conversion of marrow cells to skeletal muscle.
We have studied conversion of marrow cells to skeletal muscle in cardiotoxin-injured anterior tibialis muscle in a green fluorescent protein (GFP) to C57BL/6 transplantation model and ascertained that total body irradiation (TBI) with establishment of chimerism is a critical factor. Local irradiation has little effect in lower doses and was detrimental at higher doses. Whole body (1000 cGy) with shielding of the leg or a combination of 500 cGy TBI and 500 cGy local radiations was found to give the best results. In non-obese diabetic-severe combined immunodeficient (NOD-SCID) recipients, we were able to show that conversion could occur without radiation, albeit at relatively lower levels. Within 3 days of cardiotoxin injury, GFP-positive mononuclear cells were seen in the muscle, and within 2 weeks GFP-positive muscle fibers were identified. Conversion rates were increased by increasing donor-cell dose. Timing of the cardiotoxin injury relative to the transplantation was critical. These studies show that variables in transplantation and injury are critical features of marrow-to-muscle conversions. Irradiation primarily effects conversion by promoting chimerism. These data may explain the differences in the literature for the frequency of marrow-to-skeletal muscle conversion and can set a platform for future models and perhaps clinical protocols.


Enhanced l-Malic Acid Production by Aspergillus oryzae DSM 1863 Using Repeated-Batch Cultivation.
l-Malic acid is a C4-dicarboxylic acid and a potential key building block for a bio-based economy. At present, malic acid is synthesized petrochemically and its major market is the food and beverages industry. In future, malic acid might also serve as a building block for biopolymers or even replace the commodity chemical maleic anhydride. For a sustainable production of l-malic acid from renewable resources, the microbial synthesis by the mold Aspergillus oryzae is one possible route. As CO2 fixation is involved in the biosynthesis, high yields are possible, and at the same time greenhouse gases can be reduced. In order to enhance the production potential of the wild-type strain Aspergillus oryzae DSM 1863, process characteristics were studied in shake flasks, comparing batch, fed-batch, and repeated-batch cultivations. In the batch process, a prolonged cultivation time led to malic acid consumption. Keeping carbon source concentration on a high level by pulsed feeding could prolong cell viability and cultivation time, however, did not result in significant higher product levels. In contrast, continuous malic acid production could be achieved over six exchange cycles and a total fermentation time of 19 days in repeated-batch cultivations. Up to 178 g/L l-malic acid was produced. The maximum productivity (0.90 ± 0.05 g/L/h) achieved in the repeated-batch cultivation had more than doubled than that achieved in the batch process and also the average productivity (0.42 ± 0.03 g/L/h for five exchange cycles and 16 days) was increased considerably. Further repeated-batch experiments confirmed a positive effect of regular calcium carbonate additions on pH stability and malic acid synthesis. Besides calcium carbonate, nitrogen supplementation proved to be essential for the prolonged malic acid production in repeated-batch. As prolonged malic acid production was only observed in cultivations with product removal, product inhibition seems to be the major limiting factor for malic acid production by the wild-type strain. This study provides a systematic comparison of different process strategies under consideration of major influencing factors and thereby delivers important insights into natural l-malic acid production.


Multiplanar imaging properties of Theon sieves.
Greek ladders with diffraction-limited array foci provide a probability to realize array imaging with equal intensity. Here, taking the ancient Theon sequence as an example, we design the optical structure and have measured the focusing properties by digital holography. Then, we verify the multiplanar imaging with different magnifications by experiment. The experimental results agree well with the theoretical analysis. In addition, bi-Fourier planes filtering technology is proposed to solve the problem of crosstalk between different imaging planes to further improve the imaging resolution. Therefore, we can freely design the focal length of the bifocal lens to achieve high-quality imaging at different resolutions. As a kind of amplitude-only diffractive lens, multifocal imaging provides a possibility of application in array biological imaging, ophthalmology, and an optical zoom system.


Evidence for chronic morbillivirus infection in the Mediterranean striped dolphin (Stenella coeruleoalba).
In the summer of 1990 an epizootic infection caused by a morbillivirus (DMV) killed several thousand striped dolphins (Stenella coeruleoalba) in the Mediterranean Sea. In 1991 and 1992 the epizootic reached Italian and Greek waters. The infection by DMV in the acute period of the epizootic caused encephalitis, pneumonia and depletion of lymph nodes. After 1990, the systemic infection apparently disappeared from the Catalonian coast, giving way to cases of chronic infection of the CNS. Dolphins that died between 1991 and May 1994 were necropsied, and investigated for lesions due to DMV, and for the presence of morbillivirus antigen in tissues. Encephalitis occurred in 6 dolphins in which DMV antigen was demonstrated in the CNS and which were without lesions or antigen in other, non-nervous tissues. Inflammatory lesions, gliosis, and DMV antigen decreased in density and amount from cerebral grey matter, through the thalamic areas to the medulla oblongata. The cerebellum was usually spared. Lesions consisted of non-suppurative encephalitis, with diffuse gliosis and glial nodules and neuronophagia, and loss of neurons. Perivascular cuffing of lymphocytes and plasma cells was present in the cerebral cortex and the white matter beneath the cortex. Multinucleate syncytia were not detected in any of the dolphins. The haemagglutinin of DMV was detected mainly in neurons in the cerebral cortical areas. There was no clear relationship between the presence and amount of DMV antigen and the density or chronicity of lesions. Viral inclusions were seen in haematoxylin and eosin stained sections in 3/6 dolphins, principally in the nucleus and the cytoplasm of neurons. In the immunoperoxidase stained sections, dense granular deposits of chromogen, similar to viral inclusions, were evident in all 6 dolphins. The change in the distribution of lesions and of DMV antigen, from systemic to localized in the CNS, and the clustering of systemic DMV infections in the first four months of the epizootic, giving rise to sporadic occurrence of local CNS infection in the subsequent four years, as well as the chronic nature of the CNS lesions, which closely resembles subacute sclerosing panencephalitis, strongly support the existence of a chronic morbillivirus infection in the striped dolphin, as a delayed consequence of the 1990 epizootic.


NMR and EPR studies on a monoheme cytochrome c550 isolated from Bacillus halodenitrificans.
A c-type monoheme ferricytochrome c550 (9.6 kDa) was isolated from cells of Bacillus halodenitrificans sp.nov., grown anaerobically as a denitrifier. The visible absorption spectrum indicates the presence of a band at 695 nm characteristic of heme-methionine coordination. The midpoint redox potential was determined at several pH values by visible spectroscopy. The redox potential at pH 7.6 is 138 mV. When studied by 1H-NMR spectroscopy as a function of pH, the spectrum shows a pH dependence with pKa values of 6.0 and 11.0. According to these pKa values, three forms designated as I, II and III can be attributed to cytochrome c550. The first pKa is probably associated with protonation of the propionate groups. The second pKa value introduces a larger effect in the 1H-NMR spectrum and is probably due to the ionisation of the axial histidine. Studies of temperature variation of the 1H-NMR spectra for both the ferrous and ferri forms of the cytochrome were performed. Heme meso protons, the heme methyl groups, the thioether protons, two protons from a propionate and the methylene protons from the axial methionine were identified in the reduced form. The heme methyl resonances of the ferri form were also assigned. EPR spectroscopy was also used to probe the ferric heme environment. A signal at gmax approximately 3.5 at pH 7.5 was observed indicating an almost axial heme environment. At higher pH values the signal at gmax approximately 3.5 converts mainly to a signal at g approximately 2.96. The pKa associated with this change is around 11.3. The N-terminal sequence of this cytochrome was determined and compared with known amino acid sequences of other cytochromes.


Clinicians' Perceptions of Behavioral Economic Strategies to Increase the Use of Lung-Protective Ventilation.
Rationale: Lung-protective ventilation (LPV) improves outcomes in patients with acute respiratory distress syndrome (ARDS) and has also shown benefits in patients without ARDS. Despite this evidence, LPV use remains low.Objectives: To understand clinicians' perceptions of using behavioral economic strategies to improve rates of LPV use.Methods: We conducted semistructured interviews of clinicians across seven intensive care units within a university health system. We purposefully sampled clinicians of different professional backgrounds and experience levels. Each interview included descriptions of three of five strategies grounded in behavioral economic theory designed to facilitate clinicians' use of LPV: 1) an order set autopopulated with LPV settings ("default"), 2) an order set providing a choice between autopopulated LPV settings and open-ended order entry for alternative settings ("active choice"), 3) requirement of written justification if settings other than LPV were ordered or documented ("accountable justification"), 4) automated ARDS identification and clinician prompting ("alert"), and 5) provision of clinicians' and their peers' individual rates of LPV use ("peer comparison"). Descriptions were followed by open-ended questions to elicit perceptions about advantages, disadvantages, and acceptability. Initial interview transcripts were reviewed by two investigators to develop a thematic codebook, which was refined iteratively with the use of constant comparative methods.Results: We completed 17 interviews of physicians, nurse practitioners, and respiratory therapists. Strategies that prepopulated settings (default, active choice, and accountable justification) were perceived as providing benefit by reducing workloads and serving as cognitive prompts. The default and active choice strategies were more acceptable than accountable justification, which was perceived as potentially frustrating due to workflow impedance. The alert strategy was met with concerns about alert accuracy and alarm fatigue. The peer comparison strategy led to concerns about timing and fear of punitive measures. Participants believed that the default and active choice strategies would be highly acceptable, whereas few interviewees thought the alert would be acceptable. The active choice strategy was most consistently identified as potentially highly effective.Conclusions: Behavioral economic strategies have great potential as acceptable and potentially effective strategies to increase the use of LPV.


A new therapeutic association to manage relapsing experimental colitis: Doxycycline plus Saccharomyces boulardii.
Immunomodulatory antibiotics have been proposed for the treatment of multifactorial conditions such as inflammatory bowel disease. Probiotics are able to attenuate intestinal inflammation, being considered as safe when chronically administered. The aim of the study was to evaluate the anti-inflammatory effects of doxycycline, a tetracycline with immunomodulatory properties, alone and in association with the probiotic Saccharomyces boulardii CNCMI-745. Doxycycline was assayed both in vitro (Caco-2 epithelial cells and RAW 264.7 macrophages) and in vivo, in the trinitrobenzenesulfonic acid (TNBS) model of rat colitis and the dextran sodium sulfate (DSS) model of mouse colitis. In addition, the anti-inflammatory effect of the association of doxycycline and the probiotic was evaluated in vitro and in vivo in a DSS model of reactivated colitis in mice. Doxycycline displayed immunomodulatory activity in vitro, reducing IL-8 production by intestinal epithelial cells and nitric oxide by macrophages. Doxycycline administration to TNBS-colitic rats (5, 10 and 25 mg/kg) ameliorated the intestinal inflammatory process, being its efficacy comparable to that previously showed by minocycline. Doxycycline treatment was also effective in reducing acute intestinal inflammation in the DSS model of mouse colitis. The association of doxycycline and S. boulardii helped managing colitis in a reactivated model of colitis, by reducing intestinal inflammation and accelerating the recovery and attenuating the relapse. This was evidenced by a reduced disease activity index, colonic tissue damage and expression of inflammatory mediators. This study confirms the intestinal anti-inflammatory activity of doxycycline and supports the potential use of its therapeutic association with S. boulardii for the treatment of inflammatory bowel diseases, in which doxycycline is used to induce remission and long term probiotic administration helps to prevent the relapses.


Cell-intrinsic in vivo requirement for the E47-p21 pathway in long-term hematopoietic stem cells.
Major regulators of long-term hematopoietic stem cell (LT-HSC) self-renewal and proliferation have been identified, but knowledge of their in vivo interaction in a linear pathway is lacking. In this study, we show a direct genetic link between the transcription factor E47 and the major cell cycle regulator p21 in controlling LT-HSC integrity in vivo under repopulation stress. Numerous studies have shown that E47 activates p21 transcription in hematopoietic subsets in vitro, and we now reveal the in vivo relevance of the E47-p21 pathway by reducing the gene dose of each factor individually (E47(het) or p21(het)) versus in tandem (E47(het)p21(het)). E47(het)p21(het) LT-HSCs and downstream short-term hematopoietic stem cells exhibit hyperproliferation and preferential susceptibility to mitotoxin compared to wild-type or single haploinsufficient controls. In serial adoptive transfers that rigorously challenge self-renewal, E47(het)p21(het) LT-HSCs dramatically and progressively decline, indicating the importance of cell-intrinsic E47-p21 in preserving LT-HSCs under stress. Transient numeric recovery of downstream short-term hematopoietic stem cells enabled the production of functionally competent myeloid but not lymphoid cells, as common lymphoid progenitors were decreased, and peripheral lymphocytes were virtually ablated. Thus, we demonstrate a developmental compartment-specific and lineage-specific requirement for the E47-p21 pathway in maintaining LT-HSCs, B cells, and T cells under hematopoietic repopulation stress in vivo.


Growth-related changes in the intracellular distribution of succinate dehydrogenase activity in turkey muscle.
The intracellular distribution of succinate dehydrogenase (SDH) activity was measured and mapped in iliotibialis cranialis muscle fibers of growing turkeys. Two weeks after hatching, fibers with strong myofibrillar adenosine triphosphatase (ATPase) were larger in cross sectional area than fibers with weak ATPase, but fibers with weak ATPase subsequently exhibited more rapid relative growth. At two weeks, both types of fibers had stronger SDH activity in their central axis than in the subsarcolemmal region. At ten weeks this gradient was reversed so that SDH activity declined from the periphery to the axis (a negative centripetal gradient of SDH activity). Fibers with strong ATPase developed the steepest negative gradients of SDH activity. Fibers with weak ATPase tended to have more uniformly distributed SDH activity, even when fibers had grown quite large.


The Ser(186) phospho-acceptor site within ERK4 is essential for its ability to interact with and activate PRAK/MK5.
ERK (extracellular-signal-regulated kinase) 4 [MAPK (mitogen-activated protein kinase) 4] and ERK3 (MAPK6) are atypical MAPKs. One major difference between these proteins and the classical MAPKs is substitution of the conserved T-X-Y motif within the activation loop by a single phospho-acceptor site within an S-E-G motif. In the present study we report that Ser(186) of the S-E-G motif in ERK4 is phosphorylated in vivo. Kinase-dead ERK4 is also phosphorylated on Ser(186), indicating that an ERK4 kinase, rather than autophosphorylation, is responsible. Co-expression of MK5 [MAPK-activated protein kinase 5; also known as PRAK (p38-regulated/activated kinase)], a physiological target of ERK4, increases phosphorylation of Ser(186). This is not dependent on MK5 activity, but does require interaction between ERK4 and MK5 suggesting that MK5 binding either prevents ERK4 dephosphorylation or facilitates ERK4 kinase activity. ERK4 mutants in which Ser(186) is replaced with either an alanine residue or a phospho-mimetic residue (glutamate) are unable to activate MK5 and Ser(186) is also required for cytoplasmic anchoring of MK5. Both defects seem to reflect an impaired ability of the ERK4 mutants to interact with MK5. We find that there are at least two endogenous pools of wild-type ERK4. One form exhibits reduced mobility when analysed using SDS/PAGE. This is due to MK5-dependent phosphorylation and only this retarded ERK4 species is both phosphorylated on Ser(186) and co-immunoprecipitates with wild-type MK5. We conclude that binding between ERK4 and MK5 facilitates phosphorylation of Ser(186) and stabilization of the ERK4-MK5 complex. This results in phosphorylation and activation of MK5, which in turn phosphorylates ERK4 on sites other than Ser(186) resulting in the observed mobility shift.


Clinical pathology of endocrine tumors of the pancreas. Analysis of autopsy cases.
A clinicopathological analysis of endocrine tumors of the pancreas, using 800 autopsy cases (422 men, 378 women, mean age 78.7) was accomplished. The results were: (1) Endocrine tumors and similar lesions were found in 3% or 24 cases (25 lesions). Twenty lesions (20 cases) were found to be tumors and five lesions (five cases) were determined to be hyperplasia of Langerhans islets. (2) Incidence of tumor was 10% (6/60) in individuals having histological studies of all sections of the pancreas, and 1.6% (12/738) in individuals having histological studies of three random sections of the pancreas. (3) None of the cases with tumors and hyperplastic lesions showed symptoms of hormone production. (4) Immunohistochemical analysis revealed hormone production in all 20 tumor cases and multiple hormone production was found in 14 of these (70%). (5) Ductular or tubular structures were found in or adjacent to the tumors in 12 cases (60%) and hyperplasia in one case (20%). Langerhans islets, 500 microns or larger in size, were found in three lesions of the tumor (15%). Langerhans islets with the mean diameter of normal islets + 2 SD or larger also were found around five tumors (25%) and three hyperplasias (60%). The above findings suggest that endocrine tumors of the pancreas are prevalent and that they do not produce symptoms of excessive hormone production even though they do continue to produce hormones. Some of the endocrine tumors or hyperplasias develop from totipotent stem cells of the duct epithelia, and factors promoting the growth of Langerhans islets might exist.


What is the effect of diet and/or exercise interventions on behavioural compensation in non-exercise physical activity and related energy expenditure of free-living adults? A systematic review.
Non-exercise physical activity (NEPA) and/or non-exercise activity thermogenesis (NEAT) reductions may occur from diet and/or exercise-induced negative energy balance interventions, resulting in less-than-expected weight loss. This systematic review describes the effects of prescribed diet and/or physical activity (PA)/exercise on NEPA and/or NEAT in adults. Studies were identified from PubMed, web-of-knowledge, Embase, SPORTDiscus, ERIC and PsycINFO searches up to 1 March 2017. Eligibility criteria included randomised controlled trials (RCT), randomised trials (RT) and non-randomised trials (NRT); objective measures of PA and energy expenditure; data on NEPA, NEAT and spontaneous PA; ≥10 healthy male/female aged>18 years; and ≥7 d length. The trial is registered at PROSPERO-2017-CRD42017052635. In all, thirty-six articles (RCT-10, RT-9, NRT-17) with a total of seventy intervention arms (diet, exercise, combined diet/exercise), with a total of 1561 participants, were included. Compensation was observed in twenty-six out of seventy intervention arms (fifteen studies out of thirty-six reporting declines in NEAT (eight), NEPA (four) or both (three)) representing 63, 27 and 23 % of diet-only, combined diet/exercise, and exercise-only intervention arms, respectively. Weight loss observed in participants who decreased NEAT was double the weight loss found in those who did not compensate, suggesting that the energy imbalance degree may lead to energy conservation. Although these findings do not support the hypothesis that prescribed diet and/or exercise results in decreased NEAT and NEPA in healthy adults, the underpowered trial design and the lack of state-of-the-art methods may limit these conclusions. Future studies should explore the impact of weight-loss magnitude, energetic restriction degree, exercise dose and participant characteristics on NEAT and/or NEPA.


Polysarcosine-Functionalized mRNA Lipid Nanoparticles Tailored for Immunotherapy.
Lipid nanoparticles (LNPs) have gained great attention as carriers for mRNA-based therapeutics, finding applications in various indications, extending beyond their recent use in vaccines for infectious diseases. However, many aspects of LNP structure and their effects on efficacy are not well characterized. To further exploit the potential of mRNA therapeutics, better control of the relationship between LNP formulation composition with internal structure and transfection efficiency in vitro is necessary. We compared two well-established ionizable lipids, namely DODMA and MC3, in combination with two helper lipids, DOPE and DOPC, and two polymer-grafted lipids, either with polysarcosine (pSar) or polyethylene glycol (PEG). In addition to standard physicochemical characterization (size, zeta potential, RNA accessibility), small-angle X-ray scattering (SAXS) was used to analyze the structure of the LNPs. To assess biological activity, we performed transfection and cell-binding assays in human peripheral blood mononuclear cells (hPBMCs) using Thy1.1 reporter mRNA and Cy5-labeled mRNA, respectively. With the SAXS measurements, we were able to clearly reveal the effects of substituting the ionizable and helper lipid on the internal structure of the LNPs. In contrast, pSar as stealth moieties affected the LNPs in a different manner, by changing the surface morphology towards higher roughness. pSar LNPs were generally more active, where the highest transfection efficiency was achieved with the LNP formulation composition of MC3/DOPE/pSar. Our study highlights the utility of pSar for improved mRNA LNP products and the importance of pSar as a novel stealth moiety enhancing efficiency in future LNP formulation development. SAXS can provide valuable information for the rational development of such novel formulations by elucidating structural features in different LNP compositions.


Congenital cystic eye with multiple ocular and intracranial anomalies.
We describe a newborn with congenital cystic eye, contralateral persistent hyperplastic primary vitreous, and cerebrocutaneous abnormalities. The cerebrocutaneous abnormalities consisted of agenesis of the corpus callosum, midbrain deformity, malformed sphenoid bone, right upper eyelid coloboma, and a left periocular hamartoma. The results of karyotype analysis of the patient and his parents were normal. The association of congenital cystic eye with contralateral persistent hyperplastic primary vitreous has not been previously reported, to our knowledge. Although no unifying diagnosis exists for the collection of anomalies demonstrated in this patient, the term cranial ectodermopathy broadly classifies most of the defects.


Amino acid substrate specificity of Escherichia coli phenylalanyl-tRNA synthetase altered by distinct mutations.
Neither the tertiary structure nor the location of active sites are known for phenylalanyl-tRNA synthetase (PheRS; alpha 2 beta 2 structure), a member of class II aminoacyl-tRNA synthetases. In an attempt to detect the phenylalanine (Phe) binding site, two Escherichia coli PheRS mutant strains (pheS), which were resistant to p-fluorophenylalanine (p-F-Phe) were analysed genetically. The pheS mutations were found to cause Ala294 to Ser294 exchanges in the alpha subunits from both independent strains. This alteration (S294) resided in the well-conserved C-terminal part of the alpha subunit, precisely within motif 3, a typical class II tRNA synthetase sequence. We thus propose that motif 3 participates in the formation of the Phe binding site of PheRS. Mutation S294 was also the key for proposing a mechanism by which the substrate analogue p-F-Phe is excluded from the enzymatic reaction; this may be achieved by steric interactions between the para-position of the aromatic ring and the amino acid residue at position 294. The Phe binding site model was then tested by replacing the alanine at position 294 as well as the two flanking phenylalanines (positions 293 and 295) by a number of selected other amino acids. In vivo and in vitro results demonstrated that Phe293 and Phe295 are not directly involved in substrate binding, but replacements of those residues affected PheRS stability. However, exchanges at position 294 altered the binding of Phe, and certain mutants showed pronounced changes in specificity towards Phe analogues. Of particular interest was the Gly294 PheRS in which presumably an enlarged cavity for the para position of the aromatic ring allowed an increased aminoacylation of tRNA with p-F-Phe. Moreover, the larger para-chloro and para-bromo derivatives of Phe could interact with this enzyme in vitro and became highly toxic in vivo. The possible exploitation of the Gly294 mutant PheRS for the incorporation of non-proteinogenic amino acids into proteins is discussed.


Antimicrobial susceptibility patterns of Streptococcus agalactiae in a German university hospital.
Group B streptococci (GBS) are the major cause of neonatal sepsis and meningitis. GBS infection in neonates is usually treated with a combination of penicillin and gentamicin. According to consensus guidelines, pregnant women at risk receive intrapartum prophylaxis with either ampicillin or penicillin or, in case of allergy, with erythromycin or clindamycin. We investigated the susceptibility patterns of 190 GBS strains from neonates isolated from 1993-1999 and 150 GBS strains collected from adult women in 1997 and 1999. All isolates were susceptible to penicillin, ampicillin and cefotaxime. Erythromycin resistance among all isolates from neonates and from adult women in 1997 was 4.7% and 6%, respectively. In contrast, 12% of the isolates from adult women in 1999 were resistant to erythromycin and 7% were resistant to clindamycin. These findings show an increasing macrolide resistance in recent GBS strains and indicate the need for further surveillance.


A new dataset of spermatogenic vs. oogenic transcriptomes in the nematode Caenorhabditis elegans.
The nematode Caenorhabditis elegans is an important model for studies of germ cell biology, including the meiotic cell cycle, gamete specification as sperm or oocyte, and gamete development. Fundamental to those studies is a genome-level knowledge of the germline transcriptome. Here, we use RNA-Seq to identify genes expressed in isolated XX gonads, which are approximately 95% germline and 5% somatic gonadal tissue. We generate data from mutants making either sperm [fem-3(q96)] or oocytes [fog-2(q71)], both grown at 22°. Our dataset identifies a total of 10,754 mRNAs in the polyadenylated transcriptome of XX gonads, with 2748 enriched in spermatogenic gonads, 1732 enriched in oogenic gonads, and the remaining 6274 not enriched in either. These spermatogenic, oogenic, and gender-neutral gene datasets compare well with those of previous studies, but double the number of genes identified. A comparison of the additional genes found in our study with in situ hybridization patterns in the Kohara database suggests that most are expressed in the germline. We also query our RNA-Seq data for differential exon usage and find 351 mRNAs with sex-enriched isoforms. We suggest that this new dataset will prove useful for studies focusing on C. elegans germ cell biology.


Local polarity and hydrogen bonding inside the Sec14p phospholipid-binding cavity: high-field multi-frequency electron paramagnetic resonance studies.
Sec14p promotes the energy-independent transfer of either phosphatidylinositol (PtdIns) or phosphatidylcholine (PtdCho) between lipid bilayers in vitro and represents the major PtdIns/PtdCho transfer protein in the budding yeast Saccharomyces cerevisiae. Herein, we employ multi-frequency high-field electron paramagnetic resonance (EPR) to analyze the electrostatic and hydrogen-bonding microenvironments for series of doxyl-labeled PtdCho molecules bound by Sec14p in a soluble protein-PtdCho complex. A structurally similar compound, 5-doxyl stearic acid dissolved in a series of solvents, was used for experimental calibration. The experiments yielded two-component rigid limit 130- and 220-GHz EPR spectra with excellent resolution in the gx region. Those components were assigned to hydrogen-bonded and nonhydrogen-bonded nitroxide species. Partially resolved 130-GHz EPR spectra from n-doxyl-PtdCho bound to Sec14p were analyzed using this two-component model and allowed quantification of two parameters. First, the fraction of hydrogen-bonded nitroxide species for each n-doxyl-PtdCho was calculated. Second, the proticity profile along the phospholipid-binding cavity of Sec14p was characterized. The data suggest the polarity gradient inside the Sec14p cavity is a significant contributor to the driving molecular forces for extracting a phospholipid from the bilayer. Finally, the enhanced g-factor resolution of EPR at 130 and 220 GHz provides researchers with a spectroscopic tool to deconvolute two major contributions to the x-component of the nitroxide g-matrix: hydrogen-bond formation and local electrostatic effects.


Mechanisms of ATP release, the enabling step in purinergic dynamics.
The only effective intervention to slow onset and progression of glaucomatous blindness is to lower intraocular pressure (IOP). Among other modulators, adenosine receptors (ARs) exert complex regulation of IOP. Agonists of A(3)ARs in the ciliary epithelium activate Cl(-) channels, favoring increased formation of aqueous humor and elevated IOP. In contrast, stimulating A(1)ARs in the trabecular outflow pathway enhances release of matrix metalloproteinases (MMPs) from trabecular meshwork (TM) cells, reducing resistance to outflow of aqueous humor to lower IOP. These opposing actions are thought to be initiated by cellular release of ATP and its ectoenzymatic conversion to adenosine. This view is now supported by our identification of six ectoATPases in trabecular meshwork (TM) cells and by our observation that external ATP enhances TM-cell secretion of MMPs through ectoenzymatic formation of adenosine. ATP release is enhanced by cell swelling and stretch. Also, enhanced ATP release and downstream MMP secretion is one mediator of the action of actin depolymerization to reduce outflow resistance. Inflow and outflow cells share pannexin-1 and connexin hemichannel pathways for ATP release. However, vesicular release and P2X(7) release pathways were functionally limited to inflow and outflow cells, respectively, suggesting that blocking exocytosis might selectively inhibit inflow, lowering IOP.


Anti-inflammatory effect of (-)-epigallocatechin-3-gallate on Porphyromonas gingivalis lipopolysaccharide-stimulated fibroblasts and stem cells derived from human periodontal ligament.
(-)-epigallocatechin-3-gallate (EGCG) has been reported to exert anti-inflammatory and antibacterial effects in periodontitis. However, its exact mechanism of action has yet to be determined. The present in vitro study evaluated the anti-inflammatory effects of EGCG on human periodontal ligament fibroblasts (hPDLFs) and human periodontal ligament stem cells (hPDLSCs) affected by bacterial lipopolysaccharide (LPS) extracted from Porphyromonas gingivalis. hPDLFs and hPDLSCs were extracted from healthy young adults and were treated with EGCG and/or P. gingivalis LPS. After 1, 3, 5, and 7 days from treatment, cytotoxic and proliferative effects were evaluated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and bromodeoxyuridine assay, respectively. And then, the gene expressions of hPDLFs and hPDLSCs were observed for interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, osteoprotegerin (OPG), receptor activator of nuclear factor kappa-B ligand (RANKL), and RANKL/OPG using real-time polymerase chain reaction (PCR) at 0, 6, 24, and 48 hours after treatment. The experiments were performed with the following groups for hPDLFs and hPDLSCs; 1) No treat, 2) EGCG alone, 3) P. gingivalis LPS alone, 4) EGCG+P. gingivalis LPS. The 20 µM of EGCG and 20 µg/mL of P. gingivalis LPS had the lowest cytotoxic effects, so those concentrations were used for further experiments. The proliferations of hPDLFs and hPDLSCs increased in all groups, though the 'EGCG alone' showed less increase. In real-time PCR, the hPDLFs and hPDLSCs of 'EGCG alone' showed similar gene expressions to those cells of 'no treat'. The gene expressions of 'P. gingivalis LPS alone' in both hPDLFs and hPDLSCs were highly increased at 6 hours for IL-1β, IL-6, TNF-α, RANKL, and RANKL/OPG, except the RANKL/OPG in hPDLSCs. However, those increased gene expressions were down-regulated in 'EGCG+P. gingivalis LPS' by the additional treatment of EGCG. Our results demonstrate that EGCG could exert an anti-inflammatory effect in hPDLFs and hPDLSCs against a major pathogen of periodontitis, P. gingivalis LPS.


Factors associated with changes in self-reported health status in infliximab-treated inflammatory bowel disease patients: results from a case management survey.
Canadian inflammatory bowel disease patients treated with infliximab are predominantly managed through a nationwide case management system, named BioAdvance(®). A web-based survey was provided to patients currently receiving infliximab therapy within BioAdvance(®). Patients were categorized according to health trajectories: decliners, non-changers, moderate increasers and strong increasers. Factors associated with health trajectories were identified using multivariable multinomial logistic regression. 918 of 1160 respondents were inflammatory bowel disease patients reporting health status. Strong increasers were more likely to use educational tools than non-changers (adjusted odds ratio [aOR]: 1.65; 95% CI: 1.03-2.64), to be treated for ulcerative colitis (aOR: 2.05; 95% CI: 1.16-3.64) and to perceive case management as important (aOR: 2.52; 95% CI: 1.56-4.09). Younger (aOR: 0.72; 95% CI: 0.63-0.83) and French-speaking (aOR: 0.34; 95% CI: 0.18-0.63) patients were less likely to miss workdays. Patients were more likely to have missed workdays prior to joining the case management program. Inflammatory bowel disease patients receiving infliximab within the nationwide case management system report a positive impact on health status and absenteeism.


In search of healers--Southeast Asian refugees in the American health care system.
Healing is the alleviation of sickness, which includes both medically defined problems of pathophysiology (disease) and personal definitions of not being well (illness). Refugees from Southeast Asia now have a special need for healing because their health problems are changing from those of concern to public health, which are well documented and for which there are known effective treatments, to those that are primarily a personal concern and that are difficult to diagnose and treat effectively because of their chronic nature and their cultural and emotional components. The finding among refugees of physical complaints for which there is no identifiable medical cause is explained by cultural tendencies in Southeast Asia that promote focusing on somatic symptomatology, and by a delayed somatic response to refugee trauma. To prevent escalation of medical intervention, physicians need to be sensitive to Southeast Asians' attitudes toward health and their expectations and apprehensions regarding Western medicine.


Biodiversity of 52 chicken populations assessed by microsatellite typing of DNA pools.
In a project on the biodiversity of chickens funded by the European Commission (EC), eight laboratories collaborated to assess the genetic variation within and between 52 populations from a wide range of chicken types. Twenty-two di-nucleotide microsatellite markers were used to genotype DNA pools of 50 birds from each population. The polymorphism measures for the average, the least polymorphic population (inbred C line) and the most polymorphic population (Gallus gallus spadiceus) were, respectively, as follows: number of alleles per locus, per population: 3.5, 1.3 and 5.2; average gene diversity across markers: 0.47, 0.05 and 0.64; and proportion of polymorphic markers: 0.91, 0.25 and 1.0. These were in good agreement with the breeding history of the populations. For instance, unselected populations were found to be more polymorphic than selected breeds such as layers. Thus DNA pools are effective in the preliminary assessment of genetic variation of populations and markers. Mean genetic distance indicates the extent to which a given population shares its genetic diversity with that of the whole tested gene pool and is a useful criterion for conservation of diversity. The distribution of population-specific (private) alleles and the amount of genetic variation shared among populations supports the hypothesis that the red jungle fowl is the main progenitor of the domesticated chicken.


Prevalence of cardiovascular risk factors in a rural area in Southern Thailand: potential ethnic differences.
In Thepa district, Songkhla province, Southern Thailand, parents of children being followed long term from conception for their development (N 1325), as well as health workers of the same district (N 150) were surveyed with regards to anthropometry, occupation, family size, food and leisure habits, and blood levels of lipids, sugar, creatinine and hematocrit. Differences among those of Thai or Chinese extracts (Thai-Buddhists) and those of Malay extracts (Thai-Muslims) were separately evaluated for males and females. The mean age for the group (N 1475, including 636 couples) was 31.5 +/- 7.3 (SD) and ranged from 15-66 years. There were 794 females (485 Muslims) ages 29.6 +/- 6.6 (SD). The Muslim families were larger in size averaging 2.8 +/- 1.6 children while the Buddhist's averaged 1.9 +/- 0.9. Fifty four percent of the group were engaged in rubber tapping and this often included both members of the family. Sixty eight percent of the males currently smoked Differences in measured variables between Muslims and Buddhists were minimal. The most striking however was the high density lipoprotein cholesterol (HDL-C) among the males where the age-adjusted average was 51.3 +/- 0.72 (SE) among the Buddhists and 42.2 +/- 0.59 (SE) in the Muslims. This difference was significant even when adjusted for other related variables. Differences in the ethnic groups were also examined in terms of prevalence of risks (hypertension, BMI > or = 25%, waist-hip-ratio, fasting plasma glucose > or = 110 mg%, total cholesterol > or = 200 mg%, triglyceride > or = 150 mg% and low HDL-C). For hypertension (systolic > or = 140 or diastolic > or = 90 mmHg), female Muslims showed higher prevalence (4.5% vs 1.6%; OR 2.82 CI 1.04-7.64). For low HDL-C, male Muslim showed higher prevalence 23.6 vs 8.8%, OR 2.31 CI 1.27-4.22). Other risks showed no differences among the ethnic groups. The differences in parameters or in prevalence of risk between these two ethnic groups (minimal intermarriage) are distinct from differences among Malays and Chinese in Singapore where such differences were subsequently reflected in the differences in incidence and out-come of ischemic heart diseases.


Warm Cells, Hot Mitochondria: Achievements and Problems of Ultralocal Thermometry.
Temperature is a crucial regulator of the rate and direction of biochemical reactions and cell processes. The recent data indicating the presence of local thermal gradients associated with the sites of high-rate thermogenesis, on the one hand, demonstrate the possibility for the existence of "thermal signaling" in a cell and, on the other, are criticized on the basis of thermodynamic calculations and models. Here, we review the main thermometric techniques and sensors developed for the determination of temperature inside living cells and diverse intracellular compartments. A comparative analysis is conducted of the results obtained using these methods for the cytosol, nucleus, endo-/sarcoplasmic reticulum, and mitochondria, as well as their biological consistency. Special attention is given to the limitations, possible sources of errors and ambiguities of the sensor's responses. The issue of biological temperature limits in cells and organelles is considered. It is concluded that the elaboration of experimental protocols for ultralocal temperature measurements that take into account both the characteristics of biological systems, as well as the properties and limitations of each type of sensor is of critical importance for the generation of reliable results and further progress in this field.


Preparation of casein-chitosan microspheres containing diltiazem hydrochloride by an aqueous coacervation technique.
Sustained release casein-chitosan microspheres containing diltiazem hydrochloride (DTZ) were prepared with colloidal coacervation technique in a completely aqueous environment. The interaction between chitosan solution in dilute acetic acid (5% v/v) and casein solution in 0.5 M sodium hydroxide was the basis for the microspheres formation. Formaldehyde was used for the surface hardening of the droplets by cross-linking and thus fixing the shape and surface morphology of the formed microspheres. The entrapment efficiencies of the microspheres were variables (14.5-53.7%) depending on the preparation conditions. The prepared microspheres exhibited an angle of repose values between 31.9-42.0 degrees indicating good free flowing nature, whereas DTZ powder as such was non-flowable. The dissolution profiles of DTZ from casein-chitosan microspheres showed retarded release pattern of the drug into distilled water. Casein and chitosan concentrations, initial drug concentration and stirring time were found to be the main parameters that affect the properties and the performance of the prepared microspheres. The retarded release of DTZ was increased by increasing casein concentration, and stirring time. On the other hand, increasing chitosan concentration and using high initial drug loading showed a fast drug release.


Organic-free, versatile sessile droplet microfluidic device for chemical separation using an aqueous two-phase system.
This work presents a novel portable, versatile sessile droplet microfluidic (SDMF) device to perform liquid manipulation operations such as confining, splitting and colorimetric detection. Furthermore, chemical isolations based on an aqueous two-phase system (ATPS) for separating an analyte of choice from a complicated sample matrix can be carried out. ATPS extractions can replace conventional liquid-liquid extractions and take away the need for harmful organic solvents. Superhydrophobic (SH) surfaces were fabricated from a commercially available material, Ultra-Ever Dry® (UED®). On these SH surfaces, surface energy traps (SETs) were produced either by air plasma treatment (simultaneously) or laser micromachining (sequentially) to dock/pin an ATPS containing droplet onto the surface. Splitting of droplets or removing a precise volume of the top phase from a pinned extraction system was achieved with a sandwich-chip approach. For this, an additional SET patterned substrate was placed on top of the droplet and subsequently lifted. This multipurpose platform was used to isolate Cd from a mixture of several other metal ions (i.e. Mn, Ni, Cu, Pb, Fe) for its subsequent interference-free detection. An ATPS consisting of sodium sulfate and polyethylene glycol (PEG) as phase forming components and potassium iodine as extractant allowed separation of cadmium with an extraction efficiency of q(Cd2+) = 98.5%. Using a portable, cost-effective, smartphone-based UV/vis spectrometer, Cd was detected with a LoD of 3.4 ppm. Alternatively, the multipurpose platform can also be used as sampling platform for a benchtop UV/vis spectrometer, where a LoD of 0.53 ppm was obtained. Potential applications of the presented platform include sample preparation and separation that can be achieved by aqueous two-phase extractions, such as proteins, antibodies, DNA, cells, organic molecules and metal ions.


In vitro synthesis of some complement components (C1q, C3 and C4) by lymphoid tissues and circulating leucocytes in man.
Human lymphoid tissues and peripheral blood leucocytes and monocytes were studies with respect to the synthesis of complement components (C1q, C3 and C4) using an in vitro culture technique. All of the lymphoid tissues investigated (bone marrow, thymus, lymph node, spleen, tonsil, adenoid) synthesize complement components in different patterns. C3 was produced by all lymphoid tissues except the spleen, which was the only lymphoid tissue in which C4 production was regularly found. C1q synthesis was demonstrated in the spleen and adenoid cultures, and occasionally also in those of lymph node tissue. Lymphocytes in peripheral blood from normal individuals and in thoracic duct lymph, and also from patients suffering from chronic lymphatic leukaemia, do not synthesize any of these complement components. Peripheral blood leucocyte samples from normal individuals, containing 60 per cent lymphocytes and 40 per cent monocytes, do synthesize C3, however. Separation of the monocytes from these samples showed that it was in these cells that the synthesis of C3 occurred. Production of C3 by mononuclear phagocytes is also supported by the finding that peripheral blood leucocytes from patients suffering from acute monocytic leukaemia synthesize C3. C1q and C4 synthesis could not be demonstrated in any of the cultures of circulating leucocytes.


Flower power: tree flowering phenology as a settlement cue for migrating birds.
1. Neotropical migrant birds show a clear preference for stopover habitats with ample food supplies; yet, the proximate cues underlying these decisions remain unclear. 2. For insectivorous migrants, cues associated with vegetative phenology (e.g. flowering, leaf flush, and leaf loss) may reliably predict the availability of herbivorous arthropods. Here we examined whether migrants use the phenology of five tree species to choose stopover locations, and whether phenology accurately predicts food availability. 3. Using a combination of experimental and observational evidence, we show migrant populations closely track tree phenology, particularly the flowering phenology of honey mesquite (Prosopis glandulosa), and preferentially forage in trees with more flowers. Furthermore, the flowering phenology of honey mesquite reliably predicts overall arthropod abundance as well as the arthropods preferred by migrants for food. 4. Together, these results suggest that honey mesquite flowering phenology is an important cue used by migrants to assess food availability quickly and reliably, while in transit during spring migration.


TRAIL negatively regulates VEGF-induced angiogenesis via caspase-8-mediated enzymatic and non-enzymatic functions.
Solid tumors supply oxygen and nutrients required for angiogenesis by producing vascular endothelial growth factor (VEGF). Thus, inhibitors of VEGF signaling abrogate tumor angiogenesis, resulting in the suppression of tumor growth and metastasis. We here investigated the effects of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on VEGF-induced angiogenesis. TRAIL inhibited VEGF-induced in vitro angiogenesis of human umbilical vein endothelial cells (HUVECs) and in vivo neovascularization in chicken embryos and mice. TRAIL blocked VEGF-induced angiogenic signaling by inhibiting ERK, Src, FAK, paxillin, Akt, and eNOS. Further, TRAIL blocked intracellular Ca(2+) elevation and actin reorganization in HUVECs stimulated with VEGF, without inhibiting VEGF receptor-2 tyrosine phosphorylation. TRAIL increased caspase-8 activity, without inducing caspase-9/-3 activation and apoptosis. Moreover, TRAIL resulted in cleavage of FAK into FAK-related non-kinase-like fragments in VEGF-stimulated HUVECs, which was blocked by a caspase-8 inhibitor and cellular caspase-8-like inhibitory protein. Biochemical and pharmacological inhibition of caspase-8 and FAK blocked the inhibitory effects of TRAIL on VEGF-stimulated anti-angiogenic signaling and events. In addition, caspase-8 knockdown also suppressed VEGF-mediated signaling and angiogenesis, suggesting that procaspase-8 plays a role of a non-apoptotic modulator in VEGF-induced angiogenic signaling. These results suggest that TRAIL inhibits VEGF-induced angiogenesis by increasing caspase-8 activity and subsequently decreasing non-apoptotic signaling functions of procaspase-8, without inducing caspase-3 activation and endothelial cell cytotoxicity. These data indicate that caspase-8 may be used as an anti-angiogenic drug for solid tumors resistant to TRAIL and anti-tumor drugs.


Oxygenation of benzyldimethylamine by singlet oxygen. Products and mechanism.
[reaction: see text] A product study of the reaction of benzyldimethylamine (1) with thermally and photochemically generated 1O2 in MeCN was carried out. Benzaldehyde and N-benzyl-N-methylformamide are the reaction products, oxygenation representing ca. 9% of the overall quenching of 1O2 by 1. The temperature effect and the intermolecular and intramolecular kinetic deuterium isotope effects were also determined. It is suggested that the products derive from an intracomplex hydrogen atom transfer in a reversibly formed charge-transfer complex.


Economics of food intake in mice: energy yield of the reinforcer.
One of the Zeitgeists of the field for the study of ingestive behavior is that organisms are endowed with internal self-regulatory mechanisms that ensure optimal nutrition. However, the alarming increase in the prevalence of obesity challenges us to reconsider the extent to which internal regulatory mechanisms affect food intake, especially in a free market economy. Cued by the pioneering work of George Collier and his students, we have been examining food intake (demand) in mice when the effort or price of food is manipulated. We present two new experiments in mice that investigate the effect of energy yield per unit of food earned on working for food. The first experiment shows that when the nominal energy yield of each food pellet is halved by cellulose dilution, mice show relatively inelastic calorie-related demand despite the fact the cellulose diluted diet is unpalatable. The second experiment shows that the size of the pellet reinforcer does not have a major effect on food demand except in the extreme condition of small reward and high unit price. New analyses of distributions of responding are presented which suggest that mice work for "target" numbers of food rewards with only a small influence of price or energy gain.


Metabolic Syndrome and Amyloid Accumulation in the Aging Brain.
Recent studies show links between metabolic syndrome and Alzheimer's disease (AD) neuropathology. Understanding the link between vascular-related health conditions and dementia will help target at risk populations and inform clinical strategies for early detection and prevention of AD. To determine whether metabolic syndrome is associated with global cerebral amyloid-β (Aβ) positivity and longitudinal Aβ accumulation. Prospective study of 165 participants who underwent (11)C-Pittsburgh compound B (PiB) PET neuroimaging to measure Aβ, from June 2005 to May 2016. Metabolic syndrome was defined using the revised Third Adults Treatment Panel of the National Cholesterol Education Program criteria. Participants were classified as PiB+/-. Linear mixed effects models assessed the relationships between baseline metabolic syndrome and PiB status and regional Aβ change over time. A total of 165 cognitively normal participants of the Baltimore Longitudinal Study of Aging (BLSA) Neuroimaging substudy, aged 55-92 years (mean baseline age = 76.4 years, 85 participants were male), received an average of 2.5 PET-PiB scans over an average interval of 2.6 (3.08 SD) years between first and last visits. Metabolic syndrome was not associated with baseline PiB positivity or concurrent regional Aβ. Metabolic syndrome was associated with increased rates of Aβ accumulation in superior parietal and precuneus regions over time in the PiB+ group. Elevated fasting glucose and blood pressure showed individual associations with accelerated Aβ accumulation. Metabolic syndrome was associated with accelerated Aβ accumulation in PiB+ individuals and may be an important factor in the progression of AD pathology.


Enantioselective reduction of N-alkyl ketimines with frustrated Lewis pair catalysis using chiral borenium ions.
Enantioselective reduction of ketimines was demonstrated using chiral N-heterocyclic carbene (NHC)-stabilised borenium ions in frustrated Lewis pair catalysis. High levels of enantioselectivity were achieved for substrates featuring secondary N-alkyl substituents. Comparative reactivity and mechanistic studies identify key determinants required to achieve useful enantioselectivity and represent a step forward in the further development of enantioselective FLP methodologies.


Determinants of transient and persistent hospital efficiency: The case of Italy.
In this paper, we extend the 4-random-component closed skew-normal stochastic frontier model by including exogenous determinants of hospital persistent (long-run) and transient (short-run) inefficiency, separated from unobserved heterogeneity. We apply this new model to a dataset composed by 133 Italian hospitals during the period 2008-2013. We show that average total inefficiency is about 23%, higher than previous estimates; hence, a model where the different types of inefficiency and hospital unobserved characteristics are not confounded allows us to get less biased estimates of hospital inefficiency. Moreover, we find that transient efficiency is more important than persistent efficiency, as it accounts for 60% of the total one. Last, we find that ownership (for-profit hospitals are more transiently inefficient and less persistently inefficient than not-for-profit ones, whereas public hospitals are less transiently inefficient than not-for-profit ones), specialization (specialized hospitals are more transiently inefficient than general ones; i.e., there is evidence of scope economies in short-run efficiency), and size (large-sized hospitals are better than medium and small ones in terms of transient inefficiency) are determinants of both types of inefficiency, although we do not find any statistically significant effect of multihospital systems and teaching hospitals.


Circ_0068481 affects the human pulmonary artery smooth muscle cells' progression by miR-361-3p/KLF5 axis.
Uncontrolled proliferation of pulmonary artery smooth muscle cells (PASMCs) contributes to the pathogenesis of pulmonary arterial hypertension (PAH). In this work, we defined the precise part of circ_0068481 in PASMC proliferation and migration induced by hypoxia. We hypothesized that circ_0068481 enhanced hypoxia-induced PASMC proliferation, invasion and migration through the miR-361-3p/KLF5 pathway. Human PASMCs (hPASMCs) were exposed to hypoxic (3% O2) conditions. Circ_0068481, microRNA (miR)-361-3p and Krüppel-like factor 5 (KLF5) levels were gauged by qRT-PCR and western blot. Cell viability, proliferation, invasion and migration were detected by XTT, EdU incorporation, transwell and wound-healing assays, respectively. Dual-luciferase reporter, RNA immunoprecipitation (RIP) and RNA pull-down assays were performed to confirm the direct relationship between miR-361-3p and circ_0068481 or KLF5. Circ_0068481 expression was increased in the serum of PAH patients and hypoxia-induced hPASMCs. Downregulation of circ_0068481 attenuated hypoxia-induced promotion in hPASMC proliferation, invasion and migration. Circ_0068481 directly targeted miR-361-3p, and miR-361-3p downregulation reversed the inhibitory effects of circ_0068481 silencing on hypoxia-induced hPASMC proliferation, invasion and migration. KLF5 was a direct miR-361-3p target, and miR-361-3p upregulation mitigated hypoxia-induced hPASMC proliferation, invasion and migration by inhibiting KLF5 expression. Moreover, circ_0068481 induced KLF5 expression by binding to miR-361-3p in hypoxic hPASMCs. Circ_0068481 knockdown ameliorated hypoxia-induced hPASMC proliferation, invasion and migration at least in part through the miR-361-3p/KLF5 axis.


[Microangiopathic haemolytic anaemia as a complication of mitomycin C treatment (author's transl)].
Microangiopathic haemolytic anaemia developed in 5 (out of 14) patients treated with mitomycin C combinations, having received 4 or more treatment cycles. This finding necessitates restricted use of broad and longterm use of mitomycin C, particularly in view of the occasionally fatal outcome of this toxic complication. Adjuvant treatment with mitomycin C does not appear acceptable any longer. Patients treated with mitomycin C should be monitored carefully for occurrence of schistocytosis and platelet deficiency as very early signs of microangiopathic haemolytic anaemia.


Epetraborole, a leucyl-tRNA synthetase inhibitor, demonstrates murine efficacy, enhancing the in vivo activity of ceftazidime against Burkholderia pseudomallei, the causative agent of melioidosis.
Burkholderia pseudomallei is the causative agent of melioidosis, which is increasingly being reported worldwide. Mortality rates as high as 40% have been reported based on clinical patient outcomes in the endemic areas of Australia and Thailand. Novel therapies are needed to reduce treatment duration and adverse effects and improve treatment outcomes. Epetraborole, a novel antibiotic, targets leucyl-tRNA synthetase (LeuRS), an essential enzyme that catalyzes the attachment of leucine to transfer RNA. Epetraborole was evaluated for in vitro activity and efficacy in a murine model to assess clinical relevance against Burkholderia pseudomallei infections for possible treatment of melioidosis. Epetraborole was tested against 13 clinically derived and three reference B. pseudomallei strains that have a broad spectrum of susceptibilities to the standard-of-care (SoC) drugs for melioidosis, which showed that epetraborole exhibited minimal inhibitory concentrations of 0.25-4 μg/mL. Ex vivo studies using THP-1 macrophages confirmed the potency of epetraborole and demonstrated synergy between epetraborole and ceftazidime. In the acute pulmonary murine infection model of melioidosis, epetraborole demonstrated equivalent efficacy when delivered orally or subcutaneously, which compared well with the standard-of-care drug ceftazidime. In addition, adding epetraborole to ceftazidime significantly improved antimicrobial activity in this animal model. This work warrants further exploration of epetraborole as a candidate for treating melioidosis and substantiates LeuRS as a clinically relevant drug target in B. pseudomallei.


An improved collagen zymography approach for evaluating the collagenases MMP-1, MMP-8, and MMP-13.
Collagen zymography is an SDS-PAGE-based method for detecting both the proenzyme and active forms of collagenases. Although collagen zymography is used for assessment of the matrix metalloproteinases MMP-1 and MMP-13, it can be difficult to detect these collagenases due to technical issues. Moreover, it remains unclear whether the collagenase activity of MMP-8 can be detected by this method. Here, we present an improved collagen zymography method that allows quantification of the activities of MMP-1, MMP-8, and MMP-13. Activities of recombinant collagenases could be detected in collagen zymogram gels copolymerized with 0.3 mg/mL type I collagen extracted from rat tail tendon. This improved method is sensitive enough to detect the activity of as little as 1 ng of collagenase. We generated standard curves for the three collagenases to quantify the collagenolytic activity levels of unknown samples. To validate our improved method, we investigated MMP-1 activity levels in human thyroid cancer (8505C) and normal thyroid (Nthy-ori-3-1) cell lines, finding that the proenzyme and active MMP-1 levels were greater in 8505C cells than in Nthy-ori-3-1 cells. Taken together, our data show that collagen zymography can be used in both molecular and clinical investigations to evaluate collagenase activities in various pathological conditions.


The processing of chitosan and its derivatives and their application for postoperative anti-adhesion.
The formation of peritoneal adhesions represents one of the most common complications after abdominal surgery, and it increases the difficulty of re-operation. Thus, preventing postoperative adhesions is a major problem in the field of surgical medicine. Due to a lack of good predictive animal models and the complexity of adhesion pathogenesis, attempts to prevent or reduce peritoneal adhesions have been largely unsuccessful. As a result, the study of anti-adhesion drugs and materials has become a hot topic for experts and scholars. The processing and development of chitosan and its derivatives as new anti-adhesion materials is highly valued because chitosan is inexpensive, highly biocompatible, and not cytotoxic, making it a promising anti-adhesion material. Here, we review the sources and preparation of chitosan and the progress made toward producing different types of chitosan and its derivatives for preventing adhesion.


Exploring the Validity of a Modified Version of the SES-SFV with Students Attending Northern Irish Universities.
Compared to US university students, far less is known about the scale of unwanted and non-consensual sexual experiences [USEs] faced by UK university students, particularly those in Northern Ireland [NI]. The Sexual Experiences Survey (Short Form [SEF-SFV]) is considered a popular tool for measuring USEs but has not been updated since 2007; there is some indication that additional perpetrator tactics may be more inclusive of students' experiences and that certain scoring strategies may impact our understanding of data. This paper examines the USEs reported by 1033 students attending either of NI's traditional universities. Participants completed a modified version of the SES-SFV that included two additional perpetration tactics: "ignorance of refusal" and "taken by surprise." Sixty-three percent (n = 650) reported experiencing at least one USE, but this reduced to 53% (n = 546) without the new perpetrator tactics. Female and non-heterosexual students reported significantly more USEs than male and heterosexual students, respectively. "Taken by surprise" was highly endorsed (81%, n = 525) and the most commonly endorsed tactic. Whilst dichotomous scoring is the most straightforward, continuous scoring affords greater analytical opportunities whilst still retaining frequency of USEs. "Taken by surprise" may be a relevant addition but further mixed-methodological research is required to assess its validity among larger and more diverse samples. SES-SFV scoring options should be also validated using male and mixed-gender samples, particularly categorical scoring to ensure current construction is reflective of the wider student experience.


Does low-dose methotrexate deserve more respect from clinicians?
Medical errors associated with low-dose methotrexate may be life-threatening. Prescribers should be cognizant of the medication's toxicities and the persistent challenges in preventing adverse events. This article reviews the properties of methotrexate and its common drug-drug interactions. Best practices from the Institute for Safe Medication Practices, aimed at reducing methotrexate errors, are highlighted.


The early response of Atlantic salmon (Salmo salar) macrophages exposed in vitro to Aeromonas salmonicida cultured in broth and in fish.
Aeromonas salmonicida is a fish pathogen that causes furunculosis. Virulent strains of this bacterium are able to infect salmonid macrophages and survive within them, although mechanisms favouring intracellular survival are not completely understood. It is known that A. salmonicida cultured in vivo in the peritoneal cavity of the host undergoes changes in gene expression and surface architecture compared with cultures grown in vitro in broth. Therefore, in this study, the early macrophage responses to A. salmonicida grown in vivo and in vitro were compared. Macrophage-enriched cell preparations from head kidney of Atlantic salmon (Salmo salar) were infected in vitro in 96-well microtitre dishes and changes in gene expression during the infection process were monitored using a custom Atlantic salmon cDNA microarray. A. salmonicida cultures grown in tryptic soy broth and in peritoneal implants were used to infect the macrophages. The macrophages were harvested at 0.5, 1.0 and 2.0h after addition of the bacteria to the medium. Significant changes in gene expression were evident by microarray analysis at 2.0h post-infection in macrophages infected with broth-grown and implant-grown bacteria; however, qPCR analysis revealed earlier up-regulation of JunB and TNF-alpha in macrophages exposed to the implant-grown bacteria. Up-regulation of those genes and others is consistent with the effects of extracellular products of aeromonad bacteria on macrophages and also suggests initiation of the innate immune response.


Tumor-educated platelets.
Beyond traditional roles in homeostasis and coagulation, growing evidence suggests that platelets also reflect malignant transformation in cancer. Platelets are present in the tumor microenvironment where they interact with cancer cells. This interaction results in direct and indirect "education" as evident by platelet alterations in adhesion molecules, glycoproteins, nucleic acids, proteins and various receptors. Subsequently, these tumor-educated platelets (TEPs) circulate throughout the body and play pivotal roles in promotion of tumor growth and dissemination. Accordingly, platelet status can be considered a unique blood-based biomarker that can potentially predict prognosis and therapeutic success. Recently, liquid biopsies including TEPs have received much attention as safe, minimally invasive and sensitive alternatives for patient management. Herein, we provide an overview of TEPs and explore their benefits and limitations in cancer.


The interactome of KRAB zinc finger proteins reveals the evolutionary history of their functional diversification.
Krüppel-associated box (KRAB)-containing zinc finger proteins (KZFPs) are encoded in the hundreds by the genomes of higher vertebrates, and many act with the heterochromatin-inducing KAP1 as repressors of transposable elements (TEs) during early embryogenesis. Yet, their widespread expression in adult tissues and enrichment at other genetic loci indicate additional roles. Here, we characterized the protein interactome of 101 of the ~350 human KZFPs. Consistent with their targeting of TEs, most KZFPs conserved up to placental mammals essentially recruit KAP1 and associated effectors. In contrast, a subset of more ancient KZFPs rather interacts with factors related to functions such as genome architecture or RNA processing. Nevertheless, KZFPs from coelacanth, our most distant KZFP-encoding relative, bind the cognate KAP1. These results support a hypothetical model whereby KZFPs first emerged as TE-controlling repressors, were continuously renewed by turnover of their hosts' TE loads, and occasionally produced derivatives that escaped this evolutionary flushing by development and exaptation of novel functions.


Evidence for cardiolipin binding sites on the membrane-exposed surface of the cytochrome bc1.
The respiratory chain is located in the inner membrane of mitochondria and produces the major part of the ATP used by a cell. Cardiolipin (CL), a double charged phospholipid composing ~10-20% of the mitochondrial membrane, plays an important role in the function and supramolecular organization of the respiratory chain complexes. We present an extensive set of coarse-grain molecular dynamics (CGMD) simulations aiming at the determination of the preferential interfaces of CLs on the respiratory chain complex III (cytochrome bc(1), CIII). Six CL binding sites are identified, including the CL binding sites known from earlier structural studies and buried into protein cavities. The simulations revealed the importance of two subunits of CIII (G and K in bovine heart) for the structural integrity of these internal CL binding sites. In addition, new binding sites are found on the membrane-exposed protein surface. The reproducibility of these binding sites over two species (bovine heart and yeast mitochondria) points to an important role for the function of the respiratory chain. Interestingly the membrane-exposed CL binding sites are located on the matrix side of CIII in the inner membrane and thus may provide localized sources of proton ready for uptake by CIII. Furthermore, we found that CLs bound to those membrane-exposed sites bridge the proteins during their assembly into supercomplexes by sharing the binding sites.


Embracing humanity in the face of death: why do existential concerns moderate ingroup humanization?
People humanize their ingroup to address existential concerns about their mortality, but the reasons why they do so remain ambiguous. One explanation is that people humanize their ingroup to bolster their social identity in the face of their mortality. Alternatively, people might be motivated to see their ingroup as more uniquely human (UH) to distance themselves from their corporeal "animal" nature. These explanations were tested in Australia, where social identity is tied less to UH and more to human nature (HN) which does not distinguish humans from animals. Australians attributed more HN traits to the ingroup when mortality was salient, while the attribution of UH traits remained unchanged. This indicates that the mortality-buffering function of ingroup humanization lies in reinforcing the humanness of our social identity, rather than just distancing ourselves from our animal nature. Implications for (de)humanization in intergroup relations are discussed.


Transient subversion of CD40 ligand function diminishes immune responses to adenovirus vectors in mouse liver and lung tissues.
First-generation adenovirus vectors will have limited application in gene therapy for chronic diseases because of destructive host immune responses. Important immune effectors include CD8+ T cells, which mediate target cell destruction and ablate transgene expression, and B cells, which produce neutralizing antibodies that block effective readministration of vector. Previous studies indicated that activation of CD4+ T cells by virus capsid proteins is necessary for full realization of effector function of CD8+ T cells and B cells. In this paper, we present a strategy for preventing CD4+ T-cell activation by an adenovirus vector delivered to mouse liver and lung tissues which is based on interfering with T-cell priming via CD40 ligand-CD40 interactions. Adenovirus transgene expression was stabilized in mice genetically deficient in CD40 ligand (CD40L), and neutralizing antibody to adenovirus did not develop, allowing efficient readministration of vector. A transient blockade of T-cell activation with an antibody to CD40L infused into the animal at the time of adenovirus vector-mediated gene transfer led to stabilization of transgene expression and diminished production of neutralizing antibody, allowing readministration of vector. In vitro T-cell assays suggested that a block in the primary activation of CD4+ T cells was responsible for the lack of B-cell- and cytotoxic-T-cell-dependent responses. This suggests a strategy for improving the potential of adenovirus vectors based on administration of an antibody to CD40L at the time of vector administration.


Euphorbia Diterpenes: An Update of Isolation, Structure, Pharmacological Activities and Structure-Activity Relationship.
Euphorbia species have a rich history of ethnomedicinal use and ethnopharmacological applications in drug discovery. This is due to the presence of a wide range of diterpenes exhibiting great structural diversity and pharmacological activities. As a result, Euphorbia diterpenes have remained the focus of drug discovery investigations from natural products. The current review documents over 350 diterpenes, isolated from Euphorbia species, their structures, classification, biosynthetic pathways, and their structure-activity relationships for the period covering 2013-2020. Among the isolated diterpenes, over 20 skeletal structures were identified. Lathyrane, jatrophane, ingenane, ingenol, and ingol were identified as the major diterpenes in most Euphorbia species. Most of the isolated diterpenes were evaluated for their cytotoxicity activities, multidrug resistance abilities, and inhibitory activities in vitro, and reported good activities with significant half-inhibitory concentration (IC50) values ranging from 10-50 µM. The lathyranes, isopimaranes, and jatrophanes diterpenes were further found to show potent inhibition of P-glycoprotein, which is known to confer drug resistance abilities in cells leading to decreased cytotoxic effects. Structure-activity relationship (SAR) studies revealed the significance of a free hydroxyl group at position C-3 in enhancing the anticancer and anti-inflammatory activities and the negative effect it has in position C-2. Esterification of this functionality, in selected diterpenes, was found to enhance these activities. Thus, Euphorbia diterpenes offer a valuable source of lead compounds that could be investigated further as potential candidates for drug discovery.