Physico-chemical requirements and kinetics of membrane fusion of flavivirus-like particles.
Flaviviruses deliver their RNA genome into the host-cell cytoplasm by fusing their lipid envelope with a cellular membrane. Expression of the flavivirus pre-membrane and envelope glycoprotein genes in the absence of other viral genes results in the spontaneous assembly and secretion of virus-like particles (VLPs) with membrane fusion activity. Here, we examined the physico-chemical requirements for membrane fusion of VLPs from West Nile and Japanese encephalitis viruses. In a bulk fusion assay, optimal hemifusion (or lipid mixing) efficiencies were observed at 37 °C. Fusion efficiency increased with decreasing pH; half-maximal hemifusion was attained at pH 5.6. The anionic lipids bis(monoacylglycero)phosphate and phosphatidylinositol-3-phosphate, when present in the target membrane, significantly enhanced fusion efficiency, consistent with the emerging model that flaviviruses fuse with intermediate-to-late endosomal compartments, where these lipids are most abundant. In a single-particle fusion assay, VLPs catalysed membrane hemifusion, tracked as lipid mixing with the cellular membrane, on a timescale of 7-20 s after acidification. Lipid mixing kinetics suggest that hemifusion is a kinetically complex, multistep process.


Fast automatic lens testing for extended-field image quality.
Monitoring of high-volume lens manufacture for Polaroid Land photography requires an acceptance criterion that is both relevant to picture quality and suitable for automatic testing. In a hardware test based upon the modulation transfer function (MTF), the problem is to choose what not to measure, while obtaining enough information about a lens to assign it a useful figure of merit. The predominant defects of manufacture are field tilt, resulting from tilting or decentering of components, and field curvature, from small errors in spacing, thickness, radius, or index. Field tilt and curvature are not local defects of the image surface, insofar as they may be removed by a local refocus. We therefore need a test procedure that can (perhaps simultaneously) examine image quality over an extended field, but is principally called upon to evaluate focus differences. At multiple field locations a single-frequency modulation measurement adequately defines the MTF for the low spatial frequencies of interest and can be interpreted easily in terms of the rms blur of the point spread function. We will discuss the logic of 100% lens acceptance testing and two types of instrumentation we have used.


Economic evaluation of folic acid food fortification in The Netherlands.
Folic acid intake before and during pregnancy reduces neural tube defects (NTD). Therefore, several countries have enriched bulk food with folic acid resulting in a 26-48% decrease in the prevalence of NTDs. In 2000, the Dutch Health Council advised against folic acid enrichment based on literature research; yet formal cost-effectiveness information was absent. We designed our study to estimate cost-effectiveness of folic acid food fortification in the Netherlands. Prevalence of NTD at birth, life-time costs of care, and folic acid fortification costs were estimated using Dutch registrations, Dutch guidelines for costing, (inter)national literature and expert opinions. Both net cost per discounted life year gained and net cost per discounted quality adjusted life year (QALY) gained were estimated for the base case and sensitivity analyses. In the base case and most sensitivity analyses, folic acid enrichment was estimated to be cost-saving. Bulk food fortification with folic acid remains cost-effective as long as enrichment costs do not exceed euro5.5 million (threshold at euro20 000 per QALY). Our model suggests that folic acid fortification of bulk food to prevent cases of NTD in newborns might be a cost-saving intervention in the Netherlands. Additionally, besides the evidence that folic acid reduces the number of NTDs, there are indications that folic acid is associated with the prevention of other birth defects, cardiovascular diseases and cancer. Our model did not yet include these possibly beneficial effects.


Epidemiology of sports-related fatalities during organized school sports in Japanese high schools between 2009 and 2018.
Limited literature has investigated epidemiology of sports-related fatalities during high school organizes sports in Japan. Therefore, the purposes of this study are to determine the frequency and incidence rate of sports-related fatalities in Japanese high schools by cause and sports, and to examine the type of on-site first responder. Insurance claim data of sports-related fatalities in Japanese high schools reported to Japan Sports Council Injury and Accident Mutual Aid Benefit System between 2009 and 2018 were retrieved as the primary data source. All fatalities were classified into direct or indirect type by the reported etiology and further categorized into cardiac-related, head and neck injury, exertional heat stroke (EHS), or other. Frequency and incidence rate were calculated by cause of death and sports, and incidence rates were expressed per 100,000 athlete-years (AY) with 95% confidence interval (CI). Information regarding first responder to the incident was also retrieved and examined by frequency. A total of 63 sports-related fatalities were analyzed. The overall incidence rate was 0.45 (95%CI = 0.25-0.65) per 100,000AY. The incidence rates of direct and indirect fatalities declined from 0.36 and 0.50 per 100,000AY to 0.28 and 0.00 per 100,000AY, respectively. The leading cause of deaths was cardiac-related (n = 30/63, 47.6%), followed by head and neck injury (n = 15/63, 23.8%) and EHS (n = 14/63, 22.2%). The number of fatalities was highest in male baseball (n = 12/63, 19.0%) and the incidence rate was highest in male judo (4.79 per 100,000 AY, 95%CI: 0.68-8.15). Coach was the most frequently reported first responder onsite (n = 52/63, 82.5%). Medically trained personnel were involved in onsite care in two cases (3.2%). In conclusion, the occurrence of sports-related fatalities has declined over time from 2009 to 2018. To deliver appropriate medical care onsite for better survival, employment of medically trained personnel should be promoted in high school sports setting in Japan.


[Experimental study of optimal dose interval in antibiotic therapy].
A postantibiotic effect (PAE) was demonstrated for various antibiotics which suppressed bacterial regrowth for at least two hours beyond the termination of a short drug exposure in vitro. Rifampin, tetracycline and erythromycin already exhibit a PAE at low concentrations. In contrast, in the case of beta-lactams high concentrations - higher than those obtained in serum during oral treatment - were needed to demonstrate a PAE of these antibiotics in vitro. The PAE phenomenon may be of clinical importance with regard to dosage regimens in antimicrobial chemotherapy.


Clinical significance of monitoring circulating CD4+CD25+ regulatory T cells in kidney transplantation during the early posttransplant period.
The CD4(+)CD25(+) T regulatory cells (Tregs) play an important role in immune tolerance in experimental transplantation but the clinical significance of circulating Tregs in the peripheral blood is undetermined. In 50 kidney transplant (KT) recipients, 29 healthy controls and 32 liver transplant (LT) recipients, the frequency of Tregs was measured with flow cytometry before and after transplantation. In the KT recipients, IL-10 secretion was measured with an enzyme-linked immunospot (ELISPOT) assay. The median frequency of circulating Tregs before KT was similar to that in healthy controls but significantly lower than that in LT patients before transplantation. The frequency of Tregs was significantly decreased in patients with subclinical acute rejection compared with those without subclinical acute rejection. Calcineurin inhibitors (CNIs) and anti-CD25 antibody decreased the frequency of Tregs but mTOR inhibitor did not. The frequency of donor-specific IL-10 secreting cells did not correlate with the number of Tregs. The frequency of circulating Tregs in KT recipients is strongly affected by CNIs and anti-CD25 antibody, and a low frequency of Tregs is associated with subclinical acute rejection during the early posttransplant period.


A monoterpene, unique component of thyme honeys, induces apoptosis in prostate cancer cells via inhibition of NF-κB activity and IL-6 secretion.
We have previously demonstrated that Greek thyme honey inhibits significantly the cell viability of human prostate cancer cells. Herein, 15 thyme honey samples from several regions of Greece were submitted to phytochemical analysis for the isolation, identification and determination (through modern spectral means) of the unique thyme honey monoterpene, the compound trihydroxy ketone E-4-(1,2,4-trihydroxy-2,6,6-trimethylcyclohexyl)-but-3-en-2-one. We investigated the anti-growth and apoptotic effects of the trihydroxy ketone on PC-3 human androgen independent prostate cancer cells using MTT assay and Annexin V-FITC respectively. The molecular pathways involved to such effects were further examined by evaluating its ability to inhibit (a) the NF-κB phosphorylation (S536), (b) JNK and Akt phosphorylation (Thr183/Tyr185 and S473 respectively) and (c) IL-6 production, using ELISA method. The anti-microbial effects of the trihydroxy ketone against a panel of nine pathogenic bacteria and three fungi were also assessed. The trihydroxy ketone exerted significant apoptotic activity in PC-3 prostate cancer cells at 100 μM, while it inhibited NF-κB phosphorylation and IL-6 secretion at a concentration range 10(-6)-10(-4)M. Akt and JNK signaling were not found to participate in this process. The trihydroxy ketone exerted significant anti-microbial profile against many human pathogenic bacteria and fungi (MIC values ranged from 0.04 to 0.57 mg/ml). Conclusively, the Greek thyme honey-derived monoterpene exerted significant apoptotic activity in PC-3 cells, mediated, at least in part, through reduction of NF-κB activity and IL-6 secretion and may play a key role in the anti-growth effect of thyme honey on prostate cancer cells.


Dynamics of the anaerobic process: effects of volatile fatty acids.
A complex and fast dynamic response of the anaerobic biogas system was observed when the system was subjected to pulses of volatile fatty acids (VFAs). It was shown that a pulse of specific VFAs into a well-functioning continuous stirred tank reactor (CSTR) system operating on cow manure affected both CH(4) yield, pH, and gas production and that a unique reaction pattern was seen for the higher VFAs as a result of these pulses. In this study, two thermophilic laboratory reactors were equipped with a novel VFA-sensor for monitoring specific VFAs online. Pulses of VFAs were shown to have a positive effect on process yield and the levels of all VFA were shown to stabilize at a lower level after the biomass had been subjected to several pulses. The response to pulses of propionate or acetate was different from the response to butyrate, iso-butyrate, valerate, or iso-valerate. High concentrations of propionate affected the degradation of all VFAs, while a pulse of acetate affected primarily the degradation of iso-valerate or 2-methylbutyrate. Pulses of n-butyrate, iso-butyrate, and iso-valerate yielded only acetate, while degradation of n-valerate gave both propionate and acetate. Product sensitivity or inhibition was shown for the degradation of all VFAs tested. Based on the results, it was concluded that measurements of all specific VFAs are important for control purposes and increase and decrease in a specific VFA should always be evaluated in close relationship to the conversion of other VFAs and the history of the reactor process. It should be pointed out that the observed dynamics of VFA responses were based on hourly measurements, meaning that the response duration was much lower than the hydraulic retention time, which exceeds several days in anaerobic CSTR systems.


High-throughput transcriptome sequencing reveals extremely high doses of ionizing radiation-response genes in Caenorhabditis elegans.
This study sought novel ionizing radiation-response (IR-response) genes in Caenorhabditis elegans (C. elegans). C. elegans was divided into three groups and exposed to different high doses of IR: 0 gray (Gy), 200 Gy, and 400 Gy. Total RNA was extracted from each group and sequenced. When the transcriptomes were compared among these groups, many genes were shown to be differentially expressed, and these genes were significantly enriched in IR-related biological processes and pathways, including gene ontology (GO) terms related to cellular behaviours, cellular growth and purine metabolism and kyoto encyclopedia of genes and genomes (KEGG) pathways related to ATP binding, GTPase regulator activity, and RNA degradation. Quantitative reverse-transcription PCR (qRT-PCR) confirmed that these genes displayed differential expression across the treatments. Further gene network analysis showed a cluster of novel gene families, such as the guanylate cyclase (GCY), Sm-like protein (LSM), diacylglycerol kinase (DGK), skp1-related protein (SKR), and glutathione S-transferase (GST) gene families which were upregulated. Thus, these genes likely play important roles in IR response. Meanwhile, some important genes that are well known to be involved in key signalling pathways, such as phosphoinositide-specific phospholipase C-3 (PLC-3), phosphatidylinositol 3-kinase age-1 (AGE-1), Raf homolog serine/threonine-protein kinase (LIN-45) and protein cbp-1 (CBP-1), also showed differential expression during IR response, suggesting that IR response might perturb these key signalling pathways. Our study revealed a series of novel IR-response genes in Caenorhabditis elegans that might act as regulators of IR response and represent promising markers of IR exposure.


A case of administrative conflict: observations on the dynamic framework.
A situation is described of suddenly erupting conflict in an interdisciplinary meeting of psychiatric hospital staff. The conflict is related to the systemic impact of recent changes in psychiatric hospital practice. The observed affect and interpersonal dynamics are reviewed in the context of different conceptual frameworks. These include Maslow's hierarchy of needs model, the group focal conflict paradigm and the concept of scapegoating. The use and integration of these three approaches toward understanding affectively laden administrative conflict are discussed. Specific reference is made to the utilization of such understanding in developing leadership skills in a multi-disciplinary mental health setting.


Clinical presentations and diagnostic issues in dual diagnosis disorders.
The co-occurrence of mental disorders and substance use disorders (SUDs) is very common, and associated with substantial psychiatric morbidity, functional and quality of life impairments, and societal costs. However, dual disorders are often underdetected, misdiagnosed and inadequately treated in both substance abuse and mental health settings. Individuals with dual or multiple disorders generally have worse clinical features and long-term outcomes than those with single disorders. However, findings can vary depending on factors such as type(s) of psychiatric disorder(s) and substance(s) involved, and whether the mental disorder is primary or secondary, or substance-induced or independent. Underdiagnosis or misdiagnosis of dual disorders may occur due, in part, to the use of measurement instruments and diagnostic approaches that have uncertain clinical utility. Routine, thorough, and integrated screening and diagnosis of dual disorders are needed to facilitate implementation of appropriate treatment. Results suggest that forthcoming revisions to the Diagnostic and Statistical Manual of Mental Disorders and International Classification of Diseases should categorize and define mental and SUDs such that clinicians can more readily detect and diagnose all types of substance use and mental health comorbid conditions. Adoption and widespread use of appropriate screening, assessment and diagnostic instruments, and more thorough diagnostic (clinimetric) approaches are recommended.


Size effect of mesoporous organosilica nanoparticles on tumor penetration and accumulation.
The size effect of mesoporous organosilica nanoparticles (MONs) on tumor penetration and accumulation remains poorly understood, which strongly affects the tumor therapeutic efficacy. Herein, four different-sized thioether-bridged MONs (20, 40, 60, and 100 nm) are synthesized; all the MONs have a spherical morphology, excellent dispersity, similar surface charge, uniform mesopores (3.2-3.5 nm), and large surface areas (709-1353 m2·g-1). Hematology and histopathology analyses demonstrate that the four MONs do not cause pathological changes in mice even at a dose of 20 mg kg-1 for 30 d. The penetration depth of the MONs for multicellular spheroids (MCSs) decreases with increasing particle sizes, and the 20 nm MONs are uniformly distributed in the MCSs at a depth of 60 μm, while the larger MONs are mainly restricted to peripheral areas. In vivo experiments show that the 40 nm MONs possess the longest mean residence times, leading to their highest accumulation in blood and tumors. However, the 20 nm MONs reach the deepest penetration depth of 1230 μm for xenograft tumors. In contrast, the penetration depths of 40, 60, and 100 nm MONs are 783, 105, and 40 μm, respectively. Overall, this work provides an important guideline for the rational design of nanoplatforms for tumor treatment.


Carbon-monoxide-releasing molecules for the delivery of therapeutic CO in vivo.
The development of carbon-monoxide-releasing molecules (CORMs) as pharmaceutical agents represents an attractive and safer alternative to administration of gaseous CO. Most CORMs developed to date are transition-metal carbonyl complexes. Although such CORMs have showed promising results in the treatment of a number of animal models of disease, they still lack the necessary attributes for clinical development. Described in this Minireview are the methods used for CORM selection, to date, and how new insights into the reactivity of metal-carbonyl complexes in vivo, together with advances in methods for live-cell CO detection, are driving the design and synthesis of new CORMs, CORMs that will enable controlled CO release in vivo in a spatial and temporal manner without affecting oxygen transport by hemoglobin.


Deviant expression of Rab5 on phagosomes containing the intracellular pathogens Mycobacterium tuberculosis and Legionella pneumophila is associated with altered phagosomal fate.
The intracellular human pathogens Legionella pneumophila and Mycobacterium tuberculosis reside in altered phagosomes that do not fuse with lysosomes and are only mildly acidified. The L. pneumophila phagosome exists completely outside the endolysosomal pathway, and the M. tuberculosis phagosome displays a maturational arrest at an early endosomal stage along this pathway. Rab5 plays a critical role in regulating membrane trafficking involving endosomes and phagosomes. To determine whether an alteration in the function or delivery of Rab5 could play a role in the aberrant development of L. pneumophila and M. tuberculosis phagosomes, we have examined the distribution of the small GTPase, Rab5c, in infected HeLa cells overexpressing Rab5c. Both pathogens formed phagosomes in HeLa cells with molecular characteristics similar to their phagosomes in human macrophages and multiplied in these host cells. Phagosomes containing virulent wild-type L. pneumophila never acquired immunogold staining for Rab5c, whereas phagosomes containing an avirulent mutant L. pneumophila (which ultimately fused with lysosomes) transiently acquired staining for Rab5c after phagocytosis. In contrast, M. tuberculosis phagosomes exhibited abundant staining for Rab5c throughout its life cycle. To verify that the overexpressed, recombinant Rab5c observed on the bacterial phagosomes was biologically active, we examined the phagosomes in HeLa cells expressing Rab5c Q79L, a fusion-promoting mutant. Such HeLa cells formed giant vacuoles, and after incubation with various particles, the giant vacuoles acquired large numbers of latex beads, M. tuberculosis, and avirulent L. pneumophila but not wild-type L. pneumophila, which consistently remained in tight phagosomes that did not fuse with the giant vacuoles. These results indicate that whereas Rab5 is absent from wild-type L. pneumophila phagosomes, functional Rab5 persists on M. tuberculosis phagosomes. The absence of Rab5 on the L. pneumophila phagosome may underlie its lack of interaction with endocytic compartments. The persistence of functional Rab5 on the M. tuberculosis phagosomes may enable the phagosome to retard its own maturation at an early endosomal stage.


Varying Dosages of Subcutaneous Unfractionated Heparin and Activated Partial Thromboplastin Time in Hospitalized Antepartum Patients: A Retrospective Cohort Analysis.
Venous thromboembolism (VTE) is a leading cause of maternal morbidity and mortality in the United States. Subcutanous unfractionated heparin (UFH) has been used for decades for VTE prophylaxis and under many obstetric quality of care initiatives, hospitalized antepartum patients now receive doses as high as 10,000 units every 12 hours. This practice increases the likelihood of UFH administration around the time that epidural labor analgesia is requested or neuraxial analgesia for cesarean delivery is needed. To clarify the effect of UFH on coagulation, we reviewed the care of hospitalized antepartum patients receiving VTE prophylaxis with UFH to determine the incidence of concurrent abnormal activated partial thromboplastin time (aPTT) values and associated risk factors. This retrospective cohort study used data from the University of Chicago Pharmacy database to identify hospitalized antepartum patients receiving subcutaneous UFH from June 1, 2016 to July 1, 2019. Our institutional protocol states that all patients hospitalized for antepartum conditions should receive pharmacologic prophylaxis empirically unless contraindicated. For patients receiving UFH, dosing was based on gestational age: 5000 units every 12 hours for first trimester antepartum patients, 7500 units every 12 hours for second trimester patients, and 10,000 units every 12 hours for patients in the third trimester. As per protocol, aPTT values were obtained 2 hours after the third dose of heparin, and platelet counts after 4 days. Data collection included demographics, comorbidities, heparin doses, aPTT values, platelet counts, creatinine if available, and anesthetic type and complications. Logistic regression was performed to determine the association between elevated aPTT >40 seconds and study variables. Of the 321 antepartum patients who received subcutaneous UFH, 33 (10.3%) had an aPTT >40 seconds, 4 of those 33 patients (12.1%) received 5000 units every 12 hours, 14 (42.2%) received 7500 units every 12 hours, and 15 (45.5%) received 10,000 units every 12 hours. The likelihood of a patient having aPTT >40 seconds was 2.8% with 5000 units every 12 hours, 18.9% with 7500 units every 12 hours, and 14.6% with 10,000 units every 12 hours. Elevated aPTT values are likely with total daily doses of 15,000 or 20,000 units subcutaneous UFH in hospitalized antepartum patients.


Psychotropic medication-induced rabbit syndrome.
Rabbit syndrome (RS) is an involuntary movement disorder characterized by rapid, fine movements of an individual's mouth, similar to the chewing movements of a rabbit, and has most frequently been associated with the use of antipsychotic medications. RS is often unrecognized or misdiagnosed as tardive dyskinesia or pseudoparkinsonism. Although rare, RS is easily treatable if recognized. It is essential that nurses are able to distinguish this syndrome from other movement disorders; however, a lack of information exists in the nursing literature about this syndrome. The aims of this article are to describe the clinical symptoms of RS, its prevalence and etiology, and recommended treatment. Clinical and education implications regarding RS are also provided.


Comparison of whole-blood metal ion levels in four types of metal-on-metal large-diameter femoral head total hip arthroplasty: the potential influence of the adapter sleeve.
Metal-on-metal bearings, as used in total hip arthroplasty prostheses that have a large-diameter femoral head, were proposed as an option for treating young and active patients with degenerative hip disorders. Despite the theoretical improved performance of large metal articulations with regard to wear, metal ion levels produced by total hip arthroplasty prostheses in which a large-diameter femoral head is used have not been thoroughly evaluated. From 173 eligible patients, 144 patients were allocated to undergo a unilateral total hip arthroplasty with use of metal-on-metal components and a large-diameter femoral head. The purpose of this study was to compare the amount of metal ion release (chromium, cobalt, and titanium) from four different types of prostheses from four different implant manufacturers (Biomet, DePuy, Smith & Nephew, and Zimmer). For cobalt ion levels, a significant difference was found between the different types of total hip arthroplasty prostheses with a large-diameter femoral head at three, six, twelve, and twenty-four months, but only in male patients or patients with a femoral head size of 50 mm or greater. The highest mean cobalt levels at all follow-up periods were observed with the Zimmer implant and the lowest with the Biomet implant. Titanium ion levels were highest in the Zimmer group at all follow-up periods, whereas no difference between groups was found for chromium. At the most recent follow-up, one hip was revised after the development of an adverse local tissue reaction. Of concern was the observation at the time of revision surgery of black metallic deposits inside the adapter sleeve and on the prosthetic femoral neck. This investigation revealed that metal ion release differs greatly between various total hip arthroplasty implants with a large-diameter femoral head. The sources of metal ion production are numerous, but it is hypothesized that wear and corrosion at the junction between the adapter sleeve and the femoral stem in some patients may be responsible for the elevated cobalt ion levels that were found in the Zimmer group. On the other hand, an adapter sleeve made of titanium, such as the one used with the Biomet large-diameter-head total hip arthroplasty, is an unlikely contributor to the release of cobalt ions. Current technology or design of some total hip arthroplasty systems that make use of a large-diameter femoral head may not yet allow the use of modular large heads with a metal-on-metal articulation, especially in young, active male patients whose activities generate high loads at the hip joint. Further research is needed to better understand the favorable design characteristics of modular junctions in metal-on-metal total hip arthroplasty implants that make use of large-diameter femoral heads.


ATF4-dependent transcription is a key mechanism in VEGF up-regulation by oxidized phospholipids: critical role of oxidized sn-2 residues in activation of unfolded protein response.
We have shown previously that oxidized phospholipids (OxPLs), known to accumulate in atherosclerotic vessels, stimulate angiogenesis via induction of autocrine mediators, such as vascular endothelial growth factor (VEGF). We now address the pathways mediating up-regulation of VEGF in human endothelial cells treated with OxPLs. Analysis of structure-function relationship using individual species of OxPLs demonstrated a close relation between induction of VEGF and activation of the unfolded protein response (UPR). Inducers of UPR up-regulated VEGF, whereas inhibition of UPR by chemical chaperones or knock-down of cochaperone HTJ-1 inhibited elevation of VEGF mRNA induced by OxPLs. OxPLs induced protein expression of activating transcription factor-4 (ATF4), an important effector of UPR. Expression levels of VEGF in OxPL-treated cells strongly correlated with induction of the ATF4 target genes ATF3 and TRB3. Knocking down ATF4 was paralleled by loss of VEGF induction by OxPLs. Chromatin immunoprecipitation demonstrated that OxPLs stimulated binding of ATF4 to a regulatory site in the VEGFA gene. Taken together, these data characterize UPR and more specifically its ATF4 branch as an important mechanism mediating up-regulation of VEGF by OxPLs, and allow hypothesizing that the UPR cascade might play a role in pathologic angiogenesis in atherosclerotic plaques.


[Genetic effects of destabilizing selection for adaptively important traits in Drosophila melanogaster lines].
Related lines of Drosophila melanogaster selected for reproductive activity for more than 750 generations were studied. Results of the long-term selection experiment support D.K. Belyaev's concept of destabilizing selection. Selection for a behavioral trait (male mating activity) affected the intrinsic structure of the organism. Flies of the low-activity line (LA) exhibited numerous morphological, biochemical, and physiological alterations. Variation rate in the selected lines was accelerated; appearing mutations were often allelic and nonrandomly distributed over chromosomes. Selection was shown to mediate novel sources of variability, e.g., induction and repression of the hobo hybrid dysgenesis. In the process of selection, a genetically determined program of hobo transpositions in the genome was formed.


An immunocytochemical method for localization of estrogen receptors in rat tissues using a dinitrophenyl (DNP)-labeled rat monoclonal primary antibody.
We have developed an immunocytochemical method to demonstrate estrogen receptor in hormone-sensitive tissues of the rat using a dinitrophenyl (DNP) hapten-labeled rat antihuman estrogen receptor monoclonal antibody (MAb), H222. Mouse IgM anti-DNP was used secondarily, followed by a DNP/peroxidase conjugate, diaminobenzidine/hydrogen peroxide chromogen, and silver intensification. This method was applied to tissues from intact female rats and showed that estrogen receptor was localized in the nuclei of the stromal and glandular components of the uterine endometrium. Reduced receptor staining was observed in the luminal epithelium, with minimal myometrial staining. Anterior pituitary glands showed heterogeneous immunostaining and ovaries expressed the receptor predominantly in the interstitial cells; fallopian tubes demonstrated substantial epithelial staining. Uteri from chemically castrated rats showed reduced estrogen receptor immunostaining in both stromal and luminal cells, whereas staining was enhanced in the glandular elements. Classical estrogen-unresponsive tissues (heart, lung, and spleen) were unstained. Antibody controls involved pre-blocking antibody recognition sites on the receptor with unlabeled antibodies to estrogen receptor (H222, H226, and D547), as well as use of an inappropriate DNP-labeled antibody to metallothionein. These controls illustrated the specific nature of the DNP-H222 binding.


Estimating vaccine efficacy using auxiliary outcome data and a small validation sample.
In vaccine studies, a specific diagnosis of a suspected case by culture or serology of the infectious agent is expensive and difficult. Implementing validation sets in the study is less expensive and is easier to carry out. In studies using validation sets, the non-specific or auxiliary outcome is measured on each participant while the specific outcome is measured only for a small proportion of the participants. Vaccine efficacy, defined as one minus some measure of relative risk, could be severely attenuated if based only on the auxiliary outcome. Applying missing data analysis techniques could thus correct the bias while maintaining statistical efficiency. However, when the sample size in the validation sets is small and the vaccine is highly efficacious, all specific outcomes are likely to be negative in the validation set in the vaccinated group. Two commonly used missing data analysis methods, the mean score method and multiple imputation, depend on the ad hoc continuity correction when none of the specific outcomes are positive and the normality or log-normality assumption of relative risk, which may not hold when the relative risk is highly skewed, to estimate the confidence interval. In this paper, we propose a Bayesian method to estimate vaccine efficacy and its highest probability density (HPD) credible set using Monte Carlo (MC) methods when using auxiliary outcome data and a small validation sample. Comparing the performance of these approaches using data from a field study of influenza vaccine and simulations, we recommend to use the Bayesian method in this situation.


Rates and drivers of mangrove deforestation in Southeast Asia, 2000-2012.
The mangrove forests of Southeast Asia are highly biodiverse and provide multiple ecosystem services upon which millions of people depend. Mangroves enhance fisheries and coastal protection, and store among the highest densities of carbon of any ecosystem globally. Mangrove forests have experienced extensive deforestation owing to global demand for commodities, and previous studies have identified the expansion of aquaculture as largely responsible. The proportional conversion of mangroves to different land use types has not been systematically quantified across Southeast Asia, however, particularly in recent years. In this study we apply a combined geographic information system and remote sensing method to quantify the key proximate drivers (i.e., replacement land uses) of mangrove deforestation in Southeast Asia between 2000 and 2012. Mangrove forests were lost at an average rate of 0.18% per year, which is lower than previously published estimates. In total, more than 100,000 ha of mangroves were removed during the study period, with aquaculture accounting for 30% of this total forest change. The rapid expansion of rice agriculture in Myanmar, and the sustained conversion of mangroves to oil palm plantations in Malaysia and Indonesia, are identified as additional increasing and under-recognized threats to mangrove ecosystems. Our study highlights frontiers of mangrove deforestation in the border states of Myanmar, on Borneo, and in Indonesian Papua. To implement policies that conserve mangrove forests across Southeast Asia, it is essential to consider the national and subnational variation in the land uses that follow deforestation.


A novel role for protein farnesylation in plant innate immunity.
Plants utilize tightly regulated mechanisms to defend themselves against pathogens. Initial recognition results in activation of specific Resistance (R) proteins that trigger downstream immune responses, in which the signaling networks remain largely unknown. A point mutation in SUPPRESSOR OF NPR1 CONSTITUTIVE1 (SNC1), a RESISTANCE TO PERONOSPORA PARASITICA4 R gene homolog, renders plants constitutively resistant to virulent pathogens. Genetic suppressors of snc1 may carry mutations in genes encoding novel signaling components downstream of activated R proteins. One such suppressor was identified as a novel loss-of-function allele of ENHANCED RESPONSE TO ABSCISIC ACID1 (ERA1), which encodes the beta-subunit of protein farnesyltransferase. Protein farnesylation involves attachment of C15-prenyl residues to the carboxyl termini of specific target proteins. Mutant era1 plants display enhanced susceptibility to virulent bacterial and oomycete pathogens, implying a role for farnesylation in basal defense. In addition to its role in snc1-mediated resistance, era1 affects several other R-protein-mediated resistance responses against bacteria and oomycetes. ERA1 acts partly independent of abscisic acid and additively with the resistance regulator NON-EXPRESSOR OF PR GENES1 in the signaling network. Defects in geranylgeranyl transferase I, a protein modification similar to farnesylation, do not affect resistance responses, indicating that farnesylation is most likely specifically required in plant defense signaling. Taken together, we present a novel role for farnesyltransferase in plant-pathogen interactions, suggesting the importance of protein farnesylation, which contributes to the specificity and efficacy of signal transduction events.


High Glucose-Induced TRPC6 Channel Activation Decreases Glutamate Uptake in Rat Retinal Müller Cells.
High glucose (HG) increases the production of reactive oxygen species (ROS), leading to decreased glutamate uptake in Müller cells. The transient receptor potential cation channel 6 (TRPC6) channel, an oxidative stress-sensitive Ca2+-permeable cationic channel, is readily detected in Müller cells and highly expressed under HG conditions. Yet, the effect of high glucose-induced TRPC6 channel activation in Müller cells is poorly understood. We hypothesized that TRPC6 channel activation mediates high glucose-induced decreases in Müller cell glutamate uptake. We found RNA interference (RNAi) of the TRPC6 channel abolished HG-induced decreases in glutamate uptake and cell death. HG also decreased the expression of the glutamate-aspartate transporter (GLAST), which is the most important transporter involved in glutamate uptake. The mRNA level of ciliary neurotrophic factor (CNTF) in rMC-1 cells and the release of CNTF in the culture media was decreased, but the mRNA levels of IL-6 and vascular endothelial growth factor (VEGF) were increased under HG conditions. After RNAi silencing in rMC-1 cells, the mRNA levels of CNTF increased, but IL-6 and VEGF levels decreased. Furthermore, TRPC6 knockdown (KD) decreased expression of glial fibrillary acidic protein (GFAP) and increased expression of Kir4.1, pointing to inhibition of HG-induced gliosis in rMC-1 cells. ROS and intracellular Ca2+ levels decreased after TRPC6 knockdown. Exposure to Hyp9 (10 μM), a highly selective TRPC6 channel agonist, can aggravate HG-induced pathological changes. Collectively, our results suggest TRPC6 channel activation is involved in HG-induced decreases in glutamate uptake in rMC-1 cells. These findings provide novel insights into the role of TRPC6 in HG-induced retinal neurovasculopathy and suggest TRPC6 is a promising target for drug development for diabetic retinopathy (DR).


Chronic Treatment with a Phytosomal Preparation Containing Centella asiatica L. and Curcuma longa L. Affects Local Protein Synthesis by Modulating the BDNF-mTOR-S6 Pathway.
Brain derived neurotrophic factor (Bdnf) is the most diffuse neurotrophin in the central nervous system and it is crucial for the proper brain development and maintenance. Indeed, through the binding to its high affinity receptor TRKB and the activation of different intracellular cascades, it boosts cell survival, neurite growth and spine maturations mechanisms. Here, we evaluated if the chronic oral treatment for 10 days with a phytosomal preparation containing Centella asiatica L. and Curcuma longa L. could improve Bdnf levels in the prefrontal cortex of adult rats. Interestingly we found an increased expression of Bdnf with main effect of the treatment on the mTOR-S6 downstream signaling pathway. Accordingly, we found an increase in the expression of eukaryotic elongation factor (eEF2) with a shift towards the phosphorylated form thus increasing the transcription of Oligophrenin-1, a protein carrying the upstream Open Reading Frame (uORF) which reduction is paralleled by memory dysfunctions. These results show the ability of the phytosome to enhance mTOR-S6 regulated transcription and suggest the possibility to use this preparation in subjects with impairments in neuroplastic mechanisms, memory and cognitive abilities.


[Activity of cell enzymes during treatment of bronchial asthma patients with euphylline and hydrocortisone].
Two groups of patients with bronchial asthma (of average severity and a severe course) and a group of healthy persons were examined. Changes in dehydrogenase activity in the time course of asthma development associated with the depletion of the adaptation mechanisms, and the compensation of these disorders as a result of glucocorticosteroid therapy were established. AP activity in neutrophils could serve as an informative sign indicative of asthma progression and showing the efficacy of hormonotherapy. Various shifts in the metabolic activity of enzymes ensuring the efficacy of phagocytosis with relation to a degree of asthmatic symptoms and therapeutic modalities were revealed.


Specific inhibition on PAI-1 reduces the dose of Alteplase for ischemic stroke treatment.
Administration of recombinant tPA (rtPA, or a trade name Alteplase®) is an FDA-approved therapy for acute ischemic stroke (AIS), but often poses the risk of hemorrhagic complications. Recombinant tPA can be rapidly inactivated by the endogenous inhibitor, plasminogen activator inhibitor 1 (PAI-1). In this work, we study a novel treatment approach that combines a PAI-1 inhibitor, PAItrap4, with a reduced dose of rtPA to address the hemorrhagic concern of rtPA. PAItrap4 is a highly specific and very potent protein-based inhibitor of PAI-1, comprising a mutant variant of uPA serine protease domain, human serum albumin, and cyclic RGD peptide. PAItrap4 efficiently targets and inhibits PAI-1 on activated platelets, while also possessing a long half-life in vivo. Our results demonstrate that PAItrap4 effectively counteracts the inhibitory effects of PAI-1 on rtPA, thus preserving rtPA activity based on amidolytic and clot lysis assays. In an in vivo murine stroke model, PAItrap4, together with low-dose rtPA, enhances the blood perfusion in the stroke-affected areas, reduces infarct size, and promotes neurological recovery in mice. Importantly, such treatment does not increase the amount of cerebral hemorrhage, thus reducing the risk of cerebral hemorrhage. In addition, PAItrap4 does not compromise the normal blood coagulation function in mice, demonstrating its safety as a therapeutic agent. These findings highlight this combination therapy as a promising alternative for the treatment of ischemic stroke, offering improved safety and efficacy.


Catalase activity of liver and kidney in frogs with spontaneous renal carcinoma.
The kidney carcinoma of the leopard frog has served for various studies on catalase activity, as a first step in gaining information on enzymatic properties of neoplasms in cold blooded animals. It was found that the activity level of this tumor is reduced to approximately 13 per cent of that of the normal frog's kidney. Systemic effects of the tumor on catalase activity of liver and kidney are evident. Liver catalase in tumor-bearing frogs is diminished to about 50 per cent of the normal, and kidney catalase to an even greater degree; i.e., to about 34 per cent. A positive correlation exists between levels of catalase in tumor and in liver. Frogs kept at 3 different temperatures, 8, 18, and 26.5 degrees C., for upward of 29 days exhibited no significant change in activity levels either of tumor or of livers from normal or from tumor-bearing animals. It is suggested that a seasonal variation may occur in catalase activity of the frog. During the winter months catalase activity of the tumors was found to be higher than during the summer, whereas liver catalase was below the level of normal frogs examined during the summer. Intracoelomic injections of homogenates of tumors promptly lead to diminution of liver catalase lasting for several days. Injection of normal kidney has no such effect. These results with a spontaneous tumor of a cold blooded animal are in essential agreement with the many observations made with transplanted tumors of warm blooded animals. They lend support to the view that neoplasia is a ubiquitous biological process with similar characteristics in all species or classes of vertebrates.


Trend of incidence rate of age-related diseases: results from the National Health Insurance Service-National Sample Cohort (NHIS-NSC) database in Korea: a cross- sectional study.
This study aimed to identify and select age-related diseases (ARDs) in Korea, which is about to have a super-aged society, and to elucidate patterns in their incidence rates. The National Health Insurance Service-National Sample Cohort, comprising 1 million health insurance and medical benefit beneficiaries in Korea from 2002 to 2019, was utilized. We selected 14 diseases with high disease burden and prevalence among Koreans from the 92 diseases defined in the Global Burden of Diseases, Injuries, and Risk Factors Study as ARDs. The annual incidence rate represented the number of patients newly diagnosed with an ARD each year from 2006 to 2019, excluding those with a history of ARD diagnosis from 2002 to 2005. The incidence rate by age was categorized into 10-year units based on age as of 2019. The number of patients with ARDs in each age group was used as the numerator, and the incidence rate for each age group was calculated with the age group as the denominator. Regarding the annual incidence rates of ARDs from 2006 to 2019, chronic obstructive pulmonary disease, congestive heart failure, and ischemic heart disease decreased annually, whereas dyslipidemia, chronic kidney disease, cataracts, hearing loss, and Parkinson's disease showed a significant increase. Hypertension, diabetes, cerebrovascular disease, osteoporosis, osteoarthritis, and age-related macular degeneration initially displayed a gradual decrease in incidence but exhibited a tendency to increase after 2015. Concerning age-specific incidence rates of ARDs, two types of curves emerged. The first type, characterized by an exponential increase with age, was exemplified by congestive heart failure. The second type, marked by an exponential increase peaking between ages 60 and 80, followed by stability or decrease, was observed in 13 ARDs, excluding congestive heart failure. However, hypertension, ischemic heart disease, cerebrovascular disease, chronic obstructive pulmonary disease, and hearing loss in men belonged to the first type. From an epidemiological perspective, there are similar characteristics in age-specific ARDs that increase with age, reaching a peak followed by a plateau or decrease in Koreans.


To Evaluate and Compare the Effect of Various Surface Treatment Modalities on Shear Bond Strength of Composite to Polyetherketoneketone and SEM Analysis: An In vitro Study.
It is a challenge to bond resin materials with polyetherketoneketone (PEKK). To increase the bond strength, surface treatments using chemical adhesion, mechanical adhesion, or a combination of both can be used. The objective of the present study was to evaluate and compare the shear bond strength of PEKK to the composite resin after various surface treatments and to evaluate the fracture mode analyses. One hundred and twenty PEKK specimens were randomly divided into four groups (n = 30) after three different surface treatments (95% sulfuric acid etching, airborne abrasion with 110 μm aluminum-oxide, and 99% acetone). With the help of polytetrafluorethylene tube, resin composite (3M ESPE) was bonded on all the specimens, thermocycled, and subjected to shear bond strength testing. Thereafter, 15 samples from each group were assessed for fracture mode analyses using a scanning electron microscope. Mean, Standard deviation, one-way analysis of variance (ANOVA) f-test, post hoc Tukey's test. Statistical analysis using one-way ANOVA f-test revealed that the results were significant with a P < 0.05 with the maximum value obtained in the case of air abraded group and the minimum value obtained in the case of the untreated group. Adhesive failure mode was the most common among the air-abraded group. The mechanical surface treatment group (air abrasion) showed higher shear bond strength than the chemical surface treatment groups (sulfuric acid and acetone). The mixed-type fracture mode was most commonly noted in the air-abraded group.


[New insights into the role of macrophages in tumor immunotherapy].
Macrophages are the main components of the innate immunity system, derived mainly from blood monocytes, and help the host to defend itself against many pathogens and cancers. Most established tumors can educate macrophages into tumor-associated macrophages (TAMs), which contribute to tumor growth, invasion and metastasis, as well as resistance to chemotherapeutic agents and immune checkpoint inhibitors. However, when appropriately activated, macrophages can also exert anti-tumor effects through enhanced phagocytosis and cytotoxicity against tumor cells. In addition, TAMs are associated with poor prognosis and drug resistance, including immunotherapies, suggesting that macrophages are attractive targets as part of combination therapy in cancer treatment. Herein, we review the recent findings on the role of macrophages in tumor development, metastasis and immunotherapy. We focus mainly on macrophage-centered therapy, including strategies to reduce and reshape TAMs, to represent potential targets for tumor immunotherapy.


What can we learn about acid-base transporters in cancer from studying somatic mutations in their genes?
Acidosis is a chemical signature of the tumour microenvironment that challenges intracellular pH homeostasis. The orchestrated activity of acid-base transporters of the solute-linked carrier (SLC) family is critical for removing the end-products of fermentative metabolism (lactate/H+) and maintaining a favourably alkaline cytoplasm. Given the critical role of pH homeostasis in enabling cellular activities, mutations in relevant SLC genes may impact the oncogenic process, emerging as negatively or positively selected, or as driver or passenger mutations. To address this, we performed a pan-cancer analysis of The Cancer Genome Atlas simple nucleotide variation data for acid/base-transporting SLCs (ABT-SLCs). Somatic mutation patterns of monocarboxylate transporters (MCTs) were consistent with their proposed essentiality in facilitating lactate/H+ efflux. Among all cancers, tumours of uterine corpus endometrial cancer carried more ABT-SLC somatic mutations than expected from median tumour mutation burden. Among these, somatic mutations in SLC4A3 had features consistent with meaningful consequences on cellular fitness. Definitive evidence for ABT-SLCs as 'cancer essential' or 'driver genes' will have to consider microenvironmental context in genomic sequencing because bulk approaches are insensitive to pH heterogeneity within tumours. Moreover, genomic analyses must be validated with phenotypic outcomes (i.e. SLC-carried flux) to appreciate the opportunities for targeting acid-base transport in cancers.


Transitioning to a new nursing home: one organization's experience.
Restructuring of long-term care in Western Health, a regional health authority within Newfoundland and Labrador, created a unique opportunity to study the widespread impacts of the transition. Staff and long-term-care residents were relocated from a variety of settings to a newly constructed facility. A plan was developed to assess the impact of relocation on staff, residents, and families. Indicators included fall rates, medication errors, complaints, media database, sick leave, overtime, injuries, and staff and family satisfaction. This article reports on the findings and lessons learned from an organizational perspective with such a large-scale transition. Some of the key findings included the necessity of premove and postmove strategies to minimize negative impacts, ongoing communication and involvement in decision making during transitions, tracking of key indicators, recognition from management regarding increased workload and stress experienced by staff, engagement of residents and families throughout the transition, and assessing the timing of large-scale relocations. These findings would be of interest to health care managers and leadership team in organizations planning large-scale changes.


Hierarchical Au nanoclusters electrodeposited on amine-terminated ITO glass as a SERS-active substrate for the reliable and sensitive detection of serotonin in a Tris-HCl buffer solution.
Although serotonin (5-HT) is one of the most important neurotransmitters that are responsible for critical roles in the central nervous system, only few studies have been reported on the reliable and sensitive detection of 5-HT by the SERS technique owing to the lower Raman cross-section and lack of a common extraction method of 5-HT. In this study, a SERS-active substrate was fabricated by electrodepositing hierarchical Au nanostructures on amine-terminated ITO (indium tin oxide) glass to achieve an enhanced Raman signal of 5-HT. The electrodeposition process was performed by applying the nucleation potential E1 = 0.7 V for 2 s and the growth potential E2 = -0.2 V for 1000 s for repeated cycles. As a testing analyte, 5-HT was dissolved in four different solvents i.e. PBS buffer, Tris-HCl buffer, distilled water, and ethanol solution (20%). Among them, Tris-HCl buffer produced the most sensitive and reproducible Raman signals for 5-HT along with the lowest detection limit of ∼10-9 M due to the strong adsorption interactions between the Au surface and the indole amine groups of 5-HT under alkaline conditions. Surprisingly, the standard curve between the Raman intensity at 751 cm-1 and the log-scale concentration of 5-HT showed a linear dynamic range of over five orders of magnitude in the Tris-HCl buffer solution, suggesting a possible quantification of the 5-HT level in a biological fluid with 5-HT dysfunction or deficiency.


Amyloid beta peptide (1-42)-mediated antioxidant imbalance is associated with activation of protein kinase C in red blood cells.
Glycolysis and pentose phosphate pathway (PPP) in red blood cell (RBC) are modulated by the cell oxygenation state. This metabolic modulation is connected to variations in intracellular nicotinamide adenine dinucleotide phosphate-reduced form (NADPH) and adenosine triphosphate (ATP) levels as a function of the oxygenation state of the cell, and, consequently, it should have physiologic relevance. In the present study, we analysed the effects of amyloid beta peptide (1-42) (Abeta) on RBC metabolism and its relationship with the activity of protein kinase C (PKC). Our results showed that metabolic response to Abeta depended on the degree of cell oxygenation. In particular, under high O2 pressure, in Abeta-treated RBC, glucose metabolized through PPP approached that metabolized by RBC under low O2 pressure, differently to that observed in untreated cells. The effect of Abeta on RBC metabolism was paralleled by increase in PKC enzyme activity, but cytosolic Ca2+ concentration does not seem to be involved in this mechanism. Incubation of Abeta-treated RBC with a specific inhibitor of PKC partially restores PPP flux. A possible rationalization of the different metabolic behaviours shown by RBC following Abeta treatment is proposed. It takes into account the known post-translational modifications to cytoskeleton proteins induced by PKC. The reduction in PPP flux may lead to a weakened defence system of antioxidant reserve in RBC, becoming a source of reactive species, and, consequently, its typical, structural and functional features are lost. Therefore, oxidative stress may outflow from the RBC and trigger damage events in adjacent cells and tissue, thus contributing to vascular damage.


Locoregional recurrence risk after lipofilling in breast cancer patients.
Lipofilling has been indicated for postmastectomy and postlumpectomy breast reconstruction. The clinical literatures underline its technical efficacy but experimental studies raise important questions about the potential detrimental effect of adipocytes on the stimulation of cancer growth and reappearance. We collected 321 consecutive patients operated for a primary breast cancer between 1997 and 2008 who subsequently underwent lipofilling for reconstructive purpose. For each patient, we selected two matched patients with similar characteristics who did not undergo a lipofilling. Eighty-nine percent of the tumors were invasive. Median follow-up was 56 months from the primary surgery and 26 months from the lipofilling. Eight and 19 patients had a local event in the lipofilling and control group, respectively, leading to comparable cumulative incidence curves [P = 0.792; Hazard Ratio(Lipo vs No lipo) = 1.11 (95% confidence interval 0.47-2.64)]. These results were confirmed when patients undergoing quadrantectomy and mastectomy were analyzed separately and when the analysis was limited to invasive tumors. Based on 37 cases, the lipofilling group resulted at higher risk of local events when the analysis was limited to intraepithelial neoplasia. Lipofilling seems to be a safe procedure in breast cancer patients. Longer follow-up and further experiences from oncological series are urgently required to confirm these findings.


[Side effects of hormonal contraception. Experiences with 1,702 patients and 17,228 cycles].
During 25 years 1,702 women with 17,228 cycles under oral contraceptive were observed. This report covers 1,021 women with 11,648 cycles and the frequency of side-effects. The acceptability of OC's is more and more dominated by the side-effects. All subjective symptoms were asked for and not--as usually done--just noted when the woman complained spontaneously. For objective results the bleeding calendar was controlled for amenorrhea, spottings and break-through bleedings. Lowering the ethinylestradiol dose below 50 micrograms (with monophasic OCs, micropills) resulted in a worse cycle control as far as bleeding and silent menstruation were concerned. The genuine normophasic method showed generally a far better cycle control. Combining this method with a new gestagen (Desogestrel) resulted a still better cycle control, especially with amenorrhea. The events of nervosity, gastrointestinal disturbances, changes of libido and fatigue were also less frequent with this OC (Ovidol-Oviol).


Paediatric otolaryngology--a parental satisfaction study in outpatient surgery.
A parental satisfaction survey was administered to obtain information as to exactly how patients and their parents experience essential aspects of their treatment. The parents of 178 children who underwent ear, nose and throat (ENT) surgery completed a questionnaire on various aspects of treatment satisfaction during a 6-week period. Most of the respondents expressed overall satisfaction with the treatment. The factor analysis revealed three parental satisfaction factors. Accordingly, we interpreted the subscales as i) surgical staff and general treatment satisfaction, ii) nursing staff and general information satisfaction and iii) anaesthetic staff satisfaction. The internal consistency values of the subscales, as measured by Cronbach's alpha coefficient, were 0.86, 0.84 and 0.71, respectively. The three scores were mutually correlated; this correlation was significant (0.46-0.67). The duration of general anaesthesia and demographic factors had a small, but significant impact on the satisfaction subscores. In conclusion, the satisfaction study encourages improvement in the evaluation and administration of treatment.


Modulation of growth hormone, luteinizing hormone, and testosterone secretion by excitatory amino acids in boars.
Ketamine hydrochloride, an n-methyl-d-aspartate (NMDA) receptor antagonist was used in an experiment that tested the hypothesis that fasting-induced increases in growth hormone (GH) secretion is mediated by excitatory amino acid (EAA) neurotransmission in boars. The effects of the drug on circulating concentrations of luteinizing hormone (LH) and testosterone were also evaluated. Blood was sampled at 15-min intervals for 8 h from 12 boars fitted with jugular vein catheters. At Hours 4 and 6, fasted boars (feed was withdrawn 48 h before the start of blood sampling) received i.m. injections of ketamine (19.9 mg/kg body weight; n=4) or .9% saline (n=4). Boars allowed feed on an ad libitum basis (n=4) received i.v. injections of n-methyl-d,l-aspartate (NMA; 2.5 mg/kg body weight), an NMDA receptor agonist, at Hours 4 and 6. Secretion of GH increased after NMA injections but was unaffected by treatment with ketamine or saline. Circulating concentrations of LH and testosterone were increased by injections of ketamine but were unaffected by injections of NMA or saline. Our results suggest that NMA is a potent GH secretagogue, but do not support the hypothesis that EAA neurotransmission drives the increased GH secretion displayed in fasted boars. Our finding that ketamine increased LH and testosterone release supports the notion that EAA have inhibitory effects on gonadotropin secretion in acutely fasted swine.


HIV-1 gag proteins in virions and in infected cell fractions.
The relation of the initial products of the HIV-1 gag gene to the final products was determined in virus samples and cell fractions of infected H9 and Jurkat-tat cell cultures. The proteins were identified by immunoblotting with pooled sera from AIDS patients or monoclonal antibodies. The proportion in the virions of gag precursor proteins and the products of their proteolytic cleavage varied according to the maturity of the virus particles as determined by electron microscopy. The distribution of viral gag proteins in the cell fractions was determined 2, 4, and 24 h after infection. Treatment of cells with cycloheximide to block de novo protein synthesis did not significantly affect the results. Gag proteins containing the N terminus of the precursor p55 (including p55, the intermediate precursors p41(45) and p39, and mature protein p17) were found in the cell nuclei up to 24 h after infection. The major core protein p24 was located in the cytoplasmic fraction. These data strongly suggest that gag precursors from the p55 N terminus and the matrix protein p17 enter the infected cell separately from the major core protein p24, or become separated from it in the cytoplasm.


Processing of interaural phase differences in components of harmonic and mistuned complexes in the inferior colliculus of the Mongolian gerbil.
Harmonicity and spatial location provide eminent cues for the perceptual grouping of sounds. In general, harmonicity is a strong grouping cue. In contrast, spatial cues such as interaural phase or time difference provide for strong grouping of stimulus sequences but weak grouping for simultaneously presented sounds. By studying the neuronal basis underlying the interaction of these cues in processing simultaneous sounds using van Rossum spike train distance measures, we aim at explaining the interaction observed in psychophysical experiments. Responses to interaural phase differences imposed on single components of harmonic and mistuned complex tones as well as noise delay functions were recorded as multiunit responses from the inferior colliculus of Mongolian gerbils. Results revealed a better representation of interaural phase differences if imposed on a harmonic rather than a mistuned frequency component of a complex tone. The representation of interaural phase differences was better for long integration-time windows approximately reflecting firing rates rather than short integration-time windows reflecting the temporal pattern of the stimulus-driven response. We found only a weak impact of interaural phase differences if combined with mistuning of a component in a harmonic tone complex.


"I Had to Go to the Streets to Get Love": Pathways From Parental Rejection to HIV Risk Among Young Gay and Bisexual Men.
Young, gay, and bisexual men (YGBM) are at increased risk of family rejection, which is related to HIV infection. What remains unknown is how family rejection leads to HIV risk. In this exploratory study, qualitative interviews were conducted with 21 HIV-positive YGBM aged 18 to 24. Most participants reported family rejection, which decreased instrumental and emotional support and resulted in participants using riskier ways to support themselves, such as engaging in survival sex. Conceptualizing the findings using a family systems framework, we present a tentative conceptual model to describe the potential relationships between family rejection and HIV risk for YGBM.


Biomimetic approach to potential benzodiazepine receptor agonists and antagonists.
Several beta-carbolines, isoquinolines, imidazopyridines , and canthin -6-ones prepared in biomimetic fashion were tested for their ability to bind to the benzodiazepine receptor. Methyl isoquinoline-3-carboxylate, methyl 6,7- dimethoxyisoquinoline -3-carboxylate (3b) 1-phenyl-3- carbomethoxyimidazopyridine , (6B,) and canthin -6- one ( 13a ) bound with moderate affinities, while 2- carbomethoxycanthin -6- one ( 13b ) bound to benzodiazepine receptors with an affinity comparable to several pharmacologically active benzodiazepines. The potency of 13b suggests that the benzodiazepine receptor(s) can tolerate substitution at positions 1 and 9 of a beta-carboline without loss of activity if the substituents are trigonal and maintain a planar topography. Moreover, displacement of the carbonyl group by two atoms from the aromatic ring (C) of the beta-carboline skeleton caused a marked decrease in binding to the benzodiazepine receptor. This observation supports the hypothesis that maximum binding affinity of beta-carbolines is achieved when the carbonyl group at position 3 is attached directly to the aromatic pyridine ring.


Identification of thiamine diphosphate-binding proteins from rat liver supernatant.
Transketolase (EC 188.8.131.52) was shown to be the sole enzyme protein bound to thiamine diphosphate (ThDP) as coenzyme in the soluble fraction of rat liver. No new ThDP-binding proteins with the molar ratio of ThDP to protein of 1.0 were detected in the rat liver supernatant under the purification and assay conditions employed.


Bleeding during antithrombotic therapy in patients with atrial fibrillation. The Stroke Prevention in Atrial Fibrillation Investigators.
The Stroke Prevention in Atrial Fibrillation II study compared warfarin vs aspirin for stroke prevention in atrial fibrillation. Bleeding complications importantly detracted from warfarin's net effectiveness, particularly among older patients. To analyze bleeding complications according to assigned therapy. To identify risk factors for bleeding during anticoagulation. Eleven hundred patients (mean age, 70 years) were randomized to 325 mg of aspirin daily (enteric coated) vs warfarin (target prothrombin time ratio, 1.3 to 1.8; approximate international normalized ratio, 2.0 to 4.5). Major hemorrhages were defined prospectively. The rate of major bleeding while receiving warfarin was 2.3% per year (95% confidence interval [CI], 1.7 to 3.2) vs 1.1% per year (95% CI, 0.7 to 1.8) while receiving aspirin (relative risk, 2.1; 95% CI, 1.1 to 3.1; P = .02). Intracranial hemorrhage occurred at 0.9% per year (95% CI, 0.5 to 1.5) with warfarin and 0.3% per year (95% CI, 0.1 to 0.8) with aspirin (relative risk, 2.4; P = .08). Age (P = .006), increasing number of prescribed medications (P = .007), and intensity of anticoagulation (P = .02) were independent risks for bleeding at any site during anticoagulation. The rate of major hemorrhage was 1.7% per year in patients aged 75 years or younger who received anticoagulation vs 4.2% per year in older patients (relative risk, 2.6, P = .009); rates by age for intracranial bleeding were 0.6% per year and 1.8% per year, respectively (P = .05). Advancing age and more intense anticoagulation increase the risk of major hemorrhage in patients given warfarin for stroke prevention.


Climate change and mosquito-borne disease: knowing the horse before hitching the cart.
Speculations on the potential impacts of climate change on human health often focus on the mosquito-borne diseases but ignore the complex interplay of the multitude of factors that are generally dominant in the dynamics of their transmission. A holistic view of this complexity - particularly the ecology and behaviour of the host and the ecology and behaviour of the vector - is the only valid starting point for assessing the significance of climate in the prevalence and incidence of these diseases.


Managed care: the strategic future for education in quality.
This paper discusses the needs for future education in quality assurance, assessment and improvement, particularly in relation to managed care. The pressures for increased education about quality are derived from different components of the health care system; e.g., regulatory and governmental agencies, purchasers of care, and competitors of health institutions. The content of future education in health care quality is defined in six areas: (1) organization and management; (2) health systems; (3) quality theory and methods; (4) management information systems and research; (5) governmental policy; and (6) economics and finance. Education in health care quality in these content areas is delivered at both the primary and continuing education levels by universities, professional associations and private training and development corporations. Future oriented, strategic thinking education in health care quality is needed. The pressures for education about quality, including traditional concepts of quality assurance, methodologies for quality assessment and the newer approaches to continuing quality improvement, are clearly growing stronger. This article discusses the need for education in health care quality, the content areas and levels of education and the delivery system.


Science to practice: good fat, bad fat--does location matter?
A technique that allows rapid noninvasive imaging of abdominal fat without the potentially harmful effects of ionizing radiation would be useful in diagnosing overweight and obese children and in developing effective strategies for weight management. Siegel et al describe a quick, safe, and reproducible single-section MR method for quantifying subcutaneous and visceral abdominal fat and its distribution in preadolescents and adolescents. Although larger studies are needed, their study results confirm the use of T1-weighted MR imaging at a single level for estimating adipose tissue volumes and distribution.


A review of co-milling techniques for the production of high dose dry powder inhaler formulation.
Drug delivery by inhalation offers several advantages compared to other dosage forms, including rapid clinical onset, high bioavailability, and minimal systemic side effects. Drug delivery to the lung can be achieved as liquid suspensions or solutions in nebulizers and pressurized metered-dose inhalers (pMDI), or as dry powders in dry powder inhalers (DPIs). Compared to other delivery systems, DPIs are, in many cases, considered the most convenient as they are breath actuated and do not require the use of propellants. Currently, the delivery of low drug doses for the treatment of lung conditions such as asthma and chronic obstructive pulmonary disease are well established, with numerous commercial products available on the market. The delivery of low doses can be achieved from either standard carrier- or aggregate-based formulations, which are unsuitable in the delivery of high doses due to particle segregation associated with carrier active site saturation and the cohesiveness of micronized aggregates which have poor flow and de-agglomeration properties. High-dose delivery is required for the treatment of lung infection (i.e. antibiotics) and in the emerging application of drug delivery for the management of systemic conditions (i.e. diabetes). Therefore, there is a demand for new methods for production of high-dose dry powder formulations. This paper presents a review of co-mill processing, for the production of high-efficiency inhalation therapies, including the jet mill, mechanofusion, or ball mill methodologies. We investigate the different techniques, additives, and drugs studied, and impact on performance in DPI systems.


Comparison of VHH-Fc antibody production in Arabidopsis thaliana, Nicotiana benthamiana and Pichia pastoris.
VHHs or nanobodies are widely acknowledged as interesting diagnostic and therapeutic tools. However, for some applications, multivalent antibody formats, such as the dimeric VHH-Fc format, are desired to increase the functional affinity. The scope of this study was to compare transient expression of diagnostic VHH-Fc antibodies in Nicotiana benthamiana leaves with their stable expression in Arabidopsis thaliana seeds and Pichia pastoris. To this end, VHH-Fc antibodies targeting green fluorescent protein or the A. thaliana seed storage proteins (albumin and globulin) were produced in the three platforms. Differences were mainly observed in the accumulation levels and glycosylation patterns. Interestingly, although in plants oligomannosidic N-glycans were expected for KDEL-tagged VHH-Fcs, several VHH-Fcs with an intact KDEL-tag carried complex-type N-glycans, suggesting a dysfunctional retention in the endoplasmic reticulum. All VHH-Fcs were equally functional across expression platforms and several outperformed their corresponding VHH in terms of sensitivity in ELISA.


Adjuvant radiotherapy and radioiodine treatment for locally advanced differentiated thyroid cancer: systematic review and meta-analysis.
Treatment for locally advanced differentiated thyroid cancer is surgery followed by radioiodine while the role of adjuvant external beam radiotherapy (EBRT) is debated. The panel of the Italian Association of Radiotherapy and Clinical Oncology developed a clinical recommendation on the addition of EBRT to radioiodine after surgery for locally advanced differentiated thyroid cancer by using the Grades of Recommendation, Assessment, Development, and Evaluation methodology and the Evidence to Decision framework. A systematic review with meta-analysis about this topic was conducted with a focus on outcome of benefits and toxicity. Locoregional control was improved by EBRT while no considerable toxicity impact was reported. The panel judged uncertain the benefit/harms balance; final recommendation was conditional both for EBRT + radioiodine and radioiodine alone in the adjuvant setting.


Extracellular Vesicles Secreted by Tumor Cells Promote the Generation of Suppressive Monocytes.
Monocytes are among the first cells to infiltrate the tumor microenvironment. The conversion of monocytes to suppressor cells in the tumor microenvironment is crucial in evasion of the immune response and tumor maintenance. Tumor cells may secrete products that promote the conversion of monocytes to suppressor cells. Cells secrete extracellular vesicles (EVs) containing cargos of genetic materials and proteins as a way to communicate with neighboring cells. During pathologic conditions like cancers, tumor cells increase their EVs production containing microRNA, RNA, and proteins that may affect the immune cell response, contributing to the immunosuppressive microenvironment. Our studies show that EVs secreted by a wide range of murine tumor cells, including osteosarcoma, glioma, colon carcinoma, sarcoma, and melanoma, can be taken up by bone marrow-derived monocytes. The monocytes that took up the EVs secreted by tumor cells matured toward an immune-suppressive phenotype by upregulating the expression of suppressive cytokines and effector molecules. The monocytes also downregulated MHC class II and costimulatory molecules while increasing the expression of PD-L1 on their surface after taking up EVs from tumor cells. Most importantly, monocytes exposed to EVs secreted by tumor cells suppressed activated Ag-specific CD4+ T cells. These results show that tumor cells from several different tumor types secrete EVs which promote the conversion of monocytes into suppressor cells, thus promoting immune evasion. These studies suggest that EVs secreted by tumors are potentially a new target for future cancer therapy.


Quality Changes and Shelf-Life Prediction of Cooked Cured Ham Stored at Different Temperatures.
Cooked cured ham is a ready-to-eat food that is popular among consumers. Stored temperature has a key effect on the quality and shelf life of ham. In this work, the quality changes and shelf-life prediction of cooked cured ham stored at different temperatures were investigated. Sensory evaluation, physical and chemical indicators, and aerobic plate count were determined. Results showed that high storage temperature of cooked ham accelerates quality deterioration. Partial least squares (PLS) regression analysis based on the variable importance for projection identified nine important variables for predicting the shelf life of cooked cured ham. Compared with either PLS or back-propagation artificial neural network, the hybrid PLS-back-propagation artificial neural network model better predicts the shelf life of cooked cured ham by using the nine variables. This study provides a theoretical basis and data support for the quality control of cooked cured ham and a new idea for research on the shelf-life prediction of cooked cured ham.


Enhanced resistance to fluoroquinolones in laboratory-grown mutants & clinical isolates of Shigella due to synergism between efflux pump expression & mutations in quinolone resistance determining region.
There is a worldwide emergence of fluoroquinolone resistance in Shigella species. To understand the molecular mechanisms associated with fluoroquinolone resistance, naturally occurring fluoroquinolone-resistant strains and laboratory-induced spontaneous mutants of Shigella spp. were used and the relative contributions of acrAB-tolC efflux pumps, gyrase and topoisomerase target gene mutations towards fluoroquinolone resistance were determined. Eight Shigella flexneri and six S. dysenteriae clinical isolates were studied. Three consecutive mutants resistant to ciprofloxacin for S. flexneri SFM1 (≥ 0.25 µg/ml), SFM2 (≥ 4 µg/ml) and SFM3 (≥ 32 µg/ml) were selected in 15 steps from susceptible isolates by serial exposure to increasing concentrations of nalidixic acid and ciprofloxacin. Similarly, two mutants for S. dysenteriae SDM1 (≥ 0.25 µg/ml) and SDM2 (≥ 4 µg/ml) were selected in eight steps. After PCR amplification sequence analyses of gyrase and topoisomerase target genes were performed. Expression of efflux genes acrA, acrB, acrR and tolC was measured using real-time PCR. Mutations were observed in gyrA Ser [83]→Leu, Asp [87]→Asn/Gly, Val [196]→Ala and in parC Phe [93]→Val, Ser [80]→Ile, Asp [101]→Glu and Asp [110]→Glu. Overall, acrA and acrB overexpression was associated with fluoroquinolone resistance ( p0 <0.05); while tolC and acrR expression levels did not. Fluoroquinolone resistance in Shigella spp. is the end product of either a single or a combination of mutations in QRDRs and/ or efflux activity. Novel polymorphisms were observed at Val [196]→Ala in gyrA in clinical isolates and Phe [93]→Val, Asp [101]→Glu, Asp [110]→Glu and in parC in majority of laboratory-grown mutants.


Art Therapy Interventions for Syrian Child and Adolescent Refugees: Enhancing Mental Well-being and Resilience.
This critical review primarily aims to conduct a comprehensive assessment of recent studies conducted within the past 3 years, with a specific focus on the use of art therapy within Syrian child and adolescent refugee populations. The central objective of this inquiry is to explore the impact of art therapy interventions and provide critical insights into their effectiveness in enhancing the mental well-being and resilience of these young refugees. Recent research conducted within the past 3 years highlights the effectiveness of art therapy as an intervention strategy within Syrian child and adolescent refugee populations. These studies emphasize that art therapy offers a safe and supportive space for these young refugees to express emotions, process trauma, and improve their psychological well-being. It has shown promise in mitigating trauma-related challenges and fostering resilience among this group of refugee populations. Furthermore, art therapy plays a crucial role in preserving cultural identities and has proven to be versatile and adaptable to various artistic modalities. These findings underscore the potential of art therapy as a valuable tool for supporting the mental well-being and resilience of Syrian children and adolescent refugees, warranting further exploration and integration into their care and support programs. Recent studies on the use of art therapy within Syrian child and adolescent refugee communities reveal its potential as a powerful intervention strategy for enhancing mental well-being and resilience in this vulnerable population. The review identifies six key themes, highlighting the therapeutic benefits and versatility of art therapy. As we move forward, these findings offer promising future directions for research and practice in art therapy with this specific group of refugees, emphasizing the need for continued exploration and integration of this approach in supporting Syrian child and adolescent refugees.


Urine nicotine metabolite concentrations in relation to plasma cotinine during low-level nicotine exposure.
Plasma or saliva cotinine concentrations are used widely as biomarkers of secondhand smoke (SHS) exposure and have been associated with the risk of SHS-related disease. Concentrations of cotinine and other nicotine metabolites are considerably higher in urine than in plasma or saliva, making chemical analysis easier. In addition, urine is often more convenient to collect in some SHS exposure studies. The optimal use of nicotine metabolites in urine, singly or in combination, with or without correction for urine creatinine concentration, to estimate plasma cotinine concentration with low-level nicotine exposure has not been determined. We dosed 36 nonsmokers with 100, 200, or 400 microg deuterium-labeled nicotine (simulating exposure to SHS) by mouth daily for 5 days and then measured plasma and urine cotinine and metabolites at various intervals over 24 hr. A plasma cotinine concentration of 1 ng/ml corresponds on average to a daily intake of 100 microg nicotine. Cotinine concentrations in urine averaged four to five times those in plasma. Correction of urine cotinine for creatinine concentration improved the correlation between urine and plasma cotinine. Measuring multiple cotinine metabolites in urine did not improve the correlation with plasma cotinine, compared with the use of urine cotinine alone. Measurement of urine cotinine corrected for creatinine concentration appears to be the best predictor of plasma cotinine.


[Pharmacological modulation of NO synthesis in patients with arterial hypertension and endothelial dysfunction].
An open, comparative, cross-over 8-week trial of beta-blockers nebivolol and metoprolol in 30 patients with arterial hypertension (AH) studied the office, ambulatory blood pressure and heart rate levels, endothelium-dependent vasorelaxation, endothelial NOS and NO production. It was found that nebivolol activated the system eNOS-NO and improved vasomotor parameters of the endothelium. Metoprolol failed to activate enzyme eNOS activity and NO production to the same level and did not improve the endothelial vasomotor function. The antihypertensive activity of nebivolol was higher than that of metoprolol. Thus, nebivolol can be used in patients with AH and cardiovascular risk factors to correct endothelial dysfunction.


A systematic review of human paternal oxytocin: Insights into the methodology and what we know so far.
With the consolidation of fathers' engagement in caregiving, understanding the neuroendocrine and hormonal mechanisms underlying fatherhood becomes a relevant topic. Oxytocin (OT) has been linked with maternal bonding and caregiving, but less is known about the role of OT in human fatherhood and paternal caregiving. A systematic review of methods and findings of previous OT research in human fathers was carried. The literature search on PubMed and Scopus yielded 133 records. Twenty-four studies were included and analyzed. Significant variability emerged in OT methodology, including laboratory tasks, assessment methods, and outcome measures. Fathers' OT levels appear to increase after childbirth. OT was significantly correlated with less hostility and with the quality of paternal physical stimulation in play interactions, but not with paternal sensitivity. Fathers' and children's OT levels were significantly correlated in a limited subset of studies, intriguingly suggesting that cross-generational OT regulation may occur during the early years of life. This study highlights relevant issues and limitations of peripheral OT assessment in human subjects, especially in fathers. Although the study of paternal neuroendocrinology appears promising, coping with these issues requires dedicated efforts and methodological suggestions are provided to guide future advances in this field.


Longitudinal monitoring of neutral and adaptive genomic diversity in a reintroduction.
Restoration programs in the form of ex-situ breeding combined with reintroductions are becoming critical to counteract demographic declines and species losses. Such programs are increasingly using genetic management to improve conservation outcomes. However, the lack of long-term monitoring of genetic indicators following reintroduction prevents assessments of the trajectory and persistence of reintroduced populations. We carried out an extensive monitoring program in the wild for a threatened small-bodied fish (southern pygmy perch, Nannoperca australis) to assess the long-term genomic effects of its captive breeding and reintroduction. The species was rescued prior to its extirpation from the terminal lakes of Australia's Murray-Darling Basin, and then used for genetically informed captive breeding and reintroductions. Subsequent annual or biannual monitoring of abundance, fitness, and occupancy over a period of 11 years, combined with postreintroduction genetic sampling, revealed survival and recruitment of reintroduced fish. Genomic analyses based on data from the original wild rescued, captive born, and reintroduced cohorts revealed low inbreeding and strong maintenance of neutral and candidate adaptive genomic diversity across multiple generations. An increasing trend in the effective population size of the reintroduced population was consistent with field monitoring data in demonstrating successful re-establishment of the species. This provides a rare empirical example that the adaptive potential of a locally extinct population can be maintained during genetically informed ex-situ conservation breeding and reintroduction into the wild. Strategies to improve biodiversity restoration via ex-situ conservation should include genetic-based captive breeding and longitudinal monitoring of standing genomic variation in reintroduced populations.


Oxygen-oxygen bond formation by the Ru-Hbpp water oxidation catalyst occurs solely via an intramolecular reaction pathway.
A thorough kinetics investigation of the Ru-Hbpp water oxidation catalyst has been carried out at temperatures in the range 10-40 degrees C. Four oxidative electron-transfer processes that take the catalyst from its initial II,II oxidation state up to the formal IV,IV oxidation state were kinetically characterized and the corresponding activation parameters determined. Once the IV,IV oxidation state is reached, two additional slower kinetic processes take place, corresponding to the formation of an intermediate that finally evolves oxygen and regenerates the initial Ru-Hbpp catalyst. These two kinetic processes were also fully characterized with respect to the evaluation of their rate constants and activation parameters. Furthermore, (18)O labeling experiments were performed with different degrees of labeled catalyst and solvent, and the (16)O(2)/(16)O(18)O/(18)O(2) isotopic distribution of the generated molecular oxygen was calculated. These results clearly point to the existence of a single intramolecular reaction pathway for the formation of the oxygen-oxygen bond in the case of the Ru-Hbpp catalyst.


Tuberculin-purified protein derivative-, MPT-64-, and ESAT-6-stimulated gamma interferon responses in medical students before and after Mycobacterium bovis BCG vaccination and in patients with tuberculosis.
QuantiFERON-TB (QIFN) (CSL Limited) is a whole-blood assay for the recognition of infection with Mycobacterium tuberculosis. QIFN measures gamma interferon (IFN-gamma) production when purified protein derivatives (PPDs) of mycobacteria are incubated with venous blood samples. The specificity of QIFN in medical students before and after BCG immunization was assessed, and sensitivity in patients with tuberculosis was assessed. Antigens were PPD derived from M. tuberculosis and two M. tuberculosis-specific proteins, ESAT-6 and MPT-64. Of 60 medical students, all of whom had 0-mm tuberculin skin tests (TSTs) at study entry, 58 (97%) were initially classified as negative for M. tuberculosis infection by PPD QIFN. Five months after BCG immunization, 7 of 54 students (13%) had a TST result of >/=10 mm and 11 of 54 students (20%) tested positive by PPD QIFN. ESAT-6- and MPT-64-stimulated IFN-gamma responses in the medical students were negative prior to and after BCG immunization. For patients with active tuberculosis, 12 of 19 (63%) were positive by PPD QIFN, 11 of 19 (58%) were positive by ESAT-6 QIFN, and 0 of 12 were positive by MPT-64 QIFN. In conclusion, PPD QIFN was negative in 97% of a low-risk population who had not received BCG and who had negative TSTs. The specificities of both the TST and PPD QIFN were reduced following BCG immunization. PPD QIFN and ESAT-6 QIFN were of similar and moderate sensitivity in patients with active tuberculosis, but ESAT-6 QIFN is likely to be more specific because it is not influenced by past BCG exposure.


ATP-binding cassette transporter G1 negatively regulates thymocyte and peripheral lymphocyte proliferation.
Cholesterol is a key component of cell membranes and is essential for cell growth and proliferation. How the accumulation of cellular cholesterol affects lymphocyte development and function is not well understood. We demonstrate that ATP-binding cassette transporter G1 (ABCG1) regulates cholesterol homeostasis in thymocytes and peripheral CD4 T cells. Our work is the first to describe a cell type in Abcg1-deficient mice with such a robust change in cholesterol content and the expression of cholesterol metabolism genes. Abcg1-deficient mice display increased thymocyte cellularity and enhanced proliferation of thymocytes and peripheral T lymphocytes in vivo. The absence of ABCG1 in CD4 T cells results in hyperproliferation in vitro, but only when cells are stimulated through the TCR. We hypothesize that cholesterol accumulation in Abcg1(-/-) T cells alters the plasma membrane structure, resulting in enhanced TCR signaling for proliferation. Supporting this idea, we demonstrate that B6 T cells pretreated with soluble cholesterol have a significant increase in proliferation. Cholesterol accumulation in Abcg1(-/-) CD4 T cells results in enhanced basal phosphorylation levels of ZAP70 and ERK1/2. Furthermore, inhibition of ERK phosphorylation in TCR-stimulated Abcg1(-/-) T cells rescues the hyperproliferative phenotype. We describe a novel mechanism by which cholesterol can alter signaling from the plasma membrane to affect downstream signaling pathways and proliferation. These results implicate ABCG1 as an important negative regulator of lymphocyte proliferation through the maintenance of cellular cholesterol homeostasis.


Resilience as a multimodal dynamic process.
Resilience is rapidly gaining momentum in mental health literature. It provides a new understanding of the highly variable trajectories of mental illness, and has consistently been linked with improved mental health outcomes. The present review aims to clarify the definition of resilience and to discuss new directions for the field. After discussing the definition of resilience, this narrative review synthesizes evidence that identifies the specific protective factors involved in this process. This review also addresses the mechanisms that underlie resilience. Recent literature has clarified the three core components of resilience, which are the presence of an adversity or specific risk for mental illness; the influence of protective factors that supersede this risk; and finally, a subsequently more positive outcome than expected. Now that these are largely agreed upon, the field should move on to addressing other topics. Resilience is a dynamic process by which individuals utilize protective factors and resources to their benefit. It can vary within one individual across time and circumstance. It can also refer to good functional outcomes in the context of diagnosable illness. While previous research has focused on psychological resilience, it is essential that resilience is conceptualized across modalities. The field should move towards the development of a multimodal model of resilience. Researchers should now focus on producing empirical research which clarifies the specific protective factors and mechanisms of the process, aligning with the core concepts of resilience. This growing, more homogeneous evidence base, can then inform new intervention strategies.


Non-radioactive dot-blot DNA reassociation for unequivocal yeast identification.
Unequivocal and rapid classification of yeast cultures cannot be accomplished exclusively on the basis of unstable phenotypic traits, but requires molecular tests relating to the whole genome (or the largest possible portion of it). DNA-DNA reassociation meets this requirement, although many procedures proposed for calculating overall sequence similarity are expensive and time-consuming, thus restricting the possibility of unequivocal classification to a few specialized laboratories. A novel method, based on non-radioactive dot-blot hybridization of whole genomic DNA, has shown high and reproducible proportionality between the detected signal and the amount of double-stranded DNA effectively present on the membrane. This procedure has been optimized to obtain, within two working days, DNA relatedness values between unknown cultures and the type strains of the species previously indicated by a few conventional tests. The effective ability of the method to discriminate strains belonging to different species has been tested within taxonomic models consisting of yeast type cultures already certified by spectrophotometric reassociation.


Overcoming the Variability of iPSCs in the Manufacturing of Cell-Based Therapies.
Various factors are known to contribute to the diversity of human induced pluripotent stem cells (hiPSCs). Among these are the donor's genetic background and family history, the somatic cell source, the iPSC reprogramming method, and the culture system of choice. Moreover, variability is seen even in iPSC clones, generated in a single reprogramming event, where the donor, somatic cell type, and reprogramming platform are the same. The diversity seen in iPSC lines often translates to epigenetic differences, as well as to differences in the expansion rate, iPSC line culture robustness, and their ability to differentiate into specific cell types. As such, the diversity of iPSCs presents a hurdle to standardizing iPSC-based cell therapy manufacturing. In this review, we will expand on the various factors that impact iPSC diversity and the strategies and tools that could be taken by the industry to overcome the differences amongst various iPSC lines, therefore enabling robust and reproducible iPSC-based cell therapy manufacturing processes.


Role of the microbiology laboratory in monitoring and identifying resistance: use of molecular biology.
Antimicrobial resistance is an increasing problem in the United States. Early detection of emerging trends in antimicrobial resistance may facilitate implementation of effective control measures. Most antimicrobial susceptibility testing is qualitative, in that it categorizes isolates as susceptible, intermediate, or resistant. This approach is relatively inexpensive and generally adequate for clinical purposes. Qualitative susceptibility testing has some limitations for monitoring for emerging resistance. Selective quantitative susceptibility testing may be useful in detecting early trends toward elevated minimal inhibitory concentrations. Molecular methods have a role also in characterizing mechanisms of resistance and in the typing of resistant strains to determine patterns of spread. Laboratory monitoring of emerging resistance must be associated with an effective infection control policy and a willingness to modify practice in a rational manner based upon the trends detected.


Increased Reactive Oxygen Species and Cell Cycle Defects Contribute to Anemia in the RASA3 Mutant Mouse Model scat.
RASA3 is a Ras GTPase activating protein that plays a critical role in blood formation. The autosomal recessive mouse model scat (severe combined anemia and thrombocytopenia) carries a missense mutation in Rasa3. Homozygotes present with a phenotype characteristic of bone marrow failure that is accompanied by alternating episodes of crisis and remission. The mechanism leading to impaired erythropoiesis and peripheral cell destruction as evidenced by membrane fragmentation in scat is unclear, although we previously reported that the mislocalization of RASA3 to the cytosol of reticulocytes and mature red cells plays a role in the disease. In this study, we further characterized the bone marrow failure in scat and found that RASA3 plays a central role in cell cycle progression and maintenance of reactive oxygen species (ROS) levels during terminal erythroid differentiation, without inducing apoptosis of the precursors. In scat mice undergoing crises, there is a consistent pattern of an increased proportion of cells in the G0/G1 phase at the basophilic and polychromatophilic stages of erythroid differentiation, suggesting that RASA3 is involved in the G1 checkpoint. However, this increase in G1 is transient, and either resolves or becomes indiscernible by the orthochromatic stage. In addition, while ROS levels are normal early in erythropoiesis, there is accumulation of superoxide levels at the reticulocyte stage (DHE increased 40% in scat; p = 0.02) even though mitochondria, a potential source for ROS, are eliminated normally. Surprisingly, apoptosis is significantly decreased in the scat bone marrow at the proerythroblastic (15.3%; p = 0.004), polychromatophilic (8.5%; p = 0.01), and orthochromatic (4.2%; p = 0.02) stages. Together, these data indicate that ROS accumulation at the reticulocyte stage, without apoptosis, contributes to the membrane fragmentation observed in scat. Finally, the cell cycle defect and increased levels of ROS suggest that scat is a model of bone marrow failure with characteristics of aplastic anemia.