Meaning of family reunification as interpreted by young Chinese immigrants.
This paper is a narrative analysis of young Chinese immigrants' subjective interpretation of their family reunification in Hong Kong. The major objectives of the study were to understand what family reunification actually meant to them and how it affected their subsequent adjustment. It also tried to examine the similarities and differences in the experience and interpretation of family reunification among young immigrants who had different levels of adjustment. Participants in this study included 45 immigrants, aged between 12 and 17. Research findings show that family reunification was their long-cherished wish and the main reason for their migration. It was not only their primary concern before coming to Hong Kong but also the stance from which separation with close relatives in their native place and the subsequent hardships as a new comer in the competitive host society were justified. Reunification of the family and harmonious and caring relationship among family members had been the source of comfort and support for those who had successful adjustment. However, for those who did not experience warmth and concern from family members, the disillusionment with a happy family reunification and the experience of distant or even conflictive family relationship had not only created in them a sense of alienation but also resulted in poor adjustment in the new environment.


Pre-excitation syndrome in infants and children. Effect of digoxin, verapamil, and amiodarone.
Clinical and electrocardiographic findings for 30 patients with the pre-excitation syndrome are described together with details of treatment. Nineteen (63%) were younger than 2 years, 14 of whom were under 2 months. Sixteen infants and 7 children (77%) presented with paroxysmal supraventricular tachycardia, 14 (61%) of whom had the electrocardiographic pattern of type A Wolff-Parkinson-White (WPW) syndrome. During paroxysmal bouts the QRS complex was normal in 21 patients and wide in two. Six (20%) patients had congenital heart disease often associated with WPW syndrome type B. Seventeen patients were treated with either digoxin or verapamil intravenously to stop tachyarrhythmias. Verapamil was more effective due to the immediate response and lack of adverse effects. The tachyarrhythmias resolved in all the patients and in some of them the WPW pattern resolved later indicating maturation of the conduction tissue with loss of the accessory pathways. Verapamil provides a rapid and safe form of treatment for conversion of tachyarrhythmias since it has no effect on the accessory pathways. Oral amiodarone prevents recurrent tachyarrhythmias resistant to other treatment.


Violence in psychosis: estimating the predictive validity of readily accessible clinical information in a community sample.
This study sought to assess the validity of different combinations of readily available clinical information in predicting assaults by patients with psychosis, predominantly in the community. The combinations of information were: a) age and sex, b) age, sex and history of criminality/violence c) age, sex, history of violence and drug use and d) age, sex, history of violence, drug use and personality disorder. 708 subjects were followed for 2 years. Assaults were measured using multiple sources of information. Prediction validity was measured using the area under the receiver operating curves (AUC) and the number needed to detain (NND). A simple prediction tool was developed. The AUC values using the four combinations of information were a) 0.65, b) 0.70, c) 0.71, and d) 0.73. Prediction based on combination b), c), and d) implied a NND of 3. A rule based on c), the most accessible information, is suggested as a simple screening tool. Readily available clinical information allowed the prediction of assault over 2 years, in a sample of general psychiatric patients with psychosis, with a level of predictive accuracy comparable to that described using more detailed risk assessment tools. The information used in the predictive model was: age, sex, having committed an assault in the last 2 years (self-report) and having used any drug in the last year (self-report).


Effect of major and minor surgery on plasma levels of arginine, citrulline, nitric oxide metabolites, and ornithine in humans.
To determine the effect of surgical invasiveness on plasma levels of arginine, citrulline, ornithine, and nitric oxide (NO) in humans. Surgical trauma may have a profound effect on the metabolism of NO. However, human studies reported both increased and decreased NO levels after hemorrhagic shock. Arginine, citrulline, and ornithine are key amino acids involved in NO metabolism, but studies evaluating these amino acids together with NO and during 2 types of surgery are lacking. This study tests the hypothesis that major surgery has a more profound effect on plasma levels of arginine, citrulline, NO, and ornithine than minor surgery. Fifteen patients undergoing minor surgery (vulvectomy) and 13 patients undergoing major surgery (laparotomy) were prospectively followed up for 4 days. Plasma was collected for evaluation of levels of arginine, citrulline, NO, and ornithine. Throughout the experiment, arginine levels did not significantly differ between experimental groups. Perioperative plasma citrulline levels were significantly lower in the laparotomy group than in the vulvectomy group, whereas both groups showed a decrease in citrulline levels at the end of the operation and 24 hours postoperatively. Roughly the same pattern was seen for plasma NO and ornithine levels. However, ornithine levels in the laparotomy group showed a more drastic decrease at the end of the operation and 24 hours postoperatively than citrulline and NO levels. The level of surgical invasiveness has the most profound effect on plasma levels of ornithine. In addition, heavier surgical trauma is paired with lower postoperative levels of citrulline and NO metabolites than lighter surgery. It is suggested that surgical trauma stimulates the laparotomy group to consume significantly more ornithine, possibly for use in wound healing.


CRISPR-Cas9-based functional interrogation of unconventional translatome reveals human cancer dependency on cryptic non-canonical open reading frames.
Emerging evidence suggests that cryptic translation beyond the annotated translatome produces proteins with developmental or physiological functions. However, functions of cryptic non-canonical open reading frames (ORFs) in cancer remain largely unknown. To fill this gap and systematically identify colorectal cancer (CRC) dependency on non-canonical ORFs, we apply an integrative multiomic strategy, combining ribosome profiling and a CRISPR-Cas9 knockout screen with large-scale analysis of molecular and clinical data. Many such ORFs are upregulated in CRC compared to normal tissues and are associated with clinically relevant molecular subtypes. We confirm the in vivo tumor-promoting function of the microprotein SMIMP, encoded by a primate-specific, long noncoding RNA, the expression of which is associated with poor prognosis in CRC, is low in normal tissues and is specifically elevated in CRC and several other cancer types. Mechanistically, SMIMP interacts with the ATPase-forming domains of SMC1A, the core subunit of the cohesin complex, and facilitates SMC1A binding to cis-regulatory elements to promote epigenetic repression of the tumor-suppressive cell cycle regulators encoded by CDKN1A and CDKN2B. Thus, our study reveals a cryptic microprotein as an important component of cohesin-mediated gene regulation and suggests that the 'dark' proteome, encoded by cryptic non-canonical ORFs, may contain potential therapeutic or diagnostic targets.


Short report: Leishmania tropica: etiologic agent of a case of canine visceral leishmaniasis in northern Morocco.
The domestic dog has been previously demonstrated to be the reservoir of Leishmania infantum, the etiologic agent of human visceral leishmaniasis around the Mediterranean Basin. It can also be infected with L. tropica, the etiologic agent of anthroponotic cutaneous leishmaniasis in Morocco. We report a canine L. tropica visceral infection for the first time in Morocco.


Sexual orientation and gender identity/expression related peer victimization in adolescence: a systematic review of associated psychosocial and health outcomes.
This article reviews research on psychosocial and health outcomes associated with peer victimization related to adolescent sexual orientation and gender identity or expression. Using four electronic databases and supplementary methods, we identified 39 relevant studies. These studies were published between 1995 and 2012 and conducted in 12 different countries. The studies were diverse in terms of their approaches to sampling participants, assessing participants' sexual orientation, operationalizing peer victimization, and with regard to the psychosocial and health outcomes studied in relation to peer victimization. Despite the methodological diversity across studies, there is fairly strong evidence that peer victimization related to sexual orientation and gender identity or expression is associated with a diminished sense of school belonging and higher levels of depressive symptoms; findings regarding the relationship between peer victimization and suicidality have been more mixed. Peer victimization related to sexual orientation and gender identity or expression is also associated with disruptions in educational trajectories, traumatic stress, and alcohol and substance use. Recommendations for future research and interventions are discussed.


Maternal sensitivity and the security of infant-mother attachment: a Q-sort study.
40 mothers and their 12-month-old infants were observed twice at home by 2 observers for 2 hours. After the second visit, the observers described the infant using the Waters Attachment Behavior Q-sort and the mother's interactive behavior with the Maternal Behavior Q-sort developed by the present authors and Ainsworth's rating scales. Maternal sensitivity was unrelated to maternal age, income, or SES, but correlated positively with maternal education. Mothers of more difficult children were less sensitive. A strong relation was found between infant attachment and maternal sensitivity as measured by the Maternal Behavior Q-sort and by the Ainsworth scales. Using the Q-sort procedure, mothers of more secure infants were more frequently characterized as noticing their babies' signals and using these signals to guide their behavior; they also were more knowledgeable about their infant and appeared to enjoy them more than mothers of less secure infants.


[Mycoparasites effect on reproductive ability of Sclerotinia sclerotiorum sclerotia.].
The ability to parasitise Sclerotinia sclerotiorum and the effect on apothecia production was evaluated for the following antagonists: Trichoderma harzianum; Trichoderma koningii; Gliocladium roseum and Chaetomium globosum. Plastic trays were filled with of steam-sterilized soil. Each one of them was infested with sclerotia of S. sclerotiorum and the culture of the antagonists. The trays were kept in a greenhouse and after 30, 60 and 90 days, evaluations were made. The rates of carpogenic germination, myceliogenic germination, mycoparasitism and destruction were evaluated. To assess carpogenic germination, the sclerotia were put in a growth chamber over moistened filter paper at 20 -/+ 2 degrees C and 12 light hours. The rates of myceliogenic germination and mycoparasitism were evaluated on Petri dishes with 2% APD. Antagonists effect on carpogenic germination was observed one month after the start of the assay. In the evaluation made at 60 and 90 days, T. harzianum; T. koningii and G. roseum kept inhibitory properties. Such inhibition was not observed in the trays containing C. globosum. In the evaluations made at 30 days, mycoparasitism rate was high in the trays with T. harzianum; T. koningii and G. roseum. G. roseum and T. harzianum destroy S. sclerotiorum sclerotia.


Role of Mass Spectrometry in Establishing Safety and Efficacy of Botanical Dietary Supplements.
As in drug discovery and development, mass spectrometry has become essential at all stages for establishing the safety and efficacy of botanical dietary supplements. Applications of mass spectrometry to the development of botanical dietary supplements include preclinical studies of the mechanisms of action (e.g., proteomic target identification and validation); identification of active natural products using high resolution tandem mass spectrometry; chemical standardization using UHPLC-MS/MS; and studies of metabolism, absorption and toxicity of active compounds using high resolution and UHPLC-MS/MS. Clinical applications of mass spectrometry include evaluation of the potential for drug-botanical interactions; investigation of the pharmacokinetics of active compounds; and quantitative analysis of biomarkers of efficacy during Phase I and II and clinical trials of safety and efficacy of botanical dietary supplements.


A tailored approach to fusion transcript identification increases diagnosis of rare inherited disease.
RNA sequencing has been proposed as a means of increasing diagnostic rates in studies of undiagnosed rare inherited disease. Recent studies have reported diagnostic improvements in the range of 7.5-35% by profiling splicing, gene expression quantification and allele specific expression. To-date however, no study has systematically assessed the presence of gene-fusion transcripts in cases of germline disease. Fusion transcripts are routinely identified in cancer studies and are increasingly recognized as having diagnostic, prognostic or therapeutic relevance. Isolated reports exist of fusion transcripts being detected in cases of developmental and neurological phenotypes, and thus, systematic application of fusion detection to germline conditions may further increase diagnostic rates. However, current fusion detection methods are unsuited to the investigation of germline disease due to performance biases arising from their development using tumor, cell-line or in-silico data. We describe a tailored approach to fusion candidate identification and prioritization in a cohort of 47 undiagnosed, suspected inherited disease patients. We modify an existing fusion transcript detection algorithm by eliminating its cell line-derived filtering steps, and instead, prioritize candidates using a custom workflow that integrates genomic and transcriptomic sequence alignment, biological and technical annotations, customized categorization logic, and phenotypic prioritization. We demonstrate that our approach to fusion transcript identification and prioritization detects genuine fusion events excluded by standard analyses and efficiently removes phenotypically unimportant candidates and false positive events, resulting in a reduced candidate list enriched for events with potential phenotypic relevance. We describe the successful genetic resolution of two previously undiagnosed disease cases through the detection of pathogenic fusion transcripts. Furthermore, we report the experimental validation of five additional cases of fusion transcripts with potential phenotypic relevance. The approach we describe can be implemented to enable the detection of phenotypically relevant fusion transcripts in studies of rare inherited disease. Fusion transcript detection has the potential to increase diagnostic rates in rare inherited disease and should be included in RNA-based analytical pipelines aimed at genetic diagnosis.


Corn and Soybean Host Root Endophytic Fungi with Toxicity Toward the Soybean Cyst Nematode.
Although fungal endophytes are commonly investigated for their ability to deter microbial plant pathogens, few studies have examined the activity of fungal root endophytes against nematodes. The soybean cyst nematode (SCN; Heterodera glycines), the most severe yield-limiting pathogen of soybean (Glycine max), is commonly managed through rotation of soybean with corn (Zea mays), a nonhost of the SCN. A total of 626 fungal endophytes were isolated from surface-sterilized corn and soybean roots from experimental plots in which soybean and corn had been grown under annual rotation and under 1, 3, 5, and 35 years of continuous monoculture. Fungal isolates were grouped into 401 morphotypes, which were clustered into 108 operational taxonomic units (OTUs) based on 99% sequence similarity of the full internal transcribed spacer region. Morphotype representatives within each OTU were grown in malt extract broth and in a secondary metabolite-inducing medium buffered with ammonium tartrate, and their culture filtrates were tested for nematicidal activity against SCN juveniles. A majority of OTUs containing isolates with nematicidal culture filtrates were in the order Hypocreales, with the genus Fusarium being the most commonly isolated nematicidal genus from corn and soybean roots. Less commonly isolated taxa from soybean roots included the nematophagous fungi Hirsutella rhossiliensis, Metacordyceps chlamydosporia, and Arthrobotrys iridis. Root endophytic fungal diversity in soybean was positively correlated with SCN density, suggesting that the SCN plays a role in shaping the soybean root endophytic community.


Site of origin of and sex differences in the vasopressin innervation of the mouse (Mus musculus) brain.
Defining how arginine vasopressin (AVP) acts centrally to regulate homeostasis and behavior is problematic, as AVP is made in multiple nuclei in the hypothalamus (i.e., paraventricular [PVN], supraoptic [SON], and suprachiasmatic [SCN]) and extended amygdala (i.e., bed nucleus of the stria terminalis [BNST] and medial amygdala [MeA]), and these groups of neurons have extensive projections throughout the brain. To understand the function of AVP, it is essential to know the site of origin of various projections. In mice, we used gonadectomy to eliminate gonadal steroid hormone-dependent expression of AVP in the BNST and MeA and electrolytic lesions to eliminate the SCN, effectively eliminating those AVP-immunoreactive projections; we also quantified AVP-immunoreactive fiber density in gonadectomized and sham-operated male and female mice to examine sex differences in AVP innervation. Our results suggest that the BNST/MeA AVP system innervates regions containing major modulatory neurotransmitters (e.g., serotonin and dopamine) and thus may be involved in regulating behavioral state. Furthermore, this system may be biased toward the regulation of male behavior, given the numerous regions in which males have a denser AVP-immunoreactive innervation than females. AVP from the SCN is found in regions important for the regulation of hormone output and behavior. Innervation from the PVN and SON is found in brain regions that likely work in concert with the well-known peripheral AVP actions of controlling homeostasis and stress response; female-biased sex differences in this system may be related to the heightened stress response observed in females.


16S rRNA sequence determination for members of the genus Carnobacterium and related lactic acid bacteria and description of Vagococcus salmoninarum sp. nov.
The phylogenetic interrelationships of members of the genus Carnobacterium and some atypical lactobacilli isolated from diseased salmonid fish were investigated by using reverse transcriptase sequencing of 16S rRNA. The four species Carnobacterium piscicola, Carnobacterium divergens, Carnobacterium gallinarum, and Carnobacterium mobile exhibited a high degree of sequence similarity with each other (ca. 96 to 98%) and formed a phylogenetically coherent group that was quite distinct from all other lactic acid bacteria. The sequence data clearly demonstrated that carnobacteria are phylogenetically closer to the genera Enterococcus and Vagococcus than to members of the genus Lactobacillus. The strains from fish were found to be phylogenetically related to the genus Vagococcus and represent a new species, Vagococcus salmoninarum. The type strain of Vagococcus salmoninarum is strain NCFB 2777.


Development and characterization of NILK-2301, a novel CEACAM5xCD3 κλ bispecific antibody for immunotherapy of CEACAM5-expressing cancers.
T-cell retargeting to eliminate CEACAM5-expressing cancer cells via CEACAM5xCD3 bispecific antibodies (BsAbs) showed limited clinical activity so far, mostly due to insufficient T-cell activation, dose-limiting toxicities, and formation of anti-drug antibodies (ADA). We present here the generation and preclinical development of NILK-2301, a BsAb composed of a common heavy chain and two different light chains, one kappa and one lambda, determining specificity (so-called κλ body format). NILK-2301 binds CD3ɛ on T-cells with its lambda light chain arm with an affinity of ≈100 nM, and the CEACAM5 A2 domain on tumor cells by its kappa light chain arm with an affinity of ≈5 nM. FcγR-binding is abrogated by the "LALAPA" mutation (Leu234Ala, Leu235Ala, Pro329Ala). NILK-2301 induced T-cell activation, proliferation, cytokine release, and T-cell dependent cellular cytotoxicity of CEACAM5-positive tumor cell lines (5/5 colorectal, 2/2 gastric, 2/2 lung), e.g., SK-CO-1 (Emax = 89%), MKN-45 (Emax = 84%), and H2122 (Emax = 97%), with EC50 ranging from 0.02 to 0.14 nM. NILK-2301 binds neither to CEACAM5-negative or primary colon epithelial cells nor to other CEACAM family members. NILK-2301 alone or in combination with checkpoint inhibition showed activity in organotypic tumor tissue slices and colorectal cancer organoid models. In vivo, NILK-2301 at 10 mg/kg significantly delayed tumor progression in colon- and a pancreatic adenocarcinoma model. Single-dose pharmacokinetics (PK) and tolerability in cynomolgus monkeys at 0.5 or 10 mg/kg intravenously or 20 mg subcutaneously showed dose-proportional PK, bioavailability ≈100%, and a projected half-life in humans of 13.1 days. NILK-2301 was well-tolerated. Data were confirmed in human FcRn TG32 mice. In summary, NILK-2301 combines promising preclinical activity and safety with lower probability of ADA-generation due to its format compared to other molecules and is scheduled to enter clinical testing at the end of 2023.


Critical congenital heart disease screening practices among licensed midwives in washington state.
Since 2011, pulse oximetry screening for critical congenital heart disease (CCHD) has been recommended for newborns. Initial implementation guidelines focused on in-hospital births. Recent publications affirm the importance of universal screening, including for out-of-hospital births. No published data describe CCHD screening rates for out-of-hospital births. Licensed midwives in Washington state were surveyed regarding their current CCHD screening practices, volume of births attended annually, and typical newborn follow-up practices. For those who indicated they were screening, additional information was obtained about equipment used, timing of screening, and rationale for voluntarily initiating screening. For those who indicated that they were not screening, information regarding barriers to implementation was solicited. Of the 61 midwives in our sample, 98% indicated they were aware of published guidelines recommending universal newborn screening for CCHD utilizing pulse oximetry. Furthermore, 52% indicated that they were screening for CCHD currently. Ten percent stated they do not intend to screen, whereas the remaining respondents indicated that they plan to screen in the future. The primary barriers to screening were the cost of pulse oximetry equipment and inadequate training in screening technique and interpretation. Although voluntary implementation of CCHD screening by licensed midwives in Washington is increasing, it lags behind the implementation rates reported for in-hospital births.


Nature of linkage between the cationic headgroup and cholesteryl skeleton controls gene transfection efficiency.
Three novel cationic cholesterol derivatives with different modes of linkage between the cationic headgroup and the cholesteryl backbone have been synthesized and used as mixtures with 1, 2-dioleoyl-L-alpha-glycero-3-phosphatidyl ethanolamine (DOPE) for liposome-mediated gene transfection. A pronounced improvement in gene transfer efficiency was observed when the cationic center was appended to the cholesteryl backbone using an ether linkage as opposed to when the linkages were based on either ester or urethane groups. Amphiphiles with ether links such as cholest-5-en-3beta-oxyethane-N,N,N-trimethyl ammonium bromide (2) and cholest-5-en-3beta-oxyethane-N,N-dimethyl-N-2-hydroxyethyl ammonium bromide (3) showed transfection efficiencies considerably greater than commercially available gene transfer reagents. Notably, the transfection ability of 2 with DOPE in the presence of serum was significantly greater than Lipofectamine((R)) and Lipofectin((R)). Interestingly, 3 did not require the helper lipid DOPE for transfection. This suggests that these newly described cholesterol-based amphiphiles should be very promising in liposome-mediated gene transfection. The advantage that the ether linkage possesses would be important in the design of newer, more efficient cholesterol-based delivery reagents.


Contradictions in digital health engagement: An activity tracker's ambiguous influence on vulnerable young adults' engagement in own health.
Activity trackers are designed to support individuals in monitoring and increasing their physical activity. The use of activity trackers among individuals diagnosed with depression and anxiety has not yet been examined. This pilot study investigates how this target group engages with an activity tracker during a 10-week health intervention aimed to increase their physical activity level and improve their physical and mental health. Two groups of 11 young adults (aged 18-29 years) diagnosed with depression or anxiety participated in the digital health intervention. The study used mixed methods to investigate the research question. Quantitative health data were used to assess the intervention's influence on the participants' health and qualitative data provided insights into the participants' digital health experience. The study demonstrated an ambiguous influence from the use of an activity tracker with positive physical and mental health results, but a fading and even negative digital health engagement and counterproductive competition. The ambiguous results identify a need for (1) developing strategies for health professionals to provide supervised use of activity trackers and support the target groups' abilities to convert health information about physical activity into positive health strategies, and (2) designing alternatives for health promoting IT targeted users who face challenges and need motivation beyond self-tracking and competition.


Genetic and Environmental Influences on the Affective Regulation Network: A Prospective Experience Sampling Analysis.
Background: The study of networks of affective mental states that play a role in psychopathology may help model the influence of genetic and environmental risks. The aim of the present paper was to examine networks of affective mental states (AMS: "cheerful," "insecure," "relaxed," "anxious," "irritated," and "down") over time, stratified by genetic liability for psychopathology and exposure to environmental risk, using momentary assessment technology. Methods: Momentary AMS, collected using the experience sampling method (ESM) as well as childhood trauma and genetic liability (based on the level of shared genes and psychopathology in the co-twin) were collected in a population-based sample of female-female twin pairs and sisters (585 individuals). Networks were generated using multilevel time-lagged regression analysis, and regression coefficients were compared across three strata of childhood trauma severity and three strata of genetic liability using permutation testing. Regression coefficients were presented as network connections. Results: Visual inspection of network graphs revealed some suggestive changes in the networks with more exposure to either childhood trauma or genetic liability (i.e., stronger reinforcing loops between the three negative AMS anxious, insecure, and down both under higher early environmental, and under higher genetic liability exposure, stronger negative association between AMS of different valences: i.e., between "anxious" at t-1 and "relaxed" at t, "relaxed" at t-1 and "down" at t, under intermediate genetic liability exposure when compared to both networks under low and high genetic liability). Yet, statistical evaluation of differences across exposure strata was inconclusive. Conclusions: Although suggestive of a difference in the emotional dynamic, there was no conclusive evidence that genetic and environmental factors may impact ESM network models of individual AMS.


Integrated clinical experience: University of Nebraska Medical Center.
The Integrated Clinical Experience (ICE) at the University of Nebraska College of Medicine is a required, two-year course of study for first- and second-year students. It provides early clinical experiences in primary care settings in metropolitan and rural areas, and related instruction in the social, behavioral, and ethical foundations of medicine. The authors describe the course goals, teaching format, topics, and evaluation of students and faculty. ICE is based on the assumptions that medicine is an applied behavioral science as well as an applied biological science, that critical reflection is important in professional education, and that early exposure to primary care will promote interest in primary care careers. The authors also describe some of the challenges associated with the implementation of this new course of study. These include student dissatisfaction with behavioral and ethical topics, resistance to critical reflection about their personal attitudes and values, and discomfort with "subjective" grading. ICE has also been controversial with some basic science faculty who feel they have had to sacrifice curriculum time to make room for this new program. Also, recruiting the large number of faculty, particularly physicians, needed to run the program has been difficult. Finally, the organization of the curriculum, with basic sciences in the morning and the ICE in the afternoon, may inadvertently reinforce the conceptual split between the biomedical and psychosocial dimensions of medicine. Efforts are under way to address this problem by exploring ways to intergrate the curriculum better.


Direct correlation between a negative autoregulatory response element at the cap site of the herpes simplex virus type 1 IE175 (alpha 4) promoter and a specific binding site for the IE175 (ICP4) protein.
In transient-expression assays, the IE175 (alpha 4) promoter region of herpes simple virus is down-regulated after cotransfection with DNA encoding its own protein product (IE175 or ICP4). The inhibition by IE175 proved to be highly specific for its own promoter region and did not act on either the herpes simplex virus type 1 IE110 (alpha 0) or human cytomegalovirus major immediate-early promoters. Furthermore, the inhibition was still exhibited by IE175 effector plasmids driven by strong heterologous promoters and therefore must be a direct autoregulatory response that cannot be explained by promoter competition effects. In gel mobility retardation assays with infected-cell nuclear extracts, a prominent and specific DNA-protein complex was formed with DNA fragments containing sequences from -108 to +30 in the IE175 promoter region. This activity was not present in mock-infected samples. Even stronger binding occurred with a fragment containing sequences from -128 to +120 in the IE110 promoter, but this second locus was not associated with any detectable response phenotype in cotransfection assays. Supershift experiments with an anti-IE175 monoclonal antibody confirmed the presence of the IE175 protein in both DNA-protein complexes. In the IE175 promoter, specific binding correlated closely with the presence of an intact autoregulatory signal near the cap site as judged by the loss of both activities in a 3'-deleted promoter fragment lacking sequences from -7 to +30. Insertion of a cloned 30-mer synthetic oligonucleotide sequence from positions -8 to +18 in IE175 restored both IE175 binding activity and the down-regulation phenotype. Direct shift-up assays with a similar 30-base-pair (bp) oligonucleotide containing 21 bp from positions -75 to -55 of IE110 (which encompasses a consensus ATCGTC motif) also produced a specific DNA-protein complex containing the IE175 protein. This ATCGTC motif proved to be a necessary component of both the IE110 and IE175 binding sites, but was insufficient on its own for complex formation. Finally, deletion of 2 bp from positions -3 and -4 within the ATCGTC sequence in the IE175 cap site region abolished both binding activity and the IE175-dependent autoregulation phenotype.


Comment on "Exploiting electronic strategies to stabilize a planar tetracoordinate carbon in cyclic aromatic hydrocarbons" by O. Yañez et al., Chem. Commun., 2017, 53, 12112.
Unequivocally, the global minimum geometry for C7H2 is the linear triplet ground electronic state of heptatriynylidene (1). However, the entitled article reports that the global minimum geometry for C7H2 is the singlet ground electronic state of 1-(buta-1,3-diynyl)cyclopropenylidene (2). We disagree with this result.


Developing, Administering, and Scoring the Healthcare Ethics Consultant Certification Examination.
In November 2018, the practice of health care ethics consultation crossed a major threshold when 138 candidates took the inaugural Healthcare Ethics Consultant Certification Examination. This accomplishment, long in the making, has had and continues to have both advocates and critics. The Healthcare Ethics Consultant Certification Commission, a functionally autonomous body created and funded by the American Society for Bioethics and Humanities, was charged with overseeing creation of the certification process, developing the exam, and formulating certification standards and policies to assess candidates' qualifications. In this essay, as members of the commission, we describe the process of developing, administering, and scoring the certification examination as well as the historical context and the outlook for certification. By detailing the decisions and actions of the commission, we aim to provide a transparent account of the commission's efforts to develop a psychometrically sound, reliable, and secure examination through a deliberative, fair, and data-driven process.


HDL-cholesterol and the treatment of coronary heart disease: contrasting effects of atorvastatin and simvastatin.
Although the levels of low-density lipoprotein (LDL) cholesterol remain the main therapeutic goal when treating dyslipidaemias, there is a need to consider high-density lipoprotein (HDL) concentrations. This conclusion is based on the findings of epidemiological surveys and appropriately designed trials using statins or fibrates. The importance of HDL, as a 'protective' lipoprotein fraction, has been recognised by major treatment guidelines. This review considers the differences in HDL-raising capacity of two of the most commonly prescribed statins--atorvastatin and simvastatin. When compared with simvastatin, atorvastatin is associated with progressively decreasing rises in the levels of HDL as the dose increases (negative dose response), an effect not reported with other statins. In contrast, simvastatin shows a positive dose response (increasing concentrations of HDL with increasing dose). This effect is paralleled by changes in apolipoprotein A-I levels. Apolipoprotein A-I is the main apolipoprotein associated with HDL. This dissimilarity in HDL response is an example of several differences that have been reported when comparing various statins. If 'all statins are not created equal', we should focus prescribing on those statins that have end point evidence originating from appropriately designed trials.


Efficacy of vitamin D supplementation in major depression: A meta-analysis of randomized controlled trials.
There is a need to develop and periodically evaluate new treatment strategies in major depression due to the high burden of nonresponse and inadequate response to antidepressants. We aimed to assess the effect of vitamin D supplementation on depression symptom scores among individuals with clinically diagnosed major depression. Electronic search of databases was carried out for published randomized controlled trials in English language, peer-reviewed journals from inception till August 2017. Outcome measure used for effect size calculation was depression symptom scores. Effect sizes for the trials were computed using standardized mean difference (Cohen's d), and I2 test was used to assess sample heterogeneity. Pooled mean effect sizes were derived using both fixed and random-effects model. Critical appraisal of studies was done using the Cochrane risk of bias assessment tool. A total of four trials involving 948 participants were included in the study. In three trials, the intervention group received oral vitamin D supplementation whereas in one parenteral vitamin D was given. Pooled mean effect size for vitamin D supplementation on depressive symptom ratings in major depression was 0.58 (95% confidence interval, 0.45-0.72). The I2 value for heterogeneity was 0 suggesting low heterogeneity among studies. Egger plot intercept indicated minimal publication bias. Vitamin D supplementation favorably impacted depression ratings in major depression with a moderate effect size. These findings must be considered tentative owing to the limited number of trials available and inherent methodological bias noted in few of them.


Beating-Heart Mitral Valve Repair Using a Novel ePTFE Cordal Implantation Device: A Prospective Trial.
Conventional mitral valve (MV) operations allow direct anatomic assessment and repair on an arrested heart, but require cardiopulmonary bypass, aortic cross-clamping, sternotomy or thoracotomy, and cardioplegic cardiac arrest, and are associated with significant perioperative disability, and risks of morbidity and mortality. This study evaluated safety and performance of a transesophageal echocardiographic-guided device designed to implant artificial expanded polytetrafluoroethylene (ePTFE) cords on mitral leaflets in the beating heart. In a prospective multicenter study, 30 consecutive patients with severe degenerative mitral regurgitation (MR) were treated with a mitral valve repair system (MVRS) via small left thoracotomy. The primary (30-day) endpoint was successful implantation of cords with MR reduction to moderate or less. The primary endpoint was met in 27 of 30 patients (90%). Three patients required conversion to open mitral surgery. There were no deaths, strokes, or permanent pacemaker implantations. At 1 month, MR was mild or less in 89% (24 of 27) and was moderate in 11% (3 of 27). At 6 months, MR was mild or less in 85 % (22 of 26), moderate in 8% (2 of 26), and severe in 8% (2 of 26). Favorable cardiac remodeling at 6 months included decreases in end-diastolic (161 ± 36 ml to 122 ± 30 ml; p < 0.001) and left atrial volumes (106 ± 36 ml to 69 ± 24 ml; p < 0.001). The anterior-posterior mitral annular dimension decreased from 34.7 ± 5.8 mm to 28.2 ± 5.1 mm; p < 0.001 as did the mitral annular area (10.0 ± 2.7 cm2 vs. 6.9 ± 2.0 cm2; p < 0.0001). MVRS ePTFE cordal implantation can reduce the invasiveness and morbidity of conventional MV surgery. The device's safety profile is promising and prospective trials comparing the outcomes of the MVRS to conventional MV repair surgery are warranted. (CE Mark Study for the Harpoon Medical Device [TRACER]; NCT02768870).


Licencing of diagnostic HIV kits-to keep a sensible standpoint in an area of permanent revolution.
In vitro diagnostic medical devices or 'test kits' are important tools for diagnosing illnesses and monitoring the patients' state of health or the effects of medical treatment. It is in the public interest that commercial kits are of high quality. To review the current situation of kit evaluation in Europe and to establish practical and sensible criteria for kit evaluation in general, based on the Swiss experience with the licencing of kits for the detection of human immunodeficiency virus (HIV) infection. Kit evaluation should provide the user with a selection of sufficiently good products. In addition, it should be simple and not require rare patient materials. Also of importance is that all manufacturers are treated equally. The evaluation criteria depend on the legal situation, the intended use of the test, the state of the art and the graveness of false-negative or -positive results. In addition to diagnostic sensitivity and specificity, other important considerations include the quality control by the manufacturer, kit presentation, intra- and inter-lot reproducibility and how a test fits into a country's adopted testing system. The above goals can be achieved by setting minimal performance standards, preferably in the form of a lower limit of the confidence interval for the sensitivity/specificity that has been observed in the test's field evaluation. The better the test, the fewer samples have to be studied. For tests with no false results observed during field trials, the minimum sample number necessary for demonstration of a sensitivity or specificity >/= 99% is about 400, while it is 700 for a >/= 99.5% demonstration. Further insight into sensitivity is provided by results from well described and frequently used commercial seroconversion panels. This represents a dynamic tool that readily identifies insensitive kits that have become technically obsolete. Specificity assessment should not only involve blood donors, but also appropriate clinical controls. It is clear that the test design has an important bearing with respect to sensitivity and specificity. There are inherent advantages and disadvantages of indirect binding assays, capture assays and double antigen sandwich assays. Positive or negative results with these different test formats may not carry exactly the same message. Efficient kit evaluation does not require tens of thousands of samples but can be performed with relatively little disbursement on materials and manpower.


Quantification and mechanisms of oleic acid-induced steatosis in HepG2 cells.
Developing a quantifiable in vitro model of steatosis is critical in understanding the pathogenesis of nonalcoholic fatty liver disease (NAFLD) and searchingfor effective therapies. Using an ORO-based colorimetric measurement, we developed a convenient assay to qualify the degree of OA-induced steatosis in HepG2 cells. We demonstrated that in the absence of exogenous inflammatory mediators, OA-induced steatosis was associated with increased production and secretion of tumor necrosis factor alpha and decreased expression of peroxisome proliferators-activated receptor alpha in HepG2 cells. OA-induced steatosis was also associated with increased lipid peroxidation, apoptosis, but decreased proliferation in these cells. The increased lipid peroxidation was related to decreased SOD-1, a free radical scavenger enzyme; while increased apoptosis was related to increased active caspase-9. The decreased proliferation mediated by OA-induced steatosis was associated with increased production of p27 with unchanged alanine transaminase (ALT) level in the culture medium, indicating OA-induced steatosis alters cell cycle progression without direct toxicity to these cells. In conclusion, the present study developed a colorimetric assay that accurately quantifies OA-induced steatosis in HepG2 cells. In the absence of exogenous inflammatory mediators, OA-induced steatosis results in a series of pathophysilogical changes in HepG2 cells, indicating direct pathogenic roles of hepatocytes in NAFLD.


Basement membrane components in healing rabbit corneal epithelial wounds: immunofluorescence and ultrastructural studies.
The nature of the substrate that supports epithelial migration in vivo is of interest, particularly with respect to mechanisms of wound healing. Immunofluorescence and electron microscopy were used to search for common substrate components in prototype rabbit corneal wounds: epithelial scrape wounds, in which the corneal or conjunctival epithelium migrated over the denuded lamina densa of the corneal basement membrane (CBM), and superficial keratectomy, in which the corneal epithelium migrated over a bare stroma without CBM. The corneal epithelium moved rapidly over the CBM or stroma to cover the defect within 2-3 d, whereas the conjunctival epithelium required 1-2 wk. In all wounds, fibronectin and fibrin/fibrinogen were deposited onto the bare surface within 8 h after wounding and persisted under the migrating epithelium until migration was complete. Bullous pemphigoid antigen (BPA), a normal component of the CBM, was removed with the epithelium upon scrape wounding and reappeared in the CBM after migration was completed. In contrast, the conjunctival epithelium had a continuous subepithelial band of BPA out to the migrating tip. Laminin, also a normal component of the CBM, was not removed in the scrape wounds, indicating that the region of least resistance to shear stress was between the BPA and laminin layers. Laminin was removed by superficial keratectomy and was not detectable under the leading edge of the migrating cells. Laminin and BPA were restored in the CBM by 2-4 wk. Type IV collagen could not be detected in normal CBM, but was conspicuously present in conjunctival basement membrane and in blood vessels. Focal bands of type IV collagen did appear in the newly synthesized CBM 2-4 wk after keratectomy. These results argue that BPA, laminin, and type IV collagen are not essential for the migration of corneal epithelium during wound healing and support the hypothesis that fibronectin and fibrin/fibrinogen are the common, perhaps the essential, components of the provisional matrix that serves as a substrate until the permanent attachment components are regenerated.


The effects of distractors in multiple object tracking are modulated by the similarity of distractor and target features.
Is the effect of distractors in multiple object tracking dependent on the distractors sharing the features of the targets? In experiment 1, observers tracked five targets among five distractors that were identical to the targets and a number of additional distractors that were either identical to or featurally distinct from the targets. Results showed that distractors that are distinct from the targets in shape or color, or are stationary, impair tracking less than distractors that are identical to the targets. However, tracking performance declined as the number of distractors increased, even for featurally distinct distractors. Experiment 2 showed that distractors that differ from the targets on two features impair tracking less than distractors that differ from the targets on only one feature, but only when target tracking load is low. These results indicate that shape, color, and motion information about distractors can be used to distinguish them from targets during tracking, although even distractors with a different feature from the targets produce some interference with tracking. These findings suggest that the effect of distractors on tracking is dependent on top-down settings for target features.


The validation of a social functioning questionnaire in an African postconflict context.
This study evaluated the reliability and criterion validity of the Byumba Social Functioning Questionnaire (BSFQ), an instrument to measure social functioning in Rwanda. The instrument was locally composed in concordance with a well-described method for culture-specific and sex-specific function assessment. Respondents in a Northern Province of Rwanda (N = 393) were assessed with the BSFQ and a 19-item scale (SF-19) drawn from the Medical Outcomes Study 36-Item Short-Form (SF-36). The BSFQ's internal consistency was just acceptable for women but poor for men, while the SF-19 had good to excellent internal consisteny. BSFQ total scores showed a strong floor effect, while the SF-19 showed more variation in total score distribution. The BSFQ did not perform as well as we expected, and appears not to be suitable for measuring social functioning in the study context. This outcome seems to reflect the conceptualization of social functioning used in constructing the BSFQ. Implications for the development of culture-specific measures of functional status are discussed.


Polar functional group-containing glycolipid CD1d ligands modulate cytokine-biasing responses and prevent experimental colitis.
The MHC class I-like molecule CD1d is a nonpolymorphic antigen-presenting glycoprotein, and its ligands include glycolipids, such as α-GalCer. The complexes between CD1d and ligands activate natural killer T cells by T cell receptor recognition, leading to the secretion of various cytokines (IFN-γ, IL-4, IL-17A, etc.). Herein, we report structure-activity relationship studies of α-GalCer derivatives containing various functional groups in their lipid acyl chains. Several derivatives have been identified as potent CD1d ligands displaying higher cytokine induction levels and/or unique cytokine polarization. The studies also indicated that flexibility of the lipid moiety can affect the binding affinity, the total cytokine production level and/or cytokine biasing. Based on our immunological evaluation and investigation of physicochemical properties, we chose bisamide- and Bz amide-containing derivatives 2 and 3, and evaluated their in vivo efficacy in a DSS-induced model of ulcerative colitis. The derivative 3 that exhibits Th2- and Th17-biasing responses, demonstrated significant protective effects against intestinal inflammation in the DSS-induced model, after a single intraperitoneal injection.


Central nervous system regeneration: from leech to opossum.
A major problem of neurobiology concerns the failure of injured mammalian spinal cord to repair itself. This review summarizes work done on two preparations in which regeneration can occur: the central nervous system of an invertebrate, the leech, and the spinal cord of an immature mammal, the opossum. The aim is to understand cellular and molecular mechanisms that promote and prevent regeneration. In the leech, an individual axon regrows successfully to re-establish connections with its synaptic target, while avoiding other neurons. Functions that were lost are thereby restored. Moreover, pairs of identified neurons become re-connected with appropriate synapses in culture. It has been shown that microglial cells and nitric oxide play key roles in leech CNS regeneration. In the opossum, the neonatal brain and spinal cord are so tiny that they survive well in culture. Fibres grow across spinal cord lesions in neonatal animals and in vitro, but axon regeneration stops abruptly between postnatal days 9 and 12. A comprehensive search has been made in spinal cords that can and cannot regenerate to identify genes and establish their locations. At 9 days, growth-promoting genes, their receptors and key transcription molecules are up-regulated. By contrast at 12 days, growth-inhibitory molecules associated with myelin are prominent. The complete sequence of the opossum genome and new methods for transfecting genes offer ways to determine which molecules promote and which inhibit spinal cord regeneration. These results lead to questions about how basic research on mechanisms of regeneration could be 'translated' into effective therapies for patients with spinal cord injuries.


Intrathyroid epithelial thymoma (ITET) and carcinoma showing thymus-like differentiation (CASTLE): CD5-positive neoplasms mimicking squamous cell carcinoma of the thyroid.
Carcinoma of possible thymic epithelial origin may occur within the thyroid gland, which was first reported by Miyauchi et al. as intrathyroid epithelial thymoma (ITET). ITET is a rare tumor comprising about 0.08% of all primary thyroid malignancies. It is a low-grade thyroid carcinoma with squamous cell differentiation whose overall survival rate was found to be 71%. Lymph node metastasis at surgery was found in 40% and hematogenous metastases developed in bones, liver and lungs. This tumor grows within the thyroid gland and invades into the thyroid parenchyma as well as into the extrathyroid structures. It is a well-circumscribed solid tumor with a sharp tumor border, but is not capsulated. After fixation, the cut surface of the tumor is gray-white in color and is a solid tumor with lobulation. Tumor calcification was not detected in our 15 cases. The tumor cells show solid sheets of growth with occasional keratinization without follicular or papillary structures. Lymphocytic infiltration in the stroma is one of the most characteristic features of this tumor. The tumor cells are polygonal epithelial cells with distinct nucleoli and ill-defined cell border. Positive immunoreactivity for CD5 is a key feature to differentiate it from undifferentiated carcinoma, poorly differentiated carcinoma, medullary (C cell) carcinoma and high-grade squamous cell carcinoma (so-called primary squamous cell carcinoma) of the thyroid. Negative immunoreactivity for calcitonin, TTF1 and thyroglobulin, and positive immunoreactivity for p63 and KIT are also helpful for differential diagnosis. Nuclear atypia is mild and mitoses are less frequent, with an intermediate proliferation index (MIB-1 labeling index is usually less than 20%), which are also helpful to differentiate it from high-grade primary squamous cell carcinoma of the thyroid. The tumors in our 15 cases demonstrate 3 histological subtypes: keratinizing squamous cell carcinoma type, non-keratinizing basaloid cell carcinoma (lymphoepithelioma-like) type and neuroendocrine carcinoma type, which correspond to subtypes of the mediastinal thymic carcinomas.


Sphinx: merging knowledge-based and ab initio approaches to improve protein loop prediction.
Loops are often vital for protein function, however, their irregular structures make them difficult to model accurately. Current loop modelling algorithms can mostly be divided into two categories: knowledge-based, where databases of fragments are searched to find suitable conformations and ab initio, where conformations are generated computationally. Existing knowledge-based methods only use fragments that are the same length as the target, even though loops of slightly different lengths may adopt similar conformations. Here, we present a novel method, Sphinx, which combines ab initio techniques with the potential extra structural information contained within loops of a different length to improve structure prediction. We show that Sphinx is able to generate high-accuracy predictions and decoy sets enriched with near-native loop conformations, performing better than the ab initio algorithm on which it is based. In addition, it is able to provide predictions for every target, unlike some knowledge-based methods. Sphinx can be used successfully for the difficult problem of antibody H3 prediction, outperforming RosettaAntibody, one of the leading H3-specific ab initio methods, both in accuracy and speed. Sphinx is available at http://opig.stats.ox.ac.uk/webapps/sphinx. firstname.lastname@example.com. Supplementary data are available at Bioinformatics online.


Serum human chorionic gonadotropin: surveillance after radical and conservative management of ectopic pregnancy.
Monitoring of serum hCG concentrations has become an integral part of the postoperative management of patients with ectopic pregnancy. We monitored serial serum hCG concentrations in 25 patients to determine and compare the time for eventual clearance for two groups of patients managed by either radical or conservative surgery. Fifteen patients with ruptured ectopic pregnancy were managed by salpingectomy (group A) and 10 patients with unruptured ectopic pregnancy were managed by salpingostomy (group B). Serum samples were drawn on the first postoperative day (day 0) and at three-day intervals thereafter and followed to the limits of detectability. Serum hCG levels on day 0 were 3,542 +/- 2,480 mIU/mL (mean +/- standard deviation [SD]) with a range of 117 to 10,300 mIU/mL for group A, and 1,022 +/- 282 mIU/mL (mean +/- SD) with a range of 17 to 4,180 mIU/mL for group B. Time required for clearance to less than 10 mIU/mL was 14 +/- 4.9 days with a range of 2 to 28 days for group A and 12 +/- 3.9 days with a range of 2 to 16 days for group B. The primary factor influencing clearance was initial hCG concentration. Based on clearance data, a nomogram was developed to assist in the postoperative management and timing of serum sampling for hCG.


Artificial intelligence-guided screening for atrial fibrillation using electrocardiogram during sinus rhythm: a prospective non-randomised interventional trial.
Previous atrial fibrillation screening trials have highlighted the need for more targeted approaches. We did a pragmatic study to evaluate the effectiveness of an artificial intelligence (AI) algorithm-guided targeted screening approach for identifying previously unrecognised atrial fibrillation. For this non-randomised interventional trial, we prospectively recruited patients with stroke risk factors but with no known atrial fibrillation who had an electrocardiogram (ECG) done in routine practice. Participants wore a continuous ambulatory heart rhythm monitor for up to 30 days, with the data transmitted in near real time through a cellular connection. The AI algorithm was applied to the ECGs to divide patients into high-risk or low-risk groups. The primary outcome was newly diagnosed atrial fibrillation. In a secondary analysis, trial participants were propensity-score matched (1:1) to individuals from the eligible but unenrolled population who served as real-world controls. This study is registered with ClinicalTrials.gov, NCT04208971. 1003 patients with a mean age of 74 years (SD 8·8) from 40 US states completed the study. Over a mean 22·3 days of continuous monitoring, atrial fibrillation was detected in six (1·6%) of 370 patients with low risk and 48 (7·6%) of 633 with high risk (odds ratio 4·98, 95% CI 2·11-11·75, p=0·0002). Compared with usual care, AI-guided screening was associated with increased detection of atrial fibrillation (high-risk group: 3·6% [95% CI 2·3-5·4] with usual care vs 10·6% [8·3-13·2] with AI-guided screening, p<0·0001; low-risk group: 0·9% vs 2·4%, p=0·12) over a median follow-up of 9·9 months (IQR 7·1-11·0). An AI-guided targeted screening approach that leverages existing clinical data increased the yield for atrial fibrillation detection and could improve the effectiveness of atrial fibrillation screening. Mayo Clinic Robert D and Patricia E Kern Center for the Science of Health Care Delivery.


Growth and Detachment of Oxygen Bubbles Induced by Gold-Catalyzed Decomposition of Hydrogen Peroxide.
Whereas bubble growth out of gas-oversatured solutions has been quite well understood, including the formation and stability of surface nanobubbles, this is not the case for bubbles forming on catalytic surfaces due to catalytic reactions, though it has important implications for gas evolution reactions and self-propulsion of micro/nanomotors fueled by bubble release. In this work we have filled this gap by experimentally and theoretically examining the growth and detachment dynamics of oxygen bubbles from hydrogen peroxide decomposition catalyzed by gold. We measured the bubble radius R(t) as a function of time by confocal microscopy and find R(t) ∝ t1/2. This diffusive growth behavior demonstrates that the bubbles grow from an oxygen-oversaturated environment. For several consecutive bubbles detaching from the same position in a short period of time, a well-repeated growing behavior is obtained from which we conclude the absence of noticeable depletion effect of oxygen from previous bubbles or increasing oversaturation from the gas production. In contrast, for two bubbles far apart either in space or in time, substantial discrepancies in their growth rates are observed, which we attribute to the variation in the local gas oversaturation. The current results show that the dynamical evolution of bubbles is influenced by comprehensive effects combining chemical catalysis and physical mass transfer. Finally, we find that the size of the bubbles at the moment of detachment is determined by the balance between buoyancy and surface tension and by the detailed geometry at the bubble's contact line.


Antibody class switching differs among SJL, C57BL/6 and 129 mice.
Inbred mouse strains used for gene manipulation studies vary in many respects, including immune system function. These differences can interfere with data interpretation unless the mice are well backcrossed. Here, we show that antibody class switching to IgG3 in cultured splenic B cells from Swiss James Lambert (SJL) and 129/Sv mice is 2- to 6-fold less efficient compared with C57BL/6 (B6). Under optimal stimulation conditions, IgA switching is also 2- to 19-fold lower in SJL and 129/Sv B cells. Splenic B cells from SJL mice express higher levels of CD19 and CD21 compared with B6, and their CD21(high)CD23(low) B cells have little CD9 expression, suggesting atypical marginal zone (MZ) B cells. However, sort purification of splenic B cell subsets did not equalize in vitro class switching to IgG3 or IgA between SJL and B6. 129/Sv spleens have a 3-fold greater number of MZ B cells compared with B6, with similar CD9 expression. Poor IgG3 switching by 129/Sv B cells is specific to CD23(high) follicular B cells, whereas similar changes in IgA switching are seen in both CD21(high) and CD23(high) B cell subsets from 129/Sv. Therefore, the functions and phenotypes of mature B cells differ among three common strains of mice.


Amplification of hofmeister effect by alcohols.
We have demonstrated that Hofmeister effect can be amplified by adding alcohols to aqueous solutions. The lower critical solution temperature behavior of poly(N-isopropylacrylamide) has been employed as the model system to study the amplification of Hofmeister effect. The alcohols can more effectively amplify the Hofmeister effect following the series methanol < ethanol < 1-propanol < 2-propanol for the monohydric alcohols and following the series d-sorbitol ≈ xylitol ≈ meso-erythritol < glycerol < ethylene glycol < methanol for the polyhydric alcohols. Our study reveals that the relative extent of amplification of Hofmeister effect is determined by the stability of the water/alcohol complex, which is strongly dependent on the chemical structure of alcohols. The more stable solvent complex formed via stronger hydrogen bonds can more effectively differentiate the anions through the anion-solvent complex interactions, resulting in a stronger amplification of Hofmeister effect. This study provides an alternative method to tune the relative strength of Hofmeister effect besides salt concentration.


Celiac disease in the Turkish population.
Celiac disease (CD) is characterized by malabsorption of nutrients in the small intestine. The availability of highly specific and sensitive serologic tests has facilitated its diagnosis, increasing the disease prevalence. The aim of this study was to determine the clinical, laboratory, and histopathological features of CD in Turkish adults. Between 1968 and 2002, CD patients presenting to the Gastroenterology Unit were evaluated retrospectively. From 2002, newly diagnosed patients were prospectively followed up. Sixty patients (39 female, 21 male) were included in the study. Mean body mass index was 22.2 +/- 5.4 kg/m2. The most common symptoms were diarrhea, weight loss, and flatulence. Most common comorbidities were anemia, osteoporosis, type 1 diabetes mellitus, and steatohepatitis. Six (10.0%) patients had a family history of diabetes mellitus; one (1.7%) patient had a family history of CD. Plasma glucose and serum gamma-glutamyltransferase levels were significantly higher in females than males. Most common histopathological findings were increased lymphocytes in the lamina propria (76.2%) and villus epithelium (59.5%). Over the years, the cumulative frequency of CD increased more in females than males. This is the first study in the literature showing the characteristics of CD in Turkish adults. In our previous recent study, the prevalence of tissue transglutaminase antibody positivity in Turkish healthy blood donors was 1.3%, indicating a high prevalence of CD in our population. In this study, the cumulative frequency of CD increased more in females than males. With the better understanding and increased suspicion of the disease, more patients are being diagnosed in our population.


[Identification of Malassezia species by PCR-REA].
The genus Malassezia has acquired relevance in the last years for its pathological associations. Since 1996, the genus comprises M. pachydermatis, M. furfur, M. sympodialis, M. slooffiae, M. obtusa, M. globosa and M. restricta. The identification scheme based on morphological, physiological and biochemical characteristics does not resolve ambiguity between some species. The aim of this study was to find out a fast, specific and efficient methodology to distinguish between Malassezia species. Based on a previous technique for polymerase chain reaction-restriction enzyme analysis (PCR-REA), modifications were applied on DNA extraction and purification and the electrophoresis. These changes produced a cheaper, reliable and rapid method as identification procedure with easily defined results. The proposed modifications would allow a better knowledge on ecological and pathogenic roles of Malassezia, studies that have not yet been established.


Determination of nitrate concentration and its risk assessment in bottled water in Iran.
Bottled water is one of the sources of drinking water in many arid and semi-arid countries, including Iran. The greatest concern is the health effects of exposure to excessive nitrate concentrations in drinking water due to the development of methemoglobinemia in children. Therefore, the present study was aimed at determining the concentration of nitrate and its risk assessment in drinking water bottled in Iran. 71 different bottled water brands were identified in this study. The nitrate concentration in water samples was then measured using an Ion Chromatography No. 4110 in accordance Standard Methods for the Examination of Water and Wastewater. The hazard quotient (HQ) of nitrate was calculated using the formula based on input variables including nitrate concentration, water per capita, body weight and reference dose. The results showed that the concentration of nitrate in bottled water was in the range of 0.146-50.1 mg/L (average 10.55 mg/L) in one of which, the concentration of nitrate was higher than the WHO guideline. The mean EDI values for nitrate in different groups of infants, children, teenagers and adults were 0.0795, 0.5633, 0.3976 and 0.3186 mg/kg, respectively. The mean HQ values for nitrate in different groups of infants, children, teenagers and adults were 0.0528, 0.3737, 0.2638 and 0.2114, respectively. In general, the hazard quotient (HQ>1) for the population consuming bottled water, appropriate strategies should be considered in order to reduce the concentration of nitrate in bottled water.


Defective Fc-, CR1- and CR3-mediated monocyte phagocytosis and chemotaxis in common variable immunodeficiency and X-linked agammaglobulinemia patients.
Blood monocyte phagocytic functions were evaluated by chemotaxis, phagocytosis, and superoxide anion production in nine patients with common variable immunodeficiency (CVI), eight patients with X-linked agammaglobulinemia (XLA), and in 17 normal subjects. Further laboratory diagnosis included the determination of the Bruton's tyrosine kinase (Btk) protein expression in monocytes using flow cytometry. The analysis of monocyte phagocytic function demonstrated that CR3-, CR1-, and Fc-mediated phagocytosis (p = 0.0001) were significantly decreased in CVI and XLA patients, and chemotaxis of monocytes (p = 0.0082) was reduced in XLA patients. Superoxide anion production, however, did not differ between the CVI, XLA, and the control groups. The cytoplasmic expression of Btk protein in monocytes was normal in CVI patients and decreased or not detected in XLA patients. It is proposed that impaired chemotaxis and phagocytosis by monocytes may be a characteristic of the innate immune system in CVI and XLA patients, providing a new direction for the physiopathology of these immunodeficiencies.


High Mobility Group Box 1 Promotes Angiogenesis from Bone Marrow-derived Endothelial Progenitor Cells after Myocardial Infarction.
High mobility group box 1 (HMGB1) is a DNA-binding protein secreted into the extracellular space from necrotic cells that acts as a cytokine. We examined the role of HMGB1 in angiogenesis from bone marrow-derived cells in the heart using transgenic mice exhibiting the cardiac-specific overexpression of HMGB1 (HMGB1-TG). HMGB1-TG mice and wild-type littermate (WT) mice were lethally irradiated and injected with bone marrow cells from green fluorescent protein mice through the tail vein. After bone marrow transplantation, the left anterior descending artery was ligated to induce myocardial infarction (MI). Flow cytometry revealed that the levels of circulating endothelial progenitor cells (EPCs) mobilized from the bone marrow increased after MI in the HMGB-TG mice versus the WT mice. In addition, the size of MI was smaller in the HMGB1-TG mice than in the WT mice, and immunofluorescence staining demonstrated that the number of engrafted vascular endothelial cells derived from bone marrow in the border zones of the MI areas was increased in the HMGB1-TG mice compared to that observed in the WT mice. Moreover, the levels of cardiac vascular endothelial growth factor after MI were higher in the HMGB1-TG mice than in the WT mice. The present study demonstrated that HMGB1 promotes angiogenesis and reduces the MI size by enhancing the mobilization and differentiation of bone marrow cells to EPCs as well as their migration to the border zones of the MI areas and engraftment as vascular endothelial cells in new capillaries or arterioles in the infarcted heart.


Unidentified Factors in Jojoba Meal Prevent Oviduct Development in Broiler Breeder Females.
Supplementation of feed with jojoba meal, as a means for autonomous feed restriction, was successful in depressing feed intake and controlling body weight of broiler breeder pullets to the extent recommended by the breeder company. However, these broiler breeders never produced eggs. At the level of ovary, normal follicle development and maturation did occur. A considerable number of ovulations occurred which were not followed by oviposition. After ovulation, the ova could not be captured by the oviduct, because of the small size of the oviduct, resulting in "internal laying". The virtual absence of oviduct development cannot be explained presently but it must be due to some yet unidentified factor(s) in jojoba meal which prevent(s) the normal development of the oviduct. These factors may be acting by abnormally increasing plasma progesterone or triiodothyronin levels and/or directly by themselves interfering with oviduct development. The nature of these factors requires further investigations.


Growth properties and alloantigenic expression of murine lymphoblastoid cell lines.
Murine lymphoblastoid cell lines were evaluated for their expression of Thy-1 and thymus leukemia (TL) differentiation alloantigens. Two culture conditions were shown to affect this expression. Cells grown in fetal bovine serum (FBS)-enriched medium expressed up to 15 times the amount of TL as cells grown in horse serum (HS)-enriched medium. Thy-1 expression was less affected by the type of serum used for culture. The phase of growth when the cells were harvested, was demonstrated to affect the expression of Thy-1. The expression of Thy-1.2 for one cell line examined, L-251A, during logarithmic growth was threefold greater than cells collected during either lag or stationary growth. When culture conditions were standardized a ranking of the amount of Thy-1 and TL expressed by several cell lines was made. All cell lines, except one, L-1210, expressed Thy-1. There was a 450-fold difference in the expression of Thy-1 between the cell lines evaluated. Seven cell lines expressed TL-1,2,3 with a ninefold difference in the amount of expression. The L-251A cell line was cultured in a 14 liter fermentor for a 26 day period. During this time TL and Thy-1 expression did not vary significantly, demonstrating that lymphoblastoid cell lines can be cultured on a continuous basis and will continue to express their surface alloantigens.


[Malacoplakia-like reaction in association with colorectal adenocarcinoma].
17 cases of malacoplakia in association with an adenocarcinoma of the colon or rectum are known. In this report we describe a new case of this rare combination. An 83-year-old comatose female patient was admitted in an emergency situation because of an incipient ileus. A carcinoma had been known for months, but the patient had previously rejected any diagnostic or therapeutic measures. A tumor (6 cm in diameter) in the upper rectum was removed. This tumor had caused an obstruction of the lumen and reached the serosa. Histologically, an adenocarcinoma with suppurations in the stroma was staged pT3, G2, pN0 (clinically pT4 because of the perforated colonic wall). PAS- and von-Kossa-positive Michaelis-Gutmann-bodies were found in macrophages. Ultrastructurally, they turned out to be circular shifted early stage calcifications. Any malacoplakia-like lesion in suspicious areas of the colonic wall should be followed up by further diagnostic measures, since it may indicate the presence of an adenocarcinoma.


Are New Gender-Neutral Pronouns Difficult to Process in Reading? The Case of Hen in SWEDISH.
Hen is a Swedish gender-neutral pronoun used for non-binary individuals and as a generic singular pronoun form. Hen was added to the Swedish Academy Glossary (SAOL) in 2015, and opponents of hen have argued that gender-neutral pronouns are difficult to process, and therefore should not be used. As of yet, this has not been empirically tested. This pre-registered study used eye-tracking to experimentally test if hen has a processing cost by measuring the process of understanding whom a pronoun refers to (i.e., pronoun resolution). Participants (N = 120) read 48 sentence pairs where the first sentence included a noun referring to a person (e.g., sister, hairdresser, person) and the second included a pronoun referring to the noun. The pronouns were either gendered (she and he) or gender-neutral (hen). The nouns were either neutral (e.g., person, colleague) or gendered, either by lexically referring to gender (e.g., sister, king), or by being associated with stereotypes based on occupational gender segregation (e.g., occupational titles like hairdresser, carpenter). We tested if hen had a greater processing cost than gendered pronouns, and whether the type of noun moderated this effect. The hypotheses were that hen referring to neutral nouns would lead to a smaller processing cost than hen referring to gendered nouns. Furthermore, we hypothesized that hen referring to lexically gendered nouns would lead to larger processing costs than stereotypically gendered role nouns. The processing cost of hen was measured by reading time spent on three regions of the sentence pairs; the pronoun, the spillover region (i.e., the words following the pronoun), and the noun. The only processing cost for hen occurred in the spillover region. The processing cost in this region was greater when hen referred to neutral nouns than when hen referred to a noun associated with gender. In contrast to the hypothesis, the type of gender information associated with the noun did not interact with these effects (i.e., the same reading time for hen following e.g., the queen or carpenter). Altogether, the results do not support that gender-neutral pronouns should be avoided because they are difficult to process.


An adult learning perspective on disability and microfinance: The case of Katureebe.
Despite Uganda's progress in promoting affirmative action for persons with disabilities and its strategy of using microfinance to fight poverty, access to microfinance services by persons with disabilities is still problematic due to barriers, characterised by discrepancies between policies and practices. Regarding education, the affirmative action in favour of learners with disabilities has not translated into actual learning opportunities due to personal and environmental barriers. The study on which this article is based investigated the non-formal and informal adult learning practices regarding microfinance that persons with disabilities engaged in. This article seeks to illuminate the barriers that a person with a visual impairment encountered while learning about and engaging with microfinance and the strategies that he developed to overcome them. This was a case study, framed within the social model of disability and critical research paradigm. Data were collected through in-depth interviews of a person with visual impairment and observations of the environment in which adult learning and engagement with Savings and Credit Cooperative Organisations (SACCOs) occurred. Findings indicate that the person with a visual disability faced barriers to learning about microfinance services. He experienced barriers in an integrated manner and developed strategies to overcome these barriers. The barriers and strategies are theorised using the social model of disability. The case of a person with visual impairment suggests that persons with disabilities face multiple barriers regarding microfinance, including social, psychological and educational. However, his own agency and attitudes were also of importance as they influenced his learning. Viewing these barriers as blockades can lead to non-participation in learning and engagement with microfinance whereas viewing them as surmountable hurdles can potentially motivate participants to succeed in learning about and engaging with microfinance.


The Wisconsin epidemiologic study of diabetic retinopathy. II. Prevalence and risk of diabetic retinopathy when age at diagnosis is less than 30 years.
In a population-based study in southern Wisconsin, 996 insulin-taking, younger-onset diabetic persons were examined using standard protocols to determine the prevalence and severity of diabetic retinopathy and associated risk variables. The prevalence of diabetic retinopathy varied from 17% to 97.5% in persons with diabetes for less than five years and 15 or more years, respectively. Proliferative retinopathy varied from 1.2% to 67% in persons with diabetes for less than ten years and 35 or more years, respectively. For persons with diabetes of 10 years' duration or less, the Cox regression model relates the severity or retinopathy to longer duration, older age at examination, and higher levels of glycosylated hemoglobin. After ten years of diabetes, severity of retinopathy was related to longer duration, high levels of glycosylated hemoglobin, presence of proteinuria, higher diastolic BP, and male sex.


The effect of nontemporal stimulus size on perceived duration as assessed by the method of reproduction.
Perceived duration is assumed to be positively related to nontemporal stimulus magnitude. Most recently, the finding that larger stimuli are perceived to last longer has been challenged to represent a mere decisional bias induced by the use of comparative duration judgments. Therefore, in the present study, the method of temporal reproduction was applied as a psychophysical procedure to quantify perceived duration. Another major goal was to investigate the influence of attention on the effect of visual stimulus size on perceived duration. For this purpose, an additional dual-task paradigm was employed. Our results not only converged with previous findings in demonstrating a functional positive relationship between nontemporal stimulus size and perceived duration, but also showed that the effect of stimulus size on perceived duration was not confined to comparative duration judgments. Furthermore, the effect of stimulus size proved to be independent of attentional resources allocated to stimulus size; nontemporal visual stimulus information does not need to be processed intentionally to influence perceived duration. Finally, the effect of nontemporal stimulus size on perceived duration was effectively modulated by the duration of the target intervals, suggesting a hitherto largely unrecognized role of temporal context for the effect of nontemporal stimulus size to become evident.


Functional capacity and dyspnea during follow-up after acute pulmonary embolism.
Dissecting the determinants of functional capacity during long-term follow-up after acute pulmonary embolism (PE) can help to better characterize a patient population with persisting limitation. In a prospective cohort study, consecutive unselected survivors of acute PE underwent 3- and 12-month follow-up, including six-minute walking distance (6MWD) and dyspnea assessment with the modified Medical Research Council (mMRC) scale. We used reference equations adjusting for age, sex, and anthropometric measurements to define abnormal 6MWD. Overall, 323 of 363 (89.0%) patients had at least one recorded 6MWD value at one year. At 3 months, the prevalence of abnormal 6MWD was 21.9% and at 12 months it was 18.3%. At 3 and 12 months, 58.8% and 52.1% with abnormal 6MWD did not report dyspnea, respectively. On average and during follow-up, 6MWD significantly improved with time, while the mMRC dyspnea scale did not. Abnormal 6MWD was associated with younger age (odds ratio per decade, 0.91; 95% CI, 0.88-0.94), higher body mass index (1.10; 1.03-1.17), smoking (3.53; 1.34-9.31), intermediate- or high-risk PE (3.21; 1.21-8.56), and higher mMRC grading (2.28; 1.59-3.27). Abnormal 6MWD at 3 months was associated with the prospectively defined endpoint of post-PE impairment (3.72; 1.50-9.28) and with poor disease-specific and generic health-related quality of life. Three months after PE, 37% of patients reported dyspnea and 22% had abnormal 6MWD. After a year, 20% still had abnormal 6MWD. Dyspnea correlated with abnormal 6MWD, but over 50% of patients with abnormal 6MWD did not report dyspnea. Abnormal 6MWD predicted subsequent post-pulmonary embolism impairment and worse long-term quality of life. German Clinical Trials Register Identifier DRKS00005939.


Characterization of CDOM in saline and freshwater lakes across China using spectroscopic analysis.
Colored dissolved organic matter (CDOM) is a major component of DOM in waters, and plays a vital role in carbon cycling in inland waters. In this study, the light absorption and three-dimensional excitation-emission matrix spectra (EEMs) of CDOM of 936 water samples collected in 2014-2017 from 234 lakes in five regions across China were examined to determine relationships between lake water sources (fresh versus saline) and their fluorescence/absorption characteristics. Results indicated significant differences regarding DOC concentration and aCDOM(254) between freshwater (6.68 mg C L-1, 19.55 m-1) and saline lakes (27.4 mg C L-1, 41.17 m-1). While humic-like (F5) and fulvic-like (F3) compounds contributed to CDOM fluorescence in all lake waters significantly, their contribution to total fluorescence intensity (FT) differed between saline and freshwater lakes. Significant negative relationships were also observed between lake altitude with either F5 (R2 = 0.63, N = 306) or FT (R2 = 0.64, N = 306), suggesting that the abundance of humic-like materials in CDOM tends to decrease with increased in lakes altitude. In high-altitude lakes, strong solar irradiance and UV exposure may have induced photo-oxidation reactions resulting in decreased abundance of humic-like substances and the formation of low molecular weight compounds. These findings have important implications regarding our understanding of C dynamics in lacustrine systems and the contribution of these ecosystems to the global C cycle.


[Solid phase extraction and room temperature phosphorimetry coupling technique and its application].
The principle and application in the environmental analysis of Solid Phase Extraction (SPE) combined with Room Temperature Phosphorescence (RTP) analysis were reviewed. The principle, device, separation modes, operation procedures and application of SPE were commented. The merits and shortages in the operation procedure and quantitative analysis of classic RTP were also summarized. The potential application in the trace sample analysis and the advantage of coupling technique of SPE and RTP were indicated. The sensitivity and selectivity of the coupling technique could be greatly increased because of the strong enrichment ability of the SPE materials and the high selectivity of RTP. So far the technique has been successfully used in the analysis of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs) and polychlorinated dibenzofurans (PClDBFs) in the environmental samples according to their phosphorescence emission after preconcentration on the Whatman 1PS filter paper and C18 or C8 SPE membranes.


New sources of resistance to leafminers (Liriomyza sativae) in melon (Cucumis melo L.) germplasm.
Leafminers (Liriomyza sativae) are the main melon (Cucumis melo L.) pests in Northeast Brazil, which is the main region for the production and export of the fruit in Brazil. Of the integrated management strategies available, genetic resistance is the best method of preventing damage by these insects. The aim of this study was to select sources of resistance to leafminers in melon germplasm. Seven experiments were conducted in the laboratory, field, and greenhouse, with and without choice, using 52 melon accessions and 4 commercial hybrids as controls. Genetic variability among the accessions made it possible to select four new sources of resistance: 'CNPH 11-1072' and 'CNPH 11-1077', because they exhibited lower levels of infestation by the insect (antixenosis); and 'CNPH 00-915(R)' and 'BAGMEL 56(R)', because the pest larvae died soon after beginning to feed on the leaf mesophyll (antibiosis).


Targeting the BMK1 MAP kinase pathway in cancer therapy.
The big mitogen activated protein kinase 1 (BMK1) pathway is the most recently discovered and least-studied mammalian mitogen-activated protein (MAP) kinase cascade, ubiquitously expressed in all types of cancer cells tested so far. Mitogens and oncogenic signals strongly activate this cellular MAP kinase pathway, thereby passing down proliferative, survival, chemoresistance, invasive, and angiogenic signals in tumor cells. Recently, several pharmacologic small molecule inhibitors of this pathway have been developed. Among them, the BMK1 inhibitor XMD8-92 blocks cellular BMK1 activation and significantly suppresses tumor growth in lung and cervical tumor models and is well tolerated in animals. On the other hand, MEK5 inhibitors, BIX02188, BIX02189, and compound 6, suppress cellular MEK5 activity, but no data exist to date on their effectiveness in animals.


Resistance and Recovery of Methane-Oxidizing Communities Depends on Stress Regime and History; A Microcosm Study.
Although soil microbes are responsible for important ecosystem functions, and soils are under increasing environmental pressure, little is known about their resistance and resilience to multiple stressors. Here, we test resistance and recovery of soil methane-oxidizing communities to two different, repeated, perturbations: soil drying, ammonium addition and their combination. In replicated soil microcosms we measured methane oxidation before and after perturbations, while monitoring microbial abundance and community composition using quantitative PCR assays for the bacterial 16S rRNA and pmoA gene, and sequencing of the bacterial 16S rRNA gene. Although microbial community composition changed after soil drying, methane oxidation rates recovered, even after four desiccation events. Moreover, microcosms subjected to soil drying recovered significantly better from ammonium addition compared to microcosms not subjected to soil drying. Our results show the flexibility of microbial communities, even if abundances of dominant populations drop, ecosystem functions can recover. In addition, a history of stress may induce changes in community composition and functioning, which may in turn affect its future tolerance to different stressors.


Toward a breast cancer vaccine: work in progress.
Advances in biotechnology and basic immunology have converged to create an unprecedented opportunity to use vaccines to harness the power of the immune system in the fight against breast cancer. Cancer vaccines have several therapeutic advantages over more traditional breast cancer treatment modalities. First, targeting the antitumor immune response to critical tumor-specific antigens defines a therapy with exquisite specificity and minimal toxicity. Second, immune-mediated tumor destruction occurs by mechanisms distinct from those underlying the efficacy of chemotherapy and hormone therapy. Thus, immunotherapy offers an approach to circumventing the intrinsic drug resistance that currently underlies therapeutic failure. Third, the phenomenon of immunologic memory endows immunotherapy with the potential for creating a durable therapeutic effect that is reactivated at the onset of disease relapse. Moreover, immunologic memory also underlies the potential future use of vaccines for the prevention of breast cancer. Early clinical trials have highlighted the promise of breast cancer vaccines, and have further defined the challenges facing translational scientists and clinical investigators. The judicious application of laboratory advances to clinical trial design should facilitate the development of immunotherapy as an additional major therapeutic modality for breast cancer, with the potential for breast cancer prevention as well as treatment.


Functional characterization of Candida albicans ABC transporter Cdr1p.
In view of the importance of Candida drug resistance protein (Cdr1p) in azole resistance, we have characterized it by overexpressing it as a green fluorescent protein (GFP)-tagged fusion protein (Cdr1p-GFP). The overexpressed Cdr1p-GFP in Saccharomyces cerevisiae is shown to be specifically labeled with the photoaffinity analogs iodoarylazidoprazosin (IAAP) and azidopine, which have been used to characterize the drug-binding sites on mammalian drug-transporting P-glycoproteins. While nystatin could compete for the binding of IAAP, miconazole specifically competed for azidopine binding, suggesting that IAAP and azidopine bind to separate sites on Cdr1p. Cdr1p was subjected to site-directed mutational analysis. Among many mutant variants of Cdr1p, the phenotypes of F774A and DeltaF774 were particularly interesting. The analysis of GFP-tagged mutant variants of Cdr1p revealed that a conserved F774, in predicted transmembrane segment 6, when changed to alanine showed increased binding of both photoaffinity analogues, while its deletion (DeltaF774), as revealed by confocal microscopic analyses, led to mislocalization of the protein. The mislocalized DeltaF774 mutant Cdr1p could be rescued to the plasma membrane as a functional transporter by growth in the presence of a Cdr1p substrate, cycloheximide. Our data for the first time show that the drug substrate-binding sites of Cdr1p exhibit striking similarities with those of mammalian drug-transporting P-glycoproteins and despite differences in topological organization, the transmembrane segment 6 in Cdr1p is also a major contributor to drug substrate-binding site(s).


Genomic profiling of gastric cancer predicts lymph node status and survival.
Gastric carcinogenesis is driven by an accumulation of genetic changes that to a large extent occur at the chromosomal level. We analysed the patterns of chromosomal instability in 35 gastric carcinomas and their clinical correlations. With microarray competitive genomic hybridization, genomewide chromosomal copy number changes can be studied with high resolution and sensitivity. A genomewide scanning array with 2275 BAC and P1 clones spotted in triplicate was used. This array provided an average resolution of 1.4 Mb across the genome. Patterns of chromosomal aberrations were analysed by hierarchical cluster analysis of the normalized log(2) tumour to normal fluorescence ratios of all clones, and cluster membership was correlated to clinicopathological data including survival. Hierarchical cluster analysis revealed three groups with different genomic profiles that correlated significantly with lymph node status (P=0.02). Moreover, gastric cancer cases from cluster 3 showed a significantly better prognosis than those from clusters 1 and 2 (P=0.02). Genomic profiling of gastric adenocarcinomas based on microarray analysis of chromosomal copy number changes predicted lymph node status and survival. The possibility to discriminate between patients with a high risk of lymph node metastasis could clinically be helpful for selecting patients for extended lymph node resection.


The impact of simulation-based medical education on resident management of emergencies in pediatric anesthesiology.
Resident education in pediatric anesthesiology is challenging. Traditional curricula for anesthesiology residency programs have included a combination of didactic lectures and mentored clinical service, which can be variable. Limited pediatric medical knowledge, technical inexperience, and heightened resident anxiety further challenge patient care. We developed a pediatric anesthesia simulation-based curriculum to address crises related to hypoxemia and dysrhythmia management in the operating room as an adjunct to traditional didactic and clinical experiences. The primary objective of this trial was to evaluate the impact of a simulation curriculum designed for anesthesiology residents on their performance during the management of crises in the pediatric operating room. A secondary objective was to compare the retention of learned knowledge by assessment at the eight-week time point during the rotation. In this prospective, observational trial 30 residents were randomized to receive simulation-based education on four perioperative crises (Laryngospasm, Bronchospasm, Supraventricular Tachycardia (SVT), and Bradycardia) during the first week (Group A) or fifth week (Group B) of an eight-week rotation. Assessment sessions that included two scenarios (Laryngospasm, SVT) were performed in the first week, fifth week, and the eighth week of their rotation for all residents. The residents were assessed in real time and by video review using a 7-point checklist generated by a modified Delphi technique of senior pediatric anesthesiology faculty. Residents in Group A showed improvement between the first week and fifth week assessment as well as between first week and eighth week assessments without decrement between the fifth week and eighth week assessments for both the laryngospasm and SVT scenarios. Residents in Group B showed improvement between the first week and eighth week assessments for both scenarios and between the fifth week and eighth week assessment for the SVT scenario. This adjunctive simulation-based curriculum enhanced the learner's management of laryngospasm and SVT management and is a reasonable addition to didactic and clinical curricula for anesthesiology residents.


Functional heterogeneity of NMDA receptors in rat substantia nigra pars compacta and reticulata neurones.
The nigra substantia nigra pars compacta (SNc) and substantia pars reticulata (SNr) form two major basal ganglia components with different functional roles. SNc dopaminergic (DA) neurones are vulnerable to cell death in Parkinson's disease, and NMDA receptor activation is a potential contributing mechanism. We have investigated the sensitivity of whole-cell and synaptic NMDA responses to intracellular ATP and GTP application in the SNc and SNr from rats on postnatal day (P) 7 and P28. Both NMDA current density (pA/pF) and desensitization to prolonged or repeated NMDA application were greater in the SNr than in the SNc. When ATP levels were not supplemented, responses to prolonged NMDA administration desensitized in P7 SNc DA neurones but not at P28. At P28, SNr neurones desensitized more than SNc neurones, with or without added ATP. Responses to brief NMDA applications and synaptic NMDA currents were not sensitive to inclusion of ATP in the pipette solution. To investigate these differences between the SNc and SNr, NR2 subunit-selective antagonists were tested. NMDA currents were inhibited by ifenprodil (10 microM) and UBP141 (4 microM), but not by Zn(2+) (100 nm), in both the SNr and SNc, suggesting that SNc and SNr neurones express similar receptor subunits; NR2B and NR2D, but not NR2A. The different NMDA response properties in the SNc and SNr may be caused by differences in receptor modulation and/or trafficking. The vulnerability of SNc DA neurones to cell death is not correlated with NMDA current density or receptor subtypes, but could in part be related to inadequate NMDA receptor desensitization.


Inflammasome activation leads to cDC1-independent cross-priming of CD8 T cells by epithelial cell-derived antigen.
The innate immune system detects pathogens and initiates adaptive immune responses. Inflammasomes are central components of the innate immune system, but whether inflammasomes provide sufficient signals to activate adaptive immunity is unclear. In intestinal epithelial cells (IECs), inflammasomes activate a lytic form of cell death called pyroptosis, leading to epithelial cell expulsion and the release of cytokines. Here, we employed a genetic system to show that simultaneous antigen expression and inflammasome activation specifically in IECs is sufficient to activate CD8+ T cells. By genetic elimination of direct T cell priming by IECs, we found that IEC-derived antigens were cross-presented to CD8+ T cells. However, cross-presentation of IEC-derived antigen to CD8+ T cells only partially depended on IEC pyroptosis. In the absence of inflammasome activation, cross-priming of CD8+ T cells required Batf3+ dendritic cells (conventional type one dendritic cells [cDC1]), whereas cross-priming in the presence of inflammasome activation required a Zbtb46+ but Batf3-independent cDC population. These data suggest the existence of parallel inflammasome-dependent and inflammasome-independent pathways for cross-presentation of IEC-derived antigens.