Search

Background: Debriefing in operating theatre environments leads to benefits in mortality, efficiency, productivity, and safety culture; however, it is still not regularly performed. TALK© is a simple and widely applicable team self-debriefing method to collaboratively learn and improve., Methods: An interventional study introducing TALK© for voluntary clinical debriefing was carried out in operating theatre environments in a UK National Health Service hospital over 18 months. It explored compliance with the Five Steps to Safer Surgery and changes in behaviour in surgical teams regarding consideration and completion of debriefing., Results: Team briefing and compliance with the WHO surgical safety checklist were performed consistently (>95% and >98%, respectively) throughout the study, which included 460 surgical lists. Consideration of debriefing increased at all data collection periods after intervention, from 35.6% to 60.3-97.4% (P≤0.003). Performance of debriefing, which was 23.3% at baseline, reached 39% at 6 months (P=0.039). Team planning of actions for improvement during debriefing also increased (P<0.001). A decline in performance of debriefing and subsequent improvement actions was observed after 6 months, albeit rates were above baseline at 18 months. The most reported reason not to carry out a debriefing was 'lack of issues'. After implementation, nurses and allied healthcare professionals increased their contribution to initiating and leading debriefing. Reported barriers were <18% at baseline, and decreased after intervention., Conclusions: A simple intervention introducing TALK© for voluntary debriefing in theatres prompted significant changes in team behaviour and sustained growth regarding consideration and performance of debriefing, especially in the first 6 months., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Mutation of the GABRA1 gene is associated with neurodevelopmental defects and epilepsy. GABRA1 encodes for the α1 subunit of the γ-aminobutyric acid type A receptor (GABA A R), which regulates the fast inhibitory impulses of the nervous system. Multiple model systems have been developed to understand the function of GABRA1, but these models have produced complex and, at times, incongruent data. Thus, additional model systems are required to validate and substantiate previous results. We sought to provide initial phenotypic analysis of a novel germline mutant allele. Our analysis provides a solid foundation for the future use of this allele to characterize gabra1 functionally and pharmacologically using zebrafish. We investigated the behavioral swim patterns associated with a nonsense mutation of the zebrafish gabra1 (sa43718 allele) gene. The sa43718 allele causes a decrease in gabra1 mRNA expression, which is associated with light induced hypermotility, one phenotype previously associated with seizure like behavior in zebrafish. Mutation of gabra1 was accompanied by decreased mRNA expression of gabra2, gabra3, and gabra5, indicating a reduction in the expression of additional α sub-units of the GABA A R. Although multiple sub-units were decreased, larvae continued to respond to pentylenetetrazole (PTZ), indicating that a residual GABA A R exists in the sa43718 allele. Proteomics analysis demonstrated that mutation of gabra1 is associated with abnormal expression of proteins that regulate synaptic vesicle fusion, vesicle transport, synapse development, and mitochondrial protein complexes. These data support previous studies performed in a zebrafish nonsense allele created by CRISPR/Cas9 and validate that loss of function mutations in the gabra1 gene result in seizure-like phenotypes with abnormal development of the GABA synapse. Our results add to the existing body of knowledge as to the function of GABRA1 during development and validate that zebrafish can be used to provide complete functional characterization of the gene., (Copyright © 2024 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

Background: Using lectins to target cancer-associated modifications of PSA glycostructure for identification of clinically significant prostate cancers, e.g., Gleason score (GS) ≥ 7, from benign and indolent cancers (GS 6), is highly promising yet technically challenging. From previous findings to quantify increased PSA fucosylation in urine, we set out to construct a robust, specific test concept suitable for plasma samples., Methods: Macrophage galactose-binding lectin (MGL) coupled to 100 nm Eu3 + -nanoparticles was used to probe PSA captured from cancer cell lines, seminal plasma, and plasma samples from 249 patients with a clinical suspicion of prostate cancer onto 3 mm dense spots of free PSA antibody fab fragments. Results were compared to four kallikrein tests: tPSA, fPSA, iPSA and hK2., Results: The fPSA MGL glycovariant provided superior discrimination of the GS ≥ 7 and benign + GS 6 groups (p 0.0003) compared to fPSA (NS). The corresponding AUC in ROC analysis was 0.70 compared to 0.66 for tPSA. In contrast to all four kallikrein tests, the fPSA MGL GV was independent of prostate gland volume. Using a logistic regression analysis the fPSA MGL GV significantly improved on the four-kallikrein model., Conclusions: Due to Eu-nanoparticles and a dense fPSA capture spot, the fPSA MGL glycovariant identifies an fPSA subform with the highest cancer specificity compared to the four conventional kallikreins., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Variants in the MMACHC gene cause combined methylmalonic acidemia and homocystinuria cblC type, the most common inborn error of intracellular cobalamin (vitamin B12) metabolism. cblC is associated with neurodevelopmental, hematological, ocular, and biochemical abnormalities. In a subset of patients, mild craniofacial dysmorphia has also been described. Mouse models of Mmachc deletion are embryonic lethal but cause severe craniofacial phenotypes such as facial clefts. MMACHC encodes an enzyme required for cobalamin processing and variants in this gene result in the accumulation of two metabolites: methylmalonic acid (MMA) and homocysteine (HC). Interestingly, other inborn errors of cobalamin metabolism, such as cblX syndrome, are associated with mild facial phenotypes. However, the presence and severity of MMA and HC accumulation in cblX syndrome is not consistent with the presence or absence of facial phenotypes. Thus, the mechanisms by which mutations in MMACHC cause craniofacial defects are yet to be completely elucidated. Here we have characterized the craniofacial phenotypes in a zebrafish model of cblC (hg13) and performed restoration experiments with either a wildtype or a cobalamin binding deficient MMACHC protein. Homozygous mutants did not display gross morphological defects in facial development but did have abnormal chondrocyte nuclear organization and an increase in the average number of neighboring cell contacts, both phenotypes were fully penetrant. Abnormal chondrocyte nuclear organization was not associated with defects in the localization of neural crest specific markers, sox10 (RFP transgene) or barx1. Both nuclear angles and the number of neighboring cell contacts were fully restored by wildtype MMACHC and a cobalamin binding deficient variant of the MMACHC protein. Collectively, these data suggest that mutation of MMACHC causes mild to moderate craniofacial phenotypes that are independent of cobalamin binding., Competing Interests: Declaration of competing interest Authors have no conflicts of interests to declare., (Copyright © 2023 International Society of Differentiation. All rights reserved.)

Background: Production of SCFAs from food is a complex and dynamic saccharolytic fermentation process mediated by both human and gut microbial factors. Knowledge of SCFA production and of the relation between SCFA profiles and dietary patterns is lacking., Objectives: Temporal changes in SCFA concentrations in response to 2 contrasting diets were investigated using a novel GC-MS method., Methods: Samples were obtained from a randomized, controlled, crossover trial designed to characterize the metabolic response to 4 diets. Participants (n = 19) undertook these diets during an inpatient stay (of 72 h). Serum samples were collected 2 h after breakfast (AB), after lunch (AL), and after dinner (AD) on day 3, and a fasting sample (FA) was obtained on day 4. The 24-h urine samples were collected on day 3. In this substudy, samples from the 2 extreme diets representing a diet with high adherence to WHO healthy eating recommendations and a typical Western diet were analyzed using a bespoke GC-MS method developed to detect and quantify 10 SCFAs and precursors in serum and urine samples., Results: Considerable interindividual variation in serum SCFA concentrations was observed across all time points, and temporal fluctuations were observed for both diets. Although the sample collection timing exerted a greater magnitude of effect on circulating SCFA concentrations, the unhealthy diet was associated with a lower concentration of acetic acid (FA: coefficient: -17.0; SE: 5.8; P-trend = 0.00615), 2-methylbutyric acid (AL: coefficient: -0.1; SE: 0.028; P-trend = 4.13 × 10 -4 and AD: coefficient: -0.1; SE: 0.028; P-trend = 2.28 × 10 -3 ), and 2-hydroxybutyric acid (FA: coefficient: -15.8; SE: 5.11; P-trend: 4.09 × 10 -3 ). In contrast, lactic acid was significantly higher in the unhealthy diet (AL: coefficient: 750.2; SE: 315.2; P-trend = 0.024 and AD: coefficient: 1219.3; SE: 322.6; P-trend: 8.28 × 10 -4 )., Conclusions: The GC-MS method allowed robust mapping of diurnal patterns in SCFA concentrations, which were affected by diet, and highlighted the importance of standardizing the timing of SCFA measurements in dietary studies. This trial was registered on the NIHR UK clinical trial gateway and with ISRCTN as ISRCTN43087333., (Copyright © 2022 American Society for Nutrition.)

An experiment was conducted to compare the use of a total and digestible Ca formulation system in broilers from hatch to d 37 post-hatch. Ross 308 (n = 288) male broilers were obtained and allocated to one of 2 dietary treatments in floor pens. There were 18 birds per pen and 8 replicate pens per diet. One diet was formulated using ingredient and nutrient recommendations for total Ca and the second diet was formulated using ingredient and nutrient recommendations for standardized ileal digestible (SID) Ca. All diets were formulated to be nutrient adequate using a 2-phase feeding program and including 0.19% available P (avP) and 0.209% total Ca or 0.073% digestible Ca from 1,000 FYT/kg of phytase. On d 17 and 37, tibias and ileal contents were obtained. From hatch to d 37, birds fed diets formulated using digestible Ca gained (P < 0.05) more and were more efficient (P < 0.05) compared with birds fed diets formulated using total Ca. There was no impact of formulation system on tibia ash or minerals. Litter pH (P < 0.05) was greater and litter dry matter (P < 0.05) was lower in birds fed the diets formulated using digestible Ca compared with those fed diets formulated using total Ca. Apparent ileal digestibility (AID) of Ca (P < 0.05), AID of P (P < 0.05) and digestible P intake (P < 0.05) were lower in birds fed diets formulated using digestible Ca compared with those formulated using total Ca at d 17 or 37. However, apparent ileal digested Ca and digestible Ca intake were not different between the experimental diets on d 17 or 37. In conclusion, formulating diets using digestible Ca improved weight gain and feed conversion ratio, but reduced the AID of Ca and P compared with birds fed diets formulated using total Ca. These findings might be reflective of the higher total Ca concentration in the diets formulated using digestible Ca compared with those formulated using total Ca., Competing Interests: DISCLOSURES The authors have no conflicts of interest to report., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Arrestin binding to active phosphorylated G protein-coupled receptors terminates G protein coupling and initiates another wave of signaling. Among the effectors that bind directly to receptor-associated arrestins are extracellular signal-regulated kinases 1/2 (ERK1/2), which promote cellular proliferation and survival. Arrestins may also engage ERK1/2 in isolation in a pre- or post-signaling complex that is likely in equilibrium with the full signal initiation complex. Molecular details of these binary complexes remain unknown. Here, we investigate the molecular mechanisms whereby arrestin-2 and arrestin-3 (a.k.a. β-arrestin1 and β-arrestin2, respectively) engage ERK1/2 in pairwise interactions. We find that purified arrestin-3 binds ERK2 more avidly than arrestin-2. A combination of biophysical techniques and peptide array analysis demonstrates that the molecular basis in this difference of binding strength is that the two non-visual arrestins bind ERK2 via different parts of the molecule. We propose a structural model of the ERK2-arrestin-3 complex in solution using size-exclusion chromatography coupled to small angle X-ray scattering (SEC-SAXS). This binary complex exhibits conformational heterogeneity. We speculate that this drives the equilibrium either toward the full signaling complex with receptor-bound arrestin at the membrane or toward full dissociation in the cytoplasm. As ERK1/2 regulates cell migration, proliferation, and survival, understanding complexes that relate to its activation could be exploited to control cell fate., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Arrestins regulate a wide range of signaling events, most notably when bound to active G protein-coupled receptors (GPCRs). Among the known effectors recruited by GPCR-bound arrestins are Src family kinases, which regulate cellular growth and proliferation. Here, we focus on arrestin-3 interactions with Fgr kinase, a member of the Src family. Previous reports demonstrated that Fgr exhibits high constitutive activity, but can be further activated by both arrestin-dependent and arrestin-independent pathways. We report that arrestin-3 modulates Fgr activity with a hallmark bell-shaped concentration-dependence, consistent with a role as a signaling scaffold. We further demonstrate using NMR spectroscopy that a polyproline motif within arrestin-3 interacts directly with the SH3 domain of Fgr. To provide a framework for this interaction, we determined the crystal structure of the Fgr SH3 domain at 1.9 Å resolution and developed a model for the GPCR-arrestin-3-Fgr complex that is supported by mutagenesis. This model suggests that Fgr interacts with arrestin-3 at multiple sites and is consistent with the locations of disease-associated Fgr mutations. Collectively, these studies provide a structural framework for arrestin-dependent activation of Fgr., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

G protein coupled receptors signal through G proteins or arrestins. A long-standing mystery in the field is why vertebrates have two non-visual arrestins, arrestin-2 and arrestin-3. These isoforms are ~75% identical and 85% similar; each binds numerous receptors, and appear to have many redundant functions, as demonstrated by studies of knockout mice. We previously showed that arrestin-3 can be activated by inositol-hexakisphosphate (IP 6 ). IP 6 interacts with the receptor-binding surface of arrestin-3, induces arrestin-3 oligomerization, and this oligomer stabilizes the active conformation of arrestin-3. Here, we compared the impact of IP 6 on oligomerization and conformational equilibrium of the highly homologous arrestin-2 and arrestin-3 and found that these two isoforms are regulated differently. In the presence of IP 6 , arrestin-2 forms "infinite" chains, where each promoter remains in the basal conformation. In contrast, full length and truncated arrestin-3 form trimers and higher-order oligomers in the presence of IP 6 ; we showed previously that trimeric state induces arrestin-3 activation (Chen et al., 2017). Thus, in response to IP 6 , the two non-visual arrestins oligomerize in different ways in distinct conformations. We identified an insertion of eight residues that is conserved across arrestin-2 homologs, but absent in arrestin-3 that likely accounts for the differences in the IP 6 effect. Because IP 6 is ubiquitously present in cells, this suggests physiological consequences, including differences in arrestin-2/3 trafficking and JNK3 activation. The functional differences between two non-visual arrestins are in part determined by distinct modes of their oligomerization. The mode of oligomerization might regulate the function of other signaling proteins., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

In the nutrition field, there is a lack of understanding about the impact that dietary chiral composition may have on health, especially regarding cooked meals. Chiral amino acids (AAs) are naturally present in food and their proportion may vary quite a lot. Besides, the D-amino acids (D-AAs) are present in very low concentration compared to L-AAs, so very sensitive methods are required for their accurate quantitation. Moreover, some of them have been described as indicators of quality and different food processes. In this research, we propose a robust method for the absolute quantitation and enantiomeric ratio of 17 D-AAs in cooked meals. The AAs were extracted from 1 g of the homogenised meal with methanol, derivatised with (S)-N-(4-nitrophenoxycarbonyl) phenylalanine methoxyethyl ester ((S)-NIFE) and analysed by RP-LC-MS/MS. The separation was carried out with an Acquity BEH C18 (100 mm x 2.1 mm, 1.7 µm) column at 70 ºC, with 10 mmol/L ammonium bicarbonate in water as eluent A and acetonitrile as eluent B at a 0.3 mL/min flow rate in gradient elution. The MS operated in positive electrospray ionisation method in multiple reaction monitoring (MRM) mode. Isotopically labelled AAs were used as internal standards for the quantitation. The method was validated for 17 D-AAs in the cooked food samples in terms of specificity, linearity, precision, accuracy, matrix effect and stability. LLOQ are 2.0 ng/mL for most of them. Additionally, linearity was also studied for L-AAs. After optimization and validation, the method was applied to real breakfast, lunch and dinner samples of cooked meals (n = 18) that were part of a diet with a very high concordance with WHO dietary guidelines. Level of concentration of major and minor D-AAs have been described per total daily intake and within each of the three main meals. This method can be used for quality control purposes as well as to investigate the role of chiral composition in food and clinical outcomes., (Copyright © 2019. Published by Elsevier B.V.)

Background: Measurement of multiple food intake exposure biomarkers in urine may offer an objective method for monitoring diet. The potential of spot and cumulative urine samples that have reduced burden on participants as replacements for 24-h urine collections has not been evaluated., Objective: The aim of this study was to determine the utility of spot and cumulative urine samples for classifying the metabolic profiles of people according to dietary intake when compared with 24-h urine collections in a controlled dietary intervention study., Methods: Nineteen healthy individuals (10 male, 9 female, aged 21-65 y, BMI 20-35 kg/m2) each consumed 4 distinctly different diets, each for 1 wk. Spot urine samples were collected ∼2 h post meals on 3 intervention days/wk. Cumulative urine samples were collected daily over 3 separate temporal periods. A 24-h urine collection was created by combining the 3 cumulative urine samples. Urine samples were analyzed with metabolite fingerprinting by both high-resolution flow infusion electrospray mass spectrometry (FIE-HRMS) and proton nuclear magnetic resonance spectroscopy (1H-NMR). Concentrations of dietary intake biomarkers were measured with liquid chromatography triple quadrupole mass spectrometry and by integration of 1H-NMR data., Results: Cross-validation modeling with 1H-NMR and FIE-HRMS data demonstrated the power of spot and cumulative urine samples in predicting dietary patterns in 24-h urine collections. Particularly, there was no significant loss of information when post-dinner (PD) spot or overnight cumulative samples were substituted for 24-h urine collections (classification accuracies of 0.891 and 0.938, respectively). Quantitative analysis of urine samples also demonstrated the relation between PD spot samples and 24-h urines for dietary exposure biomarkers., Conclusions: We conclude that PD spot urine samples are suitable replacements for 24-h urine collections. Alternatively, cumulative samples collected overnight predict similarly to 24-h urine samples and have a lower collection burden for participants., (Copyright © American Society for Nutrition 2019.)

Introduction: Health at Every Size (HAES) is a weight-neutral approach focused on promoting healthy behaviors in people with different body sizes and on enhancing pleasure derived from consuming food to achieve sustainable healthy eating outcomes. However, to the best of our knowledge, there are no studies in the literature assessing the effects of the HAES approach on perceptions of eating pleasure., Objective: We qualitatively investigated the perceptions of obese women about eating pleasure before and after a new interdisciplinary, nonprescriptive intervention based on the HAES approach., Design: The intervention was a randomized controlled clinical trial, designated as Health and Wellness in Obesity, conducted over 7 months at University of São Paulo (Brazil). We used a qualitative approach to data construction and analysis of perceptions about eating pleasure. Participants were randomized to either the intervention (I-HAES) group or the control (CTRL) group. The I-HAES group featured individual nutritional counseling, group practice of enjoyable physical activity, and philosophical workshops. The CTRL group was a traditional HAES intervention group (lecture-based model). Focus group discussions eliciting perceptions of pleasure around eating were conducted at baseline and post-study. Focus group transcripts were analyzed by exploratory content analysis., Participants: Forty-three women aged 25 to 50 years with body mass index (measured in kilograms per square meter) between 30 and 39. 9 completed the intervention and the focus groups, with 32 in the I-HAES group and 11 in the CTRL group., Results: Lack of guilt about experiencing pleasure while eating and increased reflection on their own desires increased in participants of both groups after the study. The I-HAES group also displayed a greater sense of autonomy related to eating, increased pleasure in commensality, familiarity with the practice of cooking, and decreased automatic eating., Conclusion: HAES-based intervention featuring nutritional counseling, appreciation for physical activity, and philosophical engagement was shown to stimulate pleasure around eating without leading to indiscriminate eating., (Copyright © 2019 Academy of Nutrition and Dietetics. Published by Elsevier Inc. All rights reserved.)

Purpose: SUDEP is the first cause of mortality related to epilepsy. However, in Spain there are no published cases or series from Epilepsy Monitoring Units that could expose the characteristics of SUDEP in our population., Method: We reviewed all patients treated at our Spanish Epilepsy Reference Centre who died between 2010-2018. SUDEP cases were classified as definite, probable, possible or near-SUDEP. Epilepsy type, demographics and case detection issues were described., Results: From 1250 evaluated patients, 102 died during the study period. Seven patients were diagnosed with SUDEP or near-SUDEP: two definite SUDEP, one definite SUDEP plus, two probable SUDEP and two near-SUDEP. Specific problems for detection and registration of SUDEP inherent to the Spanish healthcare system and the legal framework were defined. Only 43% of cases were known by the referral neurologist. SUDEP incidence was 1.3 per 1000 patient/year, comprising 0.56% of all deaths in our cohort. Two cases were female, the average age was 36 years (18-61). All patients had focal epilepsy and suffered from generalized tonic-clonic seizures. All witnessed cases occurred after a focal to bilateral tonic-clonic seizure. Four cases occurred during sleep and all non-witnessed cases were found in prone position. One case occurred during video-EEG monitoring., Conclusions: Our casuistic represents the first Epilepsy Monitoring Unit based case series of SUDEP conducted in Spain. The incidence in our population agrees with the reported in other countries. However, in our population, SUDEP is probably underdiagnosed due to administrative and legal issues., (Copyright © 2019. Published by Elsevier Ltd.)

Competing Interests: Conflict-of-interest disclosure: The authors declare no competing financial interests.

Background: Short-chain fatty acids (SCFAs), metabolites produced through the microbial fermentation of nondigestible dietary components, have key roles in energy homeostasis. Animal research suggests that colon-derived SCFAs modulate feeding behavior via central mechanisms. In humans, increased colonic production of the SCFA propionate acutely reduces energy intake. However, evidence of an effect of colonic propionate on the human brain or reward-based eating behavior is currently unavailable., Objectives: We investigated the effect of increased colonic propionate production on brain anticipatory reward responses during food picture evaluation. We hypothesized that elevated colonic propionate would reduce both reward responses and ad libitum energy intake via stimulation of anorexigenic gut hormone secretion., Design: In a randomized crossover design, 20 healthy nonobese men completed a functional magnetic resonance imaging (fMRI) food picture evaluation task after consumption of control inulin or inulin-propionate ester, a unique dietary compound that selectively augments colonic propionate production. The blood oxygen level-dependent (BOLD) signal was measured in a priori brain regions involved in reward processing, including the caudate, nucleus accumbens, amygdala, anterior insula, and orbitofrontal cortex (n = 18 had analyzable fMRI data)., Results: Increasing colonic propionate production reduced BOLD signal during food picture evaluation in the caudate and nucleus accumbens. In the caudate, the reduction in BOLD signal was driven specifically by a lowering of the response to high-energy food. These central effects were partnered with a decrease in subjective appeal of high-energy food pictures and reduced energy intake during an ad libitum meal. These observations were not related to changes in blood peptide YY (PYY), glucagon-like peptide 1 (GLP-1), glucose, or insulin concentrations., Conclusion: Our results suggest that colonic propionate production may play an important role in attenuating reward-based eating behavior via striatal pathways, independent of changes in plasma PYY and GLP-1. This trial was registered at clinicaltrials.gov as NCT00750438.

Engagement of the T cell receptor complex reprograms T cells for proliferation, cytokine production and differentiation towards effector cells. This process depends on activating costimulatory signals and is counteracted by coinhibitory molecules. Three transcription factors, namely NF-κB, NFAT and AP-1, have a major role in inducing the transcriptional program that is required for T cell activation and differentiation. Here we describe the generation of a triple parameter reporter based on the human Jurkat T cell line, where response elements for NF-κB, NFAT and AP-1 drive the expression of the fluorescent proteins CFP, eGFP and mCherry, respectively. The emission spectra of these proteins allow simultaneous assessment of NF-κB, NFAT and AP-1 activity in response to stimulation. Ligation of the TCR complex induced moderate reporter activity, which was strongly enhanced upon coengagement of the costimulatory receptors CD2 or CD28. Moreover, we have generated and tested triple parameter reporter cells that harbor costimulatory and inhibitory receptors not endogenously expressed in the Jurkat cells. In these experiments we could show that engagement of the costimulatory molecule 4-1BB enhances NF-κB and AP-1 activity, whereas coinhibition via PD-1 or BTLA strongly reduced the activation of NF-κB and NFAT. Engagement of BTLA significantly inhibited AP-1, whereas PD-1 had little effect on the activation of this transcription factor. Our triple parameter reporter T cell line is an excellent tool to assess the effect of costimulatory and coinhibitory receptors on NF-κB, NFAT and AP-1 activity and has a wide range of applications beyond the evaluation of costimulatory pathways., (Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.)

Background & Aims: Chronic hepatitis C is a leading cause of chronic liver disease, cirrhosis and hepatocellular carcinoma. DNA methylation and histone covalent modifications constitute crucial mechanisms of genomic instability in human disease, including liver fibrosis and hepatocellular carcinoma. The present work studies the consequences of HCV-induced histone modifications in early stages of infection., Methods: Human primary hepatocytes and HuH7.5 cells were transiently transfected with the core protein of hepatitis C virus (HCV) genotypes 1a, 1b, and 2a. Infectious genotype 2a HCV in culture was also used., Results: We show that HCV and core protein inhibit the phosphorylation of Serine 10 in histone 3. The inhibition is due to the direct interaction between HCV core and Aurora B kinase (AURKB) that results in a decrease of AURKB activity. HCV and core significantly downregulate NF-κB and COX-2 transcription, two proteins with anti-apoptotic and proliferative effects implicated in the control of the inflammatory response. AURKB depletion reduced HCV and core repression of NF-κB and COX-2 gene transcription and AURKB overexpression reversed the viral effect. AURKB abrogation increased HCV specific infectivity which was decreased when AURKB was overexpressed., Conclusions: The core-mediated decrease of AURKB activity may play a role in the inflammatory pathway during the initial steps of viral infection, while ensuring HCV infectivity., (Copyright © 2015 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

We studied the extent and nature of renal involvement in a cohort of 117 adult patients with mitochondrial disease, by measuring urinary retinol-binding protein (RBP) and albumin; established markers of tubular and glomerular dysfunction, respectively. Seventy-five patients had the m.3243A>G mutation and the most frequent phenotypes within the entire cohort were 14 with MELAS, 33 with MIDD, and 17 with MERRF. Urinary RBP was increased in 29 of 75 of m.3243A>G patients, whereas albumin was increased in 23 of the 75. The corresponding numbers were 16 and 14, respectively, in the 42 non-m.3243A>G patients. RBP and albumin were higher in diabetic m.3243A>G patients than in nondiabetics, but there were no significant differences across the three major clinical phenotypes. The urine proteome (mass spectrometry) and metabonome (nuclear magnetic resonance) in a subset of the m.3243A>G patients were markedly different from controls, with the most significant alterations occurring in lysosomal proteins, calcium-binding proteins, and antioxidant defenses. Differences were also found between asymptomatic m.3243A>G carriers and controls. No patients had an elevated serum creatinine level, but 14% had hyponatremia, 10% had hypophosphatemia, and 14% had hypomagnesemia. Thus, abnormalities in kidney function are common in adults with mitochondrial disease, exist in the absence of elevated serum creatinine, and are not solely explained by diabetes.

Background & Aims: The aim of this study was to evaluate the results of treatment with pegylated interferon and ribavirin for the recurrence of hepatitis C after liver transplantation in HCV/HIV-coinfected patients., Methods: This was a prospective, multicentre cohort study, including 78 HCV/HIV-coinfected liver transplant patients who received treatment for recurrent hepatitis C. For comparison, we included 176 matched HCV-monoinfected patients who underwent liver transplantation during the same period of time at the same centres and were treated for recurrent hepatitis C., Results: Antiviral therapy was discontinued prematurely in 56% and 39% (p = 0.016), mainly because of toxicity (22% and 11%, respectively; p=0.034). Sustained virological response (SVR) was achieved in 21% of the coinfected patients and in 36% of monoinfected patients (p = 0.013). For genotype 1, SVR rates were 10% and 33% (p = 0.002), respectively; no significant differences were observed for the other genotypes. A multivariate analysis based on the whole series identified HIV-coinfection as an independent predictor of lack of SVR (OR, 0.17; 95% CI, 0.06-0.42). Other predictors of SVR were donor age, pretreatment HCV viral load, HCV genotype, and early virological response. SVR was associated with a significant improvement in survival: 5-year survival after antiviral treatment was 79% for HCV/HIV-coinfected patients with SVR vs. 43% for those without (p = 0.02) and 92% vs. 60% in HCV-monoinfected patients (p < 0.001), respectively., Conclusions: The response to pegylated interferon and ribavirin was poorer in HCV/HIV-coinfected liver recipients, particularly those with genotype 1. However, when SVR was achieved, survival of coinfected patients increased significantly., (Copyright © 2014 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Aspartyl aminopeptidase (ASP; EC 3.4.11.21) is a widely distributed and abundant cytosolic enzyme that regulates bioactive peptides such as angiotensin II. It has been demonstrated that the expression and activity of this enzyme is modified in tissue and serum of patients with several types of cancer. However, the involvement of ASP in the neoplastic development and survival of patients with colorectal cancer (CRC) has not been analyzed to date. The activity and messenger RNA expression of ASP in tumor tissue (n = 71) and plasma (n = 40) of patients with CRC was analyzed prospectively using fluorometric and quantitative real-time polymerase chain reaction methods. Data obtained from tumor tissue were compared with those from the surrounding normal mucosa. Classic pathologic parameters (grade, stage, nodal invasion, distant metastases and perineural, lymphatic, and vascular invasion) were stratified following ASP data and analyzed for 5-year survival. ASP was upregulated in CRC tissues, and greater activity correlated significantly with the absence of lymph node metastases and with better overall survival. Inversely, greater plasmatic ASP activity was associated with worse overall and disease-free survival. Data suggest that ASP is involved in colorectal neoplasia and point to this enzyme as a potential useful diagnostic tool in clinical practice., (Copyright © 2013 Mosby, Inc. All rights reserved.)

Eighty-four HCV/HIV-coinfected and 252-matched HCV-monoinfected liver transplant recipients were included in a prospective multicenter study. Thirty-six (43%) HCV/HIV-coinfected and 75 (30%) HCV-monoinfected patients died, with a survival rate at 5 years of 54% (95% CI, 42-64) and 71% (95% CI, 66 to 77; p = 0.008), respectively. When both groups were considered together, HIV infection was an independent predictor of mortality (HR, 2.202; 95% CI, 1.420-3.413 [p < 0.001]). Multivariate analysis of only the HCV/HIV-coinfected recipients, revealed HCV genotype 1 (HR, 2.98; 95% CI, 1.32-6.76), donor risk index (HR, 9.48; 95% CI, 2.75-32.73) and negative plasma HCV RNA (HR, 0.14; 95% CI, 0.03-0.62) to be associated with mortality. When this analysis was restricted to pretransplant variables, we identified three independent factors (HCV genotype 1, pretransplant MELD score and centers with <1 liver transplantation/year in HIV-infected patients) that allowed us to identify a subset of 60 (71%) patients with a similar 5-year prognosis (69%[95% CI, 54-80]) to that of HCV-monoinfected recipients. In conclusion, 5-year survival in HCV/HIV-coinfected liver recipients was lower than in HCV-monoinfected recipients, although an important subset with a favorable prognosis was identified in the former., (© Copyright 2012 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Aneuploidy is a common feature of tumours that arise by errors in chromosome segregation during mitosis. The aim of this study was to evaluate possible signaling pathways involved in sensitization to chemotherapy in cells with chromosomal instability. We designed a screen using the fission yeast Squizossaccharomyces pombe, to isolate strains showing a phenotype of chromosome mis-segregation and higher sensitivity to the antitumoral drug Bleomycin. We examined differences in gene expression using a comparative analysis of genome-wide expression of the wild type strain and one of the mutants. The results revealed a set of genes involved in cell cycle control, including Mad3/BubR1 and Chk1. We then studied the levels of these two proteins in colorectal cancer human cell lines with different genomic content. Among these, SW620 cells showed higher BubR1 and Chk1 mRNA levels than control cells under normal conditions. Since Chk1 is required for both S and G2/M checkpoints, and the microtubule-destabilizing agent, nocodazole induces mitotic arrest, we attempted to investigate the potential anticancer effects of nocodazole in combination with cisplatin. These studies showed that SW620 cells undergo synergistic cell death after spindle checkpoint activation followed by cisplatin treatment, suggesting a role of Chk1 in this checkpoint, very likely dependent on BubR1 protein. Importantly, Chk1-depleted SW620 cells lost this synergistic effect. In summary, we propose that Chk1 could be a biomarker predictive of the efficacy of chemotherapy across different types of tumors with aneuploidy. These findings may be potentially very useful for the stratification of patients for treatment., (Copyright (c) 2009 Elsevier Ltd. All rights reserved.)

Because the acquisition of in vitro transcription kits, for production of RNA standards, might not be affordable for some small laboratories, the current work describes some alternatives to the commercial Promega protocols. Yields of tested reactions were all higher than the ones reported for the standard Riboprobe kit (1mug RNA/mug template, 1x) and the optimized variant for high yields (5-10x). They were also as good as the ones described for the high RNA production kit RiboMAX (10-20x), exceeding them in approximately 70% of reactions. Our best results, achieved in a 500-mul format, were three to five times higher than the ones reported for the Promega kit.

For the investigation of the minimal structural requirements for cytokine induction, Staphylococcus aureus lipoteichoic acid derivatives with two, three, four, and five glycerophosphate backbone moieties, carrying each a d-alanyl residue, were needed. Based on two different glycerophosphate building blocks and 6b-O-phosphitylated gentiobiosyl diacylglycerol the desired target molecules (compounds 1-4) could be readily obtained and provided for biological studies.

The microbiological leaching of chalcopyrite (CuFeS(2)) is of great interest because of its potential application to many CuFeS(2)-rich ore materials. However, the efficiency of the microbiological process is very limited because this mineral is one of the most refractory to bacterial attack. Knowledge of bacterial role during chalcopyrite oxidation is very important in order to improve the efficiency of bioleaching operation. The oxidative dissolution of a massive chalcopyrite electrode by Acidithiobacillus ferrooxidans was evaluated by electrochemical impedance spectroscopy (EIS) and atomic force microscopy (AFM). A massive chalcopyrite electrode was utilized in a Tait-type electrochemical cell in acid medium for different immersion times in the presence or absence of bacterium. The differences observed in the impedance diagrams were correlated with the adhesion process of bacteria on the mineral surface.

Background: Mammograms are assigned a BI-RADS (Breast Imaging Reporting and Data System) category, which indicates the level of suspicion for cancer., Objectives: (i) To evaluate the use of BI-RADS categories in a non-academic radiology practice based in a community hospital compared with local radiology private offices; (ii) to determine positive predictive value (PPV), sensitivity and specificity of mammograms; and (iii) to explore the correlation of BI-RAD 3-5 and lesion description with diagnosis of cancer., Patients and Methods: We performed 947 SVABBs (stereotactic vacuum-assisted breast biopsies) on 911 patients with BI-RADS 3-5. Lesions were classified as: 1=microcalcifications; 2=asymmetric density; 3=circumscribed mass; and 4=spiculated mass., Results: BI-RADS category correlated with diagnosis of breast cancer (atypia excluded): category 3=4%; category 4=15%; and category 5=79%. The PPV of BI-RADS 4 and 5 for breast cancer or atypia was 20%, in contrast to 5% for BI-RADS 3. Sensitivity and specificity were 95% and 19%, respectively. For BI-RADS 3 without microcalcifications only 3% were positive, in contrast to 8% for remainder., Conclusions: First, there is a stepwise increase in cancer for each of the BI-RADS categories 3-5. Secondly, in BI-RADS 3 with microcalcifications, a biopsy is indicated according to our findings. Finally, the sensitivity of mammograms is 95% but the specificity is 19%.

Background: Stroke units reduce mortality and dependence, but the reasons are unclear. We have compared differences in management and complications of patients with acute stroke who were admitted to a stroke unit or to a general ward as part of a previously reported randomised trial., Methods: 304 patients had been randomly assigned to stroke units (n=152) or to general wards supported by a specialist stroke team (152). We used a structured format to gather prospective data on the frequency of prespecified interventions in each of the major aspects of stroke care. Observations were undertaken daily for the first week and every week for the next 3 months by independent observers. The effect of differences in management on outcome at 3 months was assessed with the modified Rankin score, dichotomised to good (0-3) and poor (4-6) outcome., Findings: Patients in the stroke unit were monitored more frequently (odds ratio 2.1 [1.3-3.4]) and more patients received oxygen (2.0 [1.3-3.2]), antipyretics (6.4 [1.5-27.5]), measures to reduce aspiration (6.0 [2.3-15.5]), and early nutrition (14.4 [5.1-40.9]) than those in general wards. Complications were less frequent in patients in the stroke unit than those in general wards (0.6 [0.2-0.7]), with fewer patients having progression of stroke, chest infection, or dehydration. Measures to prevent aspiration, early feeding, stroke unit management, and frequency of complications independently affected outcome., Interpretation: Differences in management and complications between the stroke unit and general wards differ substantially, even when specialist support is provided. Such differences could be responsible for the more favourable outcome seen in patients on stroke units than those on general wards.

Background: Organised specialist care for stroke improves outcome, but the merits of different methods of organisation are in doubt. This study compares the efficacy of stroke unit with stroke team or domiciliary care., Methods: A single-blind, randomised, controlled trial was undertaken in 457 acute-stroke patients (average age 76 years, 48% women) randomly assigned to stroke unit, general wards with stroke team support, or domiciliary stroke care, within 72 h of stroke onset. Outcome was assessed at 3, 6, and 12 months. The primary outcome measure was death or institutionalisation at 12 months. Analyses were by intention to treat., Findings: 152 patients were allocated to the stroke unit, 152 to stroke team, and 153 to domiciliary stroke care. 51 (34%) patients in the domiciliary group were admitted to hospital after randomisation. Mortality or institutionalisation at 1 year were lower in patients on a stroke unit than for those receiving care from a stroke team (21/152 [14%] vs 45/149 [30%]; p<0.001) or domiciliary care (21/152 [14%] vs 34/144 [24%]; p=0.03), mainly as a result of reduction in mortality. The proportion of patients alive without severe disability at 1 year was also significantly higher on the stroke unit compared with stroke team (129/152 [85%] vs 99/149 [66%]; p<0.001) or domiciliary care (129/152 [85%] vs 102/144 [71%]; p=0.002). These differences were present at 3 and 6 months after stroke., Interpretation: Stroke units are more effective than a specialist stroke team or specialist domiciliary care in reducing mortality, institutionalisation, and dependence after stroke.

Non-degraded thimerosal was determined in the presence of its decomposition products by directly assaying recombinant hepatitis B vaccine using a reversed-phase liquid chromatographic method. Methanol-water-orthophosphoric acid (65:35:0.9, v/v/v) was used as the eluent. Salicylic acid was employed as an internal standard. The calibration graph was linear (r = 0.99995) up to 2.5 micrograms of thimerosal. Interference from aluminium hydroxide was eliminated by centrifugation. Good stability of thimerosal in the hepatitis B vaccine was demonstrated. The results obtained were in agreement with the recently proposed mechanism of degradation.

Patients suffering from acute respiratory failure are considered poor candidates for lung transplantation (LT). We report a successful double lung transplantation performed in a patient with adult respiratory distress syndrome (ARDS). The 32-year-old woman received recombinant interleukin 2 (rIL-2) three months after an autologous bone marrow transplant for acute myelogenic leukemia as consolidation treatment. After four days of treatment with rIL-2, she developed ARDS which worsened over a three-week period, despite treatment. Lung transplantation was carried out as ultimate treatment. The postoperative course was uneventful. The patient is alive and in a good condition 11 months after LT. This case demonstrates the feasibility of LT in selected patients with ARDS. However, this case is exceptional since lung grafts should be utilized preferably for evaluated and accepted patients in transplant programs.

A 58-year-old man underwent sequential bilateral lung transplantation. On the donor heart-lung block, it was discovered that the right apical segment was supplied by a tracheal bronchus. After the separate implantation of both lungs, a right apical segmentectomy was performed and the postoperative course was uneventful. The management of this problem is discussed.

A "dot" hybridisation technique for the detection of rotavirus in stools and other biological materials is described. The assay is based on the in-situ hybridisation of labelled single-stranded RNA probes, obtained by in-vitro transcription of rotavirus particles, to heat-denatured rotavirus RNA immobilised on nitrocellulose membranes. The method is highly specific and allows for the detection of as little as 8 pg of viral RNA. Its use for the detection of rotavirus in stool suspensions and rectal swabs obtained from children with diarrhoea may facilitate epidemiological studies of rotavirus gastroenteritis.