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Attention is an important aspect of human brain function and often affected in neurological disorders. Objective assessment of attention may assist in patient care, both for diagnostics and prognostication. We present a compact test using a combination of a choice reaction time task, eye-tracking and EEG for assessment of visual attention in the clinic. The system quantifies reaction time, parameters of eye movements (i.e. saccade metrics and fixations) and event related potentials (ERPs) in a single and fast (15 min) experimental design. We present pilot data from controls, patients with mild traumatic brain injury and epilepsy, to illustrate its potential use in assessing attention in neurological patients. Reaction times and eye metrics such as fixation duration, saccade duration and latency show significant differences (p < .05) between neurological patients and controls. Late ERP components (200–800 ms) can be detected in the central line channels for all subjects, but no significant group differences could be found in the peak latencies and mean amplitudes. Our system has potential to assess key features of visual attention in the clinic. Pilot data show significant differences in reaction times and eye metrics between controls and patients, illustrating its promising use for diagnostics and prognostication.

Physical interactions between proteins are essential for most biological processes governing life1. However, the molecular determinants of such interactions have been challenging to understand, even as genomic, proteomic and structural data increase. This knowledge gap has been a major obstacle for the comprehensive understanding of cellular protein–protein interaction networks and for the de novo design of protein binders that are crucial for synthetic biology and translational applications2–9. Here we use a geometric deep-learning framework operating on protein surfaces that generates fingerprints to describe geometric and chemical features that are critical to drive protein–protein interactions10. We hypothesized that these fingerprints capture the key aspects of molecular recognition that represent a new paradigm in the computational design of novel protein interactions. As a proof of principle, we computationally designed several de novo protein binders to engage four protein targets: SARS-CoV-2 spike, PD-1, PD-L1 and CTLA-4. Several designs were experimentally optimized, whereas others were generated purely in silico, reaching nanomolar affinity with structural and mutational characterization showing highly accurate predictions. Overall, our surface-centric approach captures the physical and chemical determinants of molecular recognition, enabling an approach for the de novo design of protein interactions and, more broadly, of artificial proteins with function.

Lifestyle-related diseases like type 2 diabetes are rapidly increasing worldwide, and there is strong evidence that physical inactivity contributes to this development (1). The prediabetic and diabetic states are characterized [...], To elucidate the molecular mechanisms behind physical inactivity--induced insulin resistance in skeletal muscle, 12 young, healthy male subjects completed 7 days of bed rest with vastus lateralis muscle biopsies obtained before and after. In six of the subjects, muscle biopsies were taken from both legs before and after a 3-h hyperinsulinemic euglycemic clamp performed 3 h after a 45-min, one-legged exercise. Blood samples were obtained from one femoral artery and both femoral veins before and during the clamp. Glucose infusion rate and leg glucose extraction during the clamp were lower after than before bed rest. This bed rest-induced insulin resistance occurred together with reduced muscle GLUT4, hexokinase II, protein kinase B/Aktl, and Akt2 protein level, and a tendency for reduced 3-hydroxyacyl-CoA dehydrogenase activity. The ability of insulin to phosphorylate Akt and activate glycogen synthase (GS) was reduced with normal GS site 3 but abnormal GS site 2+2a phosphorylation after bed rest. Exercise enhanced insulIn-stimulated leg glucose extraction both before and after bed rest, which was accompanied by higher GS activity in the prior-exercised leg than the rested leg. The present findings demonstrate that physical inactivity-induced insulin resistance in muscle is associated with lower content/activity of key proteins in glucose transport/phosphorylation and storage. Diabetes 61:1090-1099, 2012

Type 2 diabetes is caused by a complicated interplay between genetic and environmental factors acting on glucose and fat metabolism involving multiple defects of peripheral (muscle) and hepatic insulin action, [...], OBJECTIVE--The aim of this study was to determine whether the type 2 diabetes-associated T-allele of transcription factor 7-like 2 (TCF7L2) rs7903146 associates with impaired insulin secretion to compensate for insulin resistance induced by bed rest. RESEARCH DESIGN AND METHODS--A total of 38 healthy young Caucasian men were studied before and after bed rest using the hyperinsulinemic-euglycemic clamp technique combined with indirect calorimetry preceded by an intravenous glucose tolerance test. The TCF7L2 rs7903146 was genotyped using allelic discrimination performed with an ABI 7900 system. The genetic analyses were done assuming a dominant model of inheritance. RESULTS--The first-phase insulin response (FPIR) was significantly lower in carriers of the T-allele compared with carriers of the CC genotype before bed rest, with and without correction for insulin resistance. The incremental rise of FPIR in response to insulin resistance induced by bed rest was lower in carriers of the T-allele (P < 0.001). Fasting plasma glucagon levels were significantly lower in carriers of the T-allele before and after bed rest. While carriers of the CC genotype developed increased hepatic insulin resistance, the TCF7L2 rs7903146 did not influence peripheral insulin action or the rate of lipolysis before or after bed rest. CONCLUSIONS--Healthy carriers of the T-allele of TCF7L2 rs7903146 exhibit a diminished increase of insulin secretion in response to intravenous glucose to compensate for insulin resistance as induced by bed rest. Reduced paracrine glucagon stimulation may contribute to the impairment of β-cell function in the carriers TCF7L2 rs7903146 T-allele associated with increased risk of type 2 diabetes.

Type 2 diabetes is caused by a complicated interplay between genetic and environmental factors that influence defects of peripheral and hepatic insulin action, insulin secretion, adipose tissue metabolism and lipolysis, [...], OBJECTIVE--The aim of this study was to investigate the impact of 9 days of bed rest on insulin secretion, insulin action, and whole-body glucose and fat metabolism in first-degree relative (FDR) and matched control (CON) subjects. RESEARCH DESIGN AND METHODS--A total of 13 FDR and 20 CON subjects participated in the study. All were studied before and after 9 days of bed rest using the clamp technique combined with indirect calorimetry preceded by an intravenous glucose tolerance test. Glucose and glycerol turnover rates were studied using stable isotope kinetics. RESULTS--Bed rest caused a significant decrease in whole-body insulin sensitivity in both groups. Hepatic insulin resistance was elevated in FDR subjects prior to bed rest and was significantly augmented by bed rest in FDR (P < 0.01) but not in CON (P = NS) subjects. The rate of whole-body lipolysis decreased during bed rest in both FDR and CON subjects, with no significant differences between the groups. Insulin resistance induced by bed rest was fully accounted for by the impairment of nonoxidative glucose metabolism in both groups (overall P < 0.001). CONCLUSIONS--Whole-body insulin action in both insulin-resistant FDR and healthy CON subjects deteriorates with 9 days of bed rest, converging toward similar degrees of whole-body insulin resistance. FDR subjects exhibit hepatic insulin resistance (HIR), which, in contrast to CON subjects, deteriorates in response to physical inactivity. FDR subjects exhibit reduced insulin secretion when seen in relation to their degree of HIR but not peripheral insulin resistance.

HIV-associated lipodystrophy (HIV lipodystrophy) is characterized by various degrees of subcutaneous fat loss, visceral fat accumulation, and metabolic disturbances, including peripheral and hepatic insulin resistance (1-3) and elevated plasma free [...], HIV-infected patients with lipodystrophy (HIV lipodystrophy) are insulin resistant and have elevated plasma free fatty acid (FFA) concentrations. We aimed to explore the mechanisms underlying FFA-induced insulin resistance in patients with HIV lipodystrophy. Using a randomized, placebo-controlled, cross-over design, we studied the effects of an overnight acipimox-induced suppression of FFAs on glucose and FFA metabolism by using stable isotope-labeled tracer techniques during basal conditions and a two-stage euglycemic-hyperinsulinemic clamp (20 and 50 mU insulin/[m.sup.2] per min, respectively) in nine patients with nondiabetic HIV lipodystrophy. All patients received antiretroviral therapy. Biopsies from the vastus lateralis muscle were obtained during each stage of the clamp. Acipimox treatment reduced basal FFA rate of appearance by 68.9% (95% CI 52.6-79.5) and decreased plasma FFA concentration by 51.6% (42.0-58.9) (both, P < 0.0001). Endogenous glucose production was not influenced by acipimox. During the clamp, the increase in glucose uptake was significantly greater after acipimox treatment compared with placebo (acipimox: 26.85 µmol x [kg.sup.-1] x [min.sup.-1] [18.09-39.86] vs. placebo: 20.30 µmol x [kg.sup.-1] x [min.sup.-1] [13.67-30.13]; P < 0.01). Insulin increased phosphorylation of Akt [Thr.sup.308] and glycogen synthase kinase-3β [Ser.sup.9], decreased phosphorylation of glycogen synthase (GS) site 3a + b, and increased GS activity (percent I-form) in skeletal muscle (P < 0.01). Acipimox decreased phosphorylation of GS (site 3a + b) (P < 0.02) and increased GS activity (P < 0.01) in muscle. The present study provides direct evidence that suppression of lipolysis in patients with HIV lipodystrophy improves insulin-stimulated peripheral glucose uptake. The increased glucose uptake may in part be explained by increased dephosphorylation of GS (site 3a + b), resulting in increased GS activity.

Pyruvate dehydogenase (PDH) complex (PDC) catalyzes the irreversible conversion of pyruvate to acetyl CoA, and dysregulation of the PDC in skeletal muscle has been suggested to be implicated in type [...], To investigate pyruvate dehydrogenase (PDH)-E1α sub-unit phosphorylation and whether free fatty acids (FFAs) regulate PDH activity, seven subjects completed two trials: saline (control) and intralipid/heparin (intralipid). Each infusion trial consisted of a 4-h rest followed by a 3-h two-legged knee extensor exercise at moderate intensity. During the 4-h resting period, activity of PDH in the active form (PDHa) did not change in either trial, yet phosphorylation of PDH-E1α site 1 (PDH-P1) and site 2 (PDH-P2) was elevated in the intralipid compared with the control trial. PDHa activity increased during exercise similarly in the two trials. After 3 h of exercise, PDHa activity remained elevated in the intralipid trial but returned to resting levels in the control trial. Accordingly, in both trials PDH-P1 and PDH-P2 decreased during exercise, and the decrease was more marked during intralipid infusion. Phosphorylation had returned to resting levels at 3 h of exercise only in the control trial. Thus, an inverse association between PDH-E1α phosphorylation and PDHa activity exists. Short-term elevation in plasma FFA at rest increases PDH-E1α phosphorylation, but exercise overrules this effect of FFA on PDH-E1α phosphorylation leading to even greater dephosphorylation during exercise with intralipid infusion than with saline. Diabetes 55:3020-3027, 2006

De novoprotein design explores uncharted sequence and structure space to generate novel proteins not sampled by evolution. A main challenge in de novodesign involves crafting “designable” structural templates to guide the sequence searches toward adopting target structures. We present a convolutional variational autoencoder that learns patterns of protein structure, dubbed Genesis. We coupled Genesis with trRosetta to design sequences for a set of protein folds and found that Genesis is capable of reconstructing native-like distance and angle distributions for five native folds and three novel, the so-called “dark-matter” folds as a demonstration of generalizability. We used a high-throughput assay to characterize the stability of the designs through protease resistance, obtaining encouraging success rates for folded proteins. Genesis enables exploration of the protein fold space within minutes, unrestricted by protein topologies. Our approach addresses the backbone designability problem, showing that small neural networks can efficiently learn structural patterns in proteins. A record of this paper’s transparent peer review process is included in the supplemental information.

ObjectivePancreatic ductal adenocarcinoma (PDAC) has the characteristics of high-density desmoplastic stroma, a distinctive immunosuppressive microenvironment and is profoundly resistant to all forms of chemotherapy and immunotherapy, leading to a 5-year survival rate of 9%. Our study aims to add novel small molecule therapeutics for the treatment of PDAC.DesignWe have studied whether TAK-981, a novel highly selective and potent small molecule inhibitor of the small ubiquitin like modifier (SUMO) activating enzyme E1 could be used to treat a preclinical syngeneic PDAC mouse model and we have studied the mode of action of TAK-981.ResultsWe found that SUMOylation, a reversible post-translational modification required for cell cycle progression, is increased in PDAC patient samples compared with normal pancreatic tissue. TAK-981 decreased SUMOylation in PDAC cells at the nanomolar range, thereby causing a G2/M cell cycle arrest, mitotic failure and chromosomal segregation defects. TAK-981 efficiently limited tumour burden in the KPC3 syngeneic mouse model without evidence of systemic toxicity. In vivo treatment with TAK-981 enhanced the proportions of activated CD8 T cells and natural killer (NK) cells but transiently decreased B cell numbers in tumour, peripheral blood, spleen and lymph nodes. Single cell RNA sequencing revealed activation of the interferon response on TAK-981 treatment in lymphocytes including T, B and NK cells. TAK-981 treatment of CD8 T cells ex vivo induced activation of STAT1 and interferon target genes.ConclusionOur findings indicate that pharmacological inhibition of the SUMO pathway represents a potential strategy to target PDAC via a dual mechanism: inhibiting cancer cell cycle progression and activating anti-tumour immunity by inducing interferon signalling.

Sézary syndrome (SS) is an aggressive leukemic form of cutaneous T-cell lymphoma with neoplastic CD4+ T cells present in skin, lymph nodes, and blood. Despite advances in therapy, prognosis remains poor, with a 5-year overall survival of 30%. The immunophenotype of Sézary cells is diverse, which hampers efficient diagnosis, sensitive disease monitoring, and accurate assessment of treatment response. Comprehensive immunophenotypic profiling of Sézary cells with an in-depth analysis of maturation and functional subsets has not been performed thus far. We immunophenotypically profiled 24 patients with SS using standardized and sensitive EuroFlow-based multiparameter flow cytometry. We accurately identified and quantified Sézary cells in blood and performed an in-depth assessment of their phenotypic characteristics in comparison with their normal counterparts in the blood CD4+ T-cell compartment. We observed inter- and intrapatient heterogeneity and phenotypic changes over time. Sézary cells exhibited phenotypes corresponding with classical and nonclassical T helper subsets with different maturation phenotypes. We combined multiparameter flow cytometry analyses with fluorescence-activated cell sorting and performed RNA sequencing studies on purified subsets of malignant Sézary cells and normal CD4+ T cells of the same patients. We confirmed pure monoclonality in Sézary subsets, compared transcriptomes of phenotypically distinct Sézary subsets, and identified novel downregulated genes, most remarkably THEMIS and LAIR1, which discriminate Sézary cells from normal residual CD4+ T cells. Together, these findings further unravel the heterogeneity of Sézary cell subpopulations within and between patients. These new data will support improved blood staging and more accurate disease monitoring.

Sediments play an essential role in the functioning of aquatic ecosystems but simultaneously retain harmful compounds. However, sediment quality assessment methods that consider the risks caused by the combined action of all sediment-associated contaminants to benthic biota are still underrepresented in water quality assessment strategies. Significant advancements have been made in the application of effect-based methods, but methodological improvements can still advance sediment risk assessment. The present study aimed to explore such improvements by integrating effect-monitoring and chemical profiling of sediment contamination. To this end, 28 day life cycle bioassays with Chironomus ripariususing intact whole sediment cores from contaminated sites were performed in tandem with explorative chemical profiling of bioavailable concentrations of groups of legacy and emerging sediment contaminants to investigate ecotoxicological risks to benthic biota. All contaminated sediments caused effects on the resilient midge C. riparius, stressing that sediment contamination is ubiquitous and potentially harmful to aquatic ecosystems. However, bioassay responses were not in line with any of the calculated toxicity indices, suggesting that toxicity was caused by unmeasured compounds. Hence, this study underlines the relevance of effect-based sediment quality assessment and provides smarter ways to do so.

Oncolytic reovirus administration has been well tolerated by cancer patients in clinical trials. However, its anti-cancer efficacy as a monotherapy remains to be augmented. We and others have previously demonstrated the feasibility of producing replication-competent reoviruses expressing a heterologous transgene. Here, we describe the production of recombinant reoviruses expressing murine (mm) or human (hs) GM-CSF (rS1-mmGMCSF and rS1-hsGMCSF, respectively). The viruses could be propagated up to 10 passages while deletion mutants occurred only occasionally. In infected cell cultures, the secretion of GM-CSF protein (up to 481 ng/106cells per day) was demonstrated by ELISA. The secreted mmGM-CSF protein was functional in cell culture, as demonstrated by the capacity to stimulate the survival and proliferation of the GM-CSF-dependent dendritic cell (DC) line D1, and by its ability to generate DCs from murine bone marrow cells. Importantly, in a murine model of pancreatic cancer we found a systemic increase in DC and T-cell activation upon intratumoral administration of rS1-mmGMCSF. These data demonstrate that reoviruses expressing functional GM-CSF can be generated and have the potential to enhance anti-tumor immune responses. The GM-CSF reoviruses represent a promising new agent for use in oncolytic virotherapy strategies.

Intrahepatic triacylglycerol (liver TG) content is associated with hepatic insulin resistance and dyslipidemia. Liver TG content can be modulated within days under hypocaloric conditions.

Most T cell–based immunotherapies of cancer depend on intact antigen presentation by HLA class I molecules (HLA-I). However, defects in the antigen-processing machinery can cause downregulation of HLA-I, rendering tumor cells resistant to CD8+ T cells. Previously, we demonstrated that a unique category of cancer antigens is selectively presented by tumor cells deficient for the peptide transporter TAP, enabling a specific attack of such tumors without causing immunopathology in mouse models. With a novel combinatorial screening approach, we now identify 16 antigens of this category in humans. These HLA-A*02:01 presented peptides do not derive from the mutanome of cancers, but are of “self” origin and therefore constitute universal neoantigens. Indeed, CD8+ T cells specific for the leader peptide of the ubiquitously expressed LRPAP1 protein recognized TAP-deficient, HLA-Ilow lymphomas, melanomas, and renal and colon carcinomas, but not healthy counterparts. In contrast to personalized mutanome-targeted therapies, these conserved neoantigens and their cognate receptors can be exploited for immune-escaped cancers across diverse histological origins.

Recent detailed genomic analysis of mycosis fungoides (MF) identified suppressor of cytokine signaling 1 (SOCS1), an inhibitor of JAK/STAT signaling, as one of the frequently deleted tumor suppressors in MF, and one-copy deletion of SOCS1 was confirmed in early-stage MF lesions. To better understand the functional role of SOCS1 in the genesis of MF, we used a genetically engineered mouse model emulating heterozygous SOCS1 loss in skin resident CD4+T cells. In these mice an experimentally induced contact-allergic reaction was maintained for 20 weeks. Ten weeks after discontinuing contact-allergic challenges, only the skin with locally one-copy deletion of Socs1 in CD4+T cells still showed high numbers of CD3+/CD4+Socs1 k.o. cells in the dermis (p ＜ 0.0001) with prevalent Stat3 activation (p ＜0.001). And in one out of 9 mice, this had progressed to far more dramatic increases, including the thickened epidermis, and with an explosive growth of Socs1 k.o. T cells in circulation; indicative of cutaneous lymphoma. Hence, we show that Socs1 mono-allelic loss in CD4+T cells locally in protractedly inflamed skin results in autonomous skin inflammation with features of early-stage MF.

Context:Oral glucose ingestion elicits a larger insulin response and delayed suppression of glucagon compared to isoglycemic IV glucose infusion (IIGI).Objective:We studied whether these differences translate into effects on endogenous glucose production (EGP) and glucose disposal in patients with type 2 diabetes and nondiabetic control subjects.Design:This was a single-blinded, randomized, crossover study.Setting:The study was conducted at a specialized research unit.Participants:Ten patients with type 2 diabetes (age, [mean ± SD] 57.1 ± 6.7 years; body mass index, 29.0 ± 4.3 kg/m2; hemoglobin A1c, 53.8 ± 11.0 mmol/mol; duration of diabetes, 9.2 ± 5.0 years) and 10 matched nondiabetic control subjects (age, 56.0±10.7 years; body mass index, 29.8 ± 2.9 kg/m2; hemoglobin A1c, 33.8 ± 5.5 mmol/mol) participated.Interventions:Three experimental days: 75 g-oral glucose tolerance test (OGTT), IIGI, and IIGI+glucagon (IIGI with a concomitant IV glucagon infusion [0.8 ng/kg/min from 0 to 25 minutes] designed to mimic portal glucagon concentrations during OGTT in the type 2 diabetic group) were undertaken.Main Outcome Measures:Glucose kinetics were assessed by tracer methodology.Results:Glucose rate of disappearance was higher during the OGTT vs IIGI in the control group, but similar on all days in the diabetic group. Surprisingly, in both groups, EGP was more suppressed during IIGI than during OGTT, and exogenous glucagon infusion during IIGI did not restore EGP to the levels observed during OGTT.Conclusion:EGP was less suppressed during OGTT than during IIGI in both patients with type 2 diabetes and in nondiabetic control subjects. Based on the present experimental design, it was not possible to attribute this difference to the delayed glucagon suppression observed in the initial phase of the OGTT.

Therapeutic vaccines preferentially stimulate T cells against tumour-specific epitopes that are created by DNA mutations or oncogenic viruses. In the setting of premalignant disease, carcinoma in situ or minimal residual disease, therapeutic vaccination can be clinically successful as monotherapy; however, in established cancers, therapeutic vaccines will require co-treatments to overcome immune evasion and to become fully effective. In this Review, we discuss the progress that has been made in overcoming immune evasion controlled by tumour cell-intrinsic factors and the tumour microenvironment. We summarize how therapeutic benefit can be maximized in patients with established cancers by improving vaccine design and by using vaccines to increase the effects of standard chemotherapies, to establish and/or maintain tumour-specific T cells that are re-energized by checkpoint blockade and other therapies, and to sustain the antitumour response of adoptively transferred T cells.

The immune checkpoint NKG2A/CD94 is a promising target for cancer immunotherapy, and its ligand major histocompatibility complex E (MHC-E) is frequently upregulated in cancer. NKG2A/CD94-mediated inhibition of lymphocytes depends on the presence of specific leader peptides in MHC-E, but when and where they are presented in situis unknown. We apply a nanobody specific for the Qdm/Qa-1bcomplex, the NKG2A/CD94 ligand in mouse, and find that presentation of Qdm peptide depends on every member of the endoplasmic reticulum-resident peptide loading complex. With a turnover rate of 30 min, the Qdm peptide reflects antigen processing capacity in real time. Remarkably, Qdm/Qa-1bcomplexes require inflammatory signals for surface expression in situ, despite the broad presence of Qa-1bmolecules in homeostasis. Furthermore, we identify LILRB1 as a functional inhibition receptor for MHC-E in steady state. These data provide a molecular understanding of NKG2A blockade in immunotherapy and assign MHC-E as a convergent ligand for multiple immune checkpoints.

Context:Patients with blocked muscle glycogen breakdown (McArdle disease) have severely reduced exercise capacity compared to healthy individuals and are not assumed to produce lactate during exercise.Objectives:The objectives were: 1) to quantify systemic and muscle lactate kinetics and oxidation rates and muscle energy utilization during exercise in patients with McArdle disease; and 2) to elucidate the role of lactate formation in muscle energy production.Design and Setting:This was a single trial in a hospital.Participants:Participants were four patients with McArdle disease and seven healthy subjects.Intervention:Patients and healthy controls were studied at rest, which was followed by 40 minutes of cycle-ergometer exercise at 60% of the patients' maximal oxygen uptake (∼35 W).Main Outcome Measures:Main outcome measures were systemic and leg skeletal muscle lactate, alanine, fatty acid, and glucose kinetics.Results:McArdle patients had a marked decrease in plasma lactate concentration at the onset of exercise, and the concentration remained suppressed during exercise. A substantial leg net lactate uptake and subsequent oxidation occurred over the entire exercise period in patients, in contrast to a net lactate release or no exchange in the healthy controls. Despite a net lactate uptake by the active leg, a simultaneous unidirectional lactate release was observed in McArdle patients at rates that were similar to the healthy controls.Conclusion:Lactate is an important energy source for contracting skeletal muscle in patients with myophosphorylase deficiency. Although McArdle patients had leg net lactate consumption, a simultaneous release of lactate was observed at rates similar to that found in healthy individuals exercising at the same very low workload, suggesting that lactate formation is mandatory for muscle energy generation during exercise.

We have found experimentally, that the exciton luminescence rise times in GaAs/AlGaAs quantum wells oscillate as a function of incident laser energies. Guided by Monte-Carlo simulations we interpret these results as the occurrence of selective LO-phonon assisted exciton formation.

ABSTRACTSuccessful multidrug-resistant clones are increasing in prevalence globally, which makes the ability to identify these clones urgent. However, adequate, easy-to-perform, and reproducible typing methods are lacking. We investigated whether DiversiLab (DL), an automated repetitive-sequence-based PCR bacterial typing system (bioMérieux), is suitable for comparing isolates analyzed at different geographic centers. A total of 39 Escherichia coliand 39 Klebsiellaspecies isolates previously typed by the coordinating center were analyzed. Pulsed-field gel electrophoresis (PFGE) confirmed the presence of one cluster of 6 isolates, three clusters of 3 isolates, and three clusters of 2 isolates for each set of isolates. DL analysis was performed in 11 centers in six different countries using the same protocol. The DL profiles of 425 E. coliand 422 Klebsiellaspp. were obtained. The DL system showed a lower discriminatory power for E. colithan did PFGE. The local DL data showed a low concordance, as indicated by the adjusted Rand and Wallace coefficients (0.132 to 0.740 and 0.070 to 1.0 [E. coli] and 0.091 to 0.864 and 0.056 to 1.0 [Klebsiellaspp.], respectively). The central analysis showed a significantly improved concordance (0.473 to 1.0 and 0.290 to 1.0 [E. coli] and 0.513 to 0.965 and 0.425 to 1.0 [Klebsiellaspp.], respectively). The misclassifications of profiles for individual isolates were mainly due to inconsistent amplification, which was most likely due to variations in the quality and amounts of the isolated DNA used for amplification. Despite local variations, the DL system has the potential to indicate the occurrence of clonal outbreaks in an international setting, provided there is strict adherence to standardized, reproducible DNA isolation methods and analysis protocols, all supported by a central database for profile comparisons.

ABSTRACTWe studied clinical characteristics, appropriateness of initial antibiotic treatment, and other factors associated with day 30 mortality in patients with bacteremia caused by extended-spectrum-β-lactamase (ESBL)-producing bacteria in eight Dutch hospitals. Retrospectively, information was collected from 232 consecutive patients with ESBL bacteremia (due to Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae) between 2008 and 2010. In this cohort (median age of 65 years; 24 patients were <18 years of age), many had comorbidities, such as malignancy (34%) or recurrent urinary tract infection (UTI) (15%). One hundred forty episodes (60%) were nosocomial, 54 (23%) were otherwise health care associated, and 38 (16%) were community acquired. The most frequent sources of infection were UTI (42%) and intra-abdominal infection (28%). Appropriate therapy within 24 h after bacteremia onset was prescribed to 37% of all patients and to 54% of known ESBL carriers. The day 30 mortality rate was 20%. In a multivariable analysis, a Charlson comorbidity index of ≥3, an age of ≥75 years, intensive care unit (ICU) stay at bacteremia onset, a non-UTI bacteremia source, and presentation with severe sepsis, but not inappropriate therapy within <24 h (adjusted odds ratio [OR], 1.53; 95% confidence interval [CI], 0.68 to 3.45), were associated with day 30 mortality. Further assessment of confounding and a stratified analysis for patients with UTI and non-UTI origins of infection did not reveal a statistically significant effect of inappropriate therapy on day 30 mortality, and these results were insensitive to the possible misclassification of patients who had received β-lactam–β-lactamase inhibitor combinations or ceftazidime as initial treatment. In conclusion, ESBL bacteremia occurs mostly in patients with comorbidities requiring frequent hospitalization, and 84% of episodes were health care associated. Factors other than inappropriate therapy within <24 h determined day 30 mortality.

This review highlights the role of cytokines, in particular tumour necrosis factor alpha (TNF-) and interleukin-6 (IL-6), in relation to the nature of human in-vivo muscle wasting in disease.

We recently demonstrated that reconstituted high-density lipoprotein (rHDL) modulates glucose metabolism in humans via both AMP-activated protein kinase (AMPK) in muscle and by increasing plasma insulin. Given the key roles of both AMPK and insulin in fatty acid metabolism, the current study investigated the effect of rHDL infusion on fatty acid oxidation and lipolysis. Thirteen patients with type 2 diabetes received separate infusions of rHDL and placebo in a randomized, cross-over study. Fatty acid metabolism was assessed using steady-state tracer methodology, and plasma lipids were measured by mass spectrometry (lipidomics). In vitro studies were undertaken in 3T3-L1 adipocytes. rHDL infusion inhibited fasting-induced lipolysis (P = 0.03), fatty acid oxidation (P < 0.01), and circulating glycerol (P = 0.04). In vitro, HDL inhibited adipocyte lipolysis in part via activation of AMPK, providing a possible mechanistic link for the apparent reductions in lipolysis observed in vivo. In contrast, circulating NEFA increased after rHDL infusion (P < 0.01). Lipidomic analyses implicated phospholipase hydrolysis of rHDL-associated phosphatidylcholine as the cause, rather than lipolysis of endogenous fat stores. rHDL infusion inhibits fasting-induced lipolysis and oxidation in patients with type 2 diabetes, potentially through both AMPK activation in adipose tissue and elevation of plasma insulin. The phospholipid component of rHDL also has the potentially undesirable effect of increasing circulating NEFA.

ABSTRACTMany bacterial typing methods are specific for one species only, time-consuming, or poorly reproducible. DiversiLab (DL; bioMe´rieux) potentially overcomes these limitations. In this study, we evaluated the DL system for the identification of hospital outbreaks of a number bacterial species. Appropriately typed clinical isolates were tested with DL. DL typing agreed with pulsed-field gel electrophoresis (PFGE) for Acinetobacter(n= 26) and Stenotrophomonas maltophilia(n= 13) isolates. With two exceptions, DL typing of Klebsiellaisolates (n= 23) also correlated with PFGE, and in addition, PFGE-nontypeable (PFGE-NT) isolates could be typed. Enterobacter(n= 28) results also correlated with PFGE results; also, PFGE-NT isolates could be clustered. In a larger study (n= 270), a cluster of 30 isolates was observed that could be subdivided by PFGE. The results for Escherichia coli(n= 38) correlated less well with an experimental multilocus variable number of tandem repeats analysis (MLVA) scheme. Pseudomonas aeruginosa(n= 52) showed only a limited number of amplification products for most isolates. When multiple Pseudomonasisolates were assigned to a single type in DL, all except one showed multiple multilocus sequence types. Methicillin-resistant Staphylococcus aureusgenerally also showed a limited number of amplification products. Isolates that belonged to different outbreaks by other typing methods, including PFGE, spatyping, and MLVA, were grouped together in a number of cases. For Enterococcus faecium, the limited variability of the amplification products obtained made interpretation difficult and correlation with MLVA and espgene typing was poor. All of the results are reflected in Simpson's index of diversity and adjusted Rand's and Wallace's coefficients. DL is a useful tool to help identify hospital outbreaks of Acinetobacterspp., S. maltophilia, the Enterobacter cloacaecomplex, Klebsiellaspp., and, to a somewhat lesser extent, E. coli. In our study, DL was inadequate for P. aeruginosa, E. faecium, and MRSA. However, it should be noted that for the identification of outbreaks, epidemiological data should be combined with typing results.

OBJECTIVE To determine whether patients with defects of the respiratory chain have metabolic adaptations that promote a preferential use of fats or carbohydrates, similar to what is observed in metabolic myopathies affecting glycolysis or fat oxidation. DESIGN Causation and case-control study. Fat metabolism was determined by means of indirect calorimetry and stable isotope technique in patients and healthy subjects. Patients carried various types and loads (mean [SE], 72% [5%]) of mitochondrial DNA (mtDNA) mutations in skeletal muscle. All subjects exercised at the same absolute workload (mean [SE], 65 [10] W), corresponding to 72% (in patients) and 30% (in healthy subjects) of maximum oxygen consumption. SETTING Neuromuscular research unit. PARTICIPANTS Ten patients with mtDNA mutations and 10 sex-matched healthy subjects. MAIN OUTCOME MEASURES Fat turnover, plasma concentrations of palmitate and total free fatty acids, glucose mobilization, and total carbohydrate oxidation. RESULTS Fat turnover and plasma concentrations of palmitate and total free fatty acids were similar in patients and healthy subjects at rest and during exercise. In line with the higher relative workload of the patients, glucose mobilization and total carbohydrate oxidation were higher in the patients compared with the healthy subjects. CONCLUSION During moderate-intensity exercise, the balance between fat and carbohydrate use in patients with mtDNA mutations matches that seen in healthy subjects, indicating that manipulating dietary fat and carbohydrate content is not a feasible therapeutic option to improve exercise intolerance in these disorders.Arch Neurol. 2009;66(3):365-370--

It is known that muscle phosphorylase deficiency restricts carbohydrate utilization, but the implications for muscle fat metabolism have not been studied. We questioned whether patients with McArdle disease can compensate for the blocked muscle glycogen breakdown by enhancing fat oxidation during exercise.

CONTEXT: Low-grade systemic inflammation is a feature of most lifestyle-related chronic diseases. Enhanced TNF- concentrations have been implicated in the development of hyperlipidemia. OBJECTIVE: We hypothesized that an acute elevation of TNF- in plasma would cause an increase in lipolysis, increasing circulatory free fatty acid (FFA) levels. Subjects and Methods: Using a randomized controlled, crossover design, healthy young male individuals (n = 10) received recombinant human (rh) TNF- (700 ng/m–2·h–1) for 4 h, and energy metabolism was evaluated using a combination of tracer dilution methodology and arterial-venous differences over the leg. RESULTS: Plasma TNF- levels increased from 0.7 ± 0.04 to 16.7 ± 1.8 pg/ml, and plasma IL-6 increased from 1.0 ± 0.2 to 9.2 ± 1.0 pg/ml (P < 0.05) after 4-h rhTNF- infusion. Here, we demonstrate that 4-h rhTNF- infusion increases whole body lipolysis by 40% (P < 0.05) with a concomitant increase in FFA clearance, with no changes in skeletal muscle FFA uptake, release, or oxidation. Of note, systemic glucose turnover and lactate and catecholamine levels were unaffected by rhTNF- infusion. CONCLUSION: This study demonstrates that a relatively low dose of rhTNF- induces systemic lipolysis and that the skeletal muscle fat metabolism is unaffected.

ABSTRACTA large outbreak with an aminoglycoside-resistant Enterobacter cloacae(AREC) clone occurred at the University Medical Center Utrecht beginning in 2001 and continued up through the time that this study was completed. This clone (genotype I) contains a conjugative R plasmid carrying the qnrA1, blaCTX-M-9, and aadBgenes, encoding resistance to quinolones, extended-spectrum ß-lactamases, and aminoglycosides, respectively. The aim of this study was to determine whether this clone was more transmissible than other AREC strains. Therefore, the dissemination of this genotype and of other E. cloacaestrains was studied. In addition, infection control measures taken were evaluated. Pulsed-field gel electrophoresis analysis divided the 191 AREC strains into 42 different genotypes, of which 5 (12%) involved at least three patients. Aside from this outbreak (133 patients), only two other small outbreaks occurred, showing that the infection control measures were successful for all strains but one. Among 324 aminoglycoside-susceptible E. cloacaestrains, 34/166 (20%) genotypes were identified from at least three patients, but only 4 involved small outbreaks. The outbreak strain was also detected in 11 of 15 other Dutch hospitals and caused outbreaks in at least 4. Evaluation of infection control measures showed that the outbreak strain disseminated throughout the hospital despite adequate implementation of internationally accepted guidelines on the control of multidrug-resistant Enterobacteriaceae(MRE). In conclusion, some MRE strains are better able to spread than others, and these strains may not be controlled by the current infection control guidelines. Strategies to identify such strains in an early phase and adapted guidelines for such “superbugs” are needed to prevent these clones from becoming endemic.

ABSTRACTA hospital-wide increase in the number of patients with aminoglycoside-resistant Enterobacter cloacae(AREC) isolated from clinical cultures was detected in December 2002 using a classical surveillance system (CSS). CSS refers to a strategy based on the recognition of an increased incidence of a species with a particular antibiogram at certain wards in a limited period. Since clonal spread was suspected, hospital records were reviewed for E. cloacaeculture-positive patients. Based upon genotyping of 139 clinical E. cloacaeisolates from 80 patients, it was concluded that 53 patients had had clinical cultures with a single AREC clone since April 2001. Determinants for unnoticed spread were investigated retrospectively, as was the possibility that a computer-assisted surveillance method would have detected this outbreak at an earlier stage. Determinants associated with late detection of clonal spread were the following: (i) the absence of a hospital-wide increase in incidence of E. cloacaecases for 1.5 years, (ii) the long time interval between cases, (iii) the hospital-wide occurrence of new cases, due to a high number of patient transfers between wards, (iv) the large variety of clinical sites, and (v) the high variability of antibiograms (n= 33). Retrospective application of a recently described computer-assisted surveillance method as well as an “in-house”-developed algorithm resulted in earlier detection of the outbreak of 6 and 12 months, respectively. These findings suggest that computerized tools for surveillance may recognize resistance trends that are too complex to be detected by manual review and indicate the need for prospective evaluation of such algorithms.

A hospital-wide increase in the number of patients with aminoglycoside-resistant Enterobacter cloacae (AREC) isolated from clinical cultures was detected in December 2002 using a classical surveillance system (CSS). CSS refers to a strategy based on the recognition of an increased incidence of a species with a particular antibiogram at certain wards in a limited period. Since clonal spread was suspected, hospital records were reviewed for E. cloacae culture-positive patients. Based upon genotyping of 139 clinical E. cloacae isolates from 80 patients, it was concluded that 53 patients had had clinical cultures with a single AREC clone since April 2001. Determinants for unnoticed spread were investigated retrospectively, as was the possibility that a computer-assisted surveillance method would have detected this outbreak at an earlier stage. Determinants associated with late detection of clonal spread were the following: (i) the absence of a hospital-wide increase in incidence of E. cloacae cases for 1.5 years, (ii) the long time interval between cases, (iii) the hospital-wide occurrence of new cases, due to a high number of patient transfers between wards, (iv) the large variety of clinical sites, and (v) the high variability of antibiograms (n = 33). Retrospective application of a recently described computer-assisted surveillance method as well as an "in-house"-developed algorithm resulted in earlier detection of the outbreak of 6 and 12 months, respectively. These findings suggest that computerized tools for surveillance may recognize resistance trends that are too complex to be detected by manual review and indicate the need for prospective evaluation of such algorithms.

ABSTRACTSeventy-four isolates of multiresistant Escherichia coliand Klebsiellaspp. recovered during a 3-year period and 17 control strains with genotypically identified beta-lactamases were tested for the production of extended-spectrum beta-lactamases (ESBLs) by using the Etest and the VITEK 1, VITEK 2, and Phoenix automated instruments. The use of the Etest was evaluated by investigating its accuracy in detecting the ESBLs of the control strains and by comparing interpretation results of laboratory technicians and experts. The accuracy of the Etest was 94%. With the Etest as the reference for the clinical strains and the genotype as the reference for the control strains, the automated instruments detected the ESBLs with accuracies of 78% (VITEK 2), 83% (VITEK 1), and 89% (Phoenix). No significant difference between the systems with regard to the control strains was detected. The VITEK 2 did, however, perform less well than the Phoenix (P= 0.03) on the collection of clinical isolates, mainly because of its high percentage of indeterminate test results (11%). No significant difference between the performances of the VITEK 1 and either the VITEK 2 or the Phoenix was found. However, because of its associated BDXpert system the Phoenix showed the best performance. The Etest was found to be an accurate test but was limited by its indeterminate results (4%), its inability to differentiate between K1 hyperproduction and ESBLs, questionable guidelines concerning mutants inside the inhibition zones, and the inability of the technicians to recognize subtle zone deformations.

ABSTRACTAn increase in multiresistant Enterobacteriaceaewas observed at one of the departments of the University Medical Center Utrecht. Nine different integrons and 17 gene cassettes were found, including the new gene cassette aadA8. This cassette was highly related to aadA3and aadA2. In addition, an unknown promoter sequence was found for two integrons.

The purpose of the present study was to investigate the degree to which peak heart rate is reduced during exhaustive exercise in acute hypoxia. Five sea-level lowlanders performed maximal exercise at normobaric normoxia and at three different levels of hypobaric hypoxia (barometric pressures of 518, 459, and 404 mmHg) in a hypobaric chamber and while breathing 9% O2 in N2. These conditions were equivalent to altitudes of 3300, 4300, 5300, and 6300 m above sea level, respectively. At 4300 m, maximal exercise was also repeated after 4 and 8 h. Peak heart rate (HR) decreased from 191 (182-202) (mean and range) at sea level to 189 (179-200), 182 (172-189), 175 (166-183), and 165 (162-169) in the acute hypoxic conditions. Peak HR did not decrease further after 4 and 8 h at 4300 m compared to the acute exposure at this altitude. Between barometric pressures of 518 and 355 mmHg (~3300 and 6300 m), peak HR decreased linearly: peak HRhypobaria = peak HRsea level - 0.135 × [hypobaria3100 - hypobaria (mmHg)]; or peak HRaltitude = peak HRsea level - 0.15 × (altitude - 3100 m). This corresponds to ~1-beat · min-1 reduction in peak HR for every 7-mmHg decrease in barometric pressure below 530 mmHg (~130 m of altitude gained above 3100 m). At termination of exercise, maximal plasma lactate and norepinephrine concentrations were similar to those observed during maximal exercise in normobaric normoxia. This study clearly demonstrates a progressive decrease in peak HR with increasing altitude, despite evidence of similar exercise effort and unchanged sympathetic excitation.

We have measured maximal heart rate during a graded maximal bicycle exercise test to exhaustion in five healthy climbers before and during an expedition to Mt. Everest. Maximal heart rates at sea level were 186 (177-204) beats/min-1 at sea level and 170 (169-182) beats/min-1 with acute hypoxia. After 1, 4 and 6 weeks of acclimatization to 5400 m, maximal heart rates were 155 (135-182), 158 (144-182), and 155 (140-183) beats/min-1, respectively. Heart rates of two of the climbers were measured during their attempt to reach the summit of Mt. Everest without the use of supplemental oxygen. The peak heart rates at 8750 m for the two climbers were 142 and 144 beats/min-1, which were similar to their maximal heart rates during exhaustive bicycle exercise at 5400 m, the values being 144 and 148 beats/min-1, respectively. The peak heart rates at 8750 m are in agreement with other field studies, but considerably higher than values reported from hypobaric chamber studies.

The proteasome is the principal provider of major histocompatibility complex (MHC) class I–presented peptides. Interferon (IFN)-γ induces expression of three catalytically active proteasome subunits (LMP2, LMP7, and MECL-1) and the proteasome-associated activator PA28. These molecules are thought to optimize the generation of MHC class I–presented peptides. However, known information on their contribution in vivo is very limited. Here, we examined the antigen processing of two murine leukemia virus-encoded cytotoxic T lymphocyte (CTL) epitopes in murine cell lines equipped with a tetracycline-controlled, IFN-γ–independent expression system. We thus were able to segregate the role of the immunosubunits from the role of PA28. The presence of either immunosubunits or PA28 did not alter the presentation of a subdominant murine leukemia virus (MuLV)-derived CTL epitope. However, the presentation of the immunodominant MuLV-derived epitope was markedly enhanced upon induction of each of these two sets of genes. Thus, the IFN-γ–inducible proteasome subunits and PA28 can independently enhance antigen presentation of some CTL epitopes. Our data show that tetracycline-regulated expression of PA28 increases CTL epitope generation without affecting the 20S proteasome composition or half-life. The differential effect of these IFN-γ–inducible proteins on MHC class I processing may have a decisive influence on the quality of the CTL immune response.

In the present study, we have investigated the effect of carbohydrate and protein hydrolysate ingestion on muscle glycogen resynthesis during 4 h of recovery from intense cycle exercise. Five volunteers were studied during recovery while they ingested, immediately after exercise, a 600-ml bolus and then every 15 min a 150-ml bolus containing 1) 1.67 g ⋅ kg body wt−1⋅ l−1of sucrose and 0.5 g ⋅ kg body wt−1⋅ l−1of a whey protein hydrolysate (CHO/protein), 2) 1.67 g ⋅ kg body wt−1⋅ l−1of sucrose (CHO), and 3) water. CHO/protein and CHO ingestion caused an increased arterial glucose concentration compared with water ingestion during 4 h of recovery. With CHO ingestion, glucose concentration was 1–1.5 mmol/l higher during the first hour of recovery compared with CHO/protein ingestion. Leg glucose uptake was initially 0.7 mmol/min with water ingestion and decreased gradually with no measurable glucose uptake observed at 3 h of recovery. Leg glucose uptake was rather constant at 0.9 mmol/min with CHO/protein and CHO ingestion, and insulin levels were stable at 70, 45, and 5 mU/l for CHO/protein, CHO, and water ingestion, respectively. Glycogen resynthesis rates were 52 ± 7, 48 ± 5, and 18 ± 6 for the first 1.5 h of recovery and decreased to 30 ± 6, 36 ± 3, and 8 ± 6 mmol ⋅ kg dry muscle−1⋅ h−1between 1.5 and 4 h for CHO/protein, CHO, and water ingestion, respectively. No differences could be observed between CHO/protein and CHO ingestion ingestion. It is concluded that coingestion of carbohydrate and protein, compared with ingestion of carbohydrate alone, did not increase leg glucose uptake or glycogen resynthesis rate further when carbohydrate was ingested in sufficient amounts every 15 min to induce an optimal rate of glycogen resynthesis.

Lack of interleukin-6 (IL-6) leads to expansion of adipose tissue mass in rodents and humans. The exact underlying mechanisms have not been identified. In this placebo-controlled, non-randomized, participant-blinded crossover study, we use the IL-6 receptor antibody tocilizumab to investigate the role of endogenous IL-6 in regulating systemic energy metabolism at rest and during exercise and recovery in lean and obese men using tracer dilution methodology. Tocilizumab reduces fatty acid appearance in the circulation under all conditions in lean and obese individuals, whereas lipolysis (the rate of glycerol appearance into the circulation) is mostly unaffected. The fact that fatty acid oxidation is unaffected by IL-6 receptor blockade suggests increased re-esterification of fatty acids. Glucose kinetics are unaffected. We find that blocking endogenous IL-6 signaling with tocilizumab impairs fat mobilization, which may contribute to expansion of adipose tissue mass and, thus, affect the health of individuals undergoing anti-IL-6 therapy (Clinicaltrials.gov: NCT03967691).