A Genetic Tool to Track Protein Aggregates and Control Prion Inheritance.

Protein aggregation is a hallmark of many diseases but also underlies a wide range of positive cellular functions. This phenomenon has been difficult to study because of a lack of quantitative and high-throughput cellular tools. Here, we develop a synthetic genetic tool to sense and control protein aggregation. We apply the technology to yeast prions, developing sensors to track their aggregation states and employing prion fusions to encode synthetic memories in yeast cells. Utilizing high-throughput screens, we identify prion-curing mutants and engineer "anti-prion drives" that reverse the non-Mendelian inheritance pattern of prions and eliminate them from yeast populations. We extend our technology to yeast RNA-binding proteins (RBPs) by tracking their propensity to aggregate, searching for co-occurring aggregates, and uncovering a group of coalescing RBPs through screens enabled by our platform. Our work establishes a quantitative, high-throughput, and generalizable technology to study and control diverse protein aggregation processes in cells.

AMP-activated protein kinase (AMPK)α2 plays a role in determining the cellular fate of glucose in insulin-resistant mouse skeletal muscle.

AIMS/HYPOTHESIS: We determined whether: (1) an acute lipid infusion impairs skeletal muscle AMP-activated protein kinase (AMPK)α2 activity, increases inducible nitric oxide synthase (iNOS) and causes peripheral insulin resistance in conscious, unstressed, lean mice; and (2) restoration of AMPKα2 activity during the lipid infusion attenuates the increase in iNOS and reverses the defect in insulin sensitivity in vivo. METHODS: Chow-fed, 18-week-old C57BL/6J male mice were surgically catheterised. After 5 days they received: (1) a 5 h infusion of 5 ml kg(-1) h(-1) Intralipid + 6 U/h heparin (Lipid treatment) or saline (Control); (2) Lipid treatment or Control, followed by a 2 h hyperinsulinaemic-euglycaemic clamp (insulin clamp; 4 mU kg(-1) min(-1)); and (3) infusion of the AMPK activator, 5-aminoimidazole-4-carboxamide 1-ß-D-ribofuranoside (AICAR) (1 mg kg(-1) min(-1)), or saline during Lipid treatment, followed by a 2 h insulin clamp. In a separate protocol, mice producing a muscle-specific kinase-dead AMPKα2 subunit (α2-KD) underwent an insulin clamp to determine the role of AMPKα2 in insulin-mediated muscle glucose metabolism. RESULTS: Lipid treatment decreased AMPKα2 activity, increased iNOS abundance/activation and reduced whole-body insulin sensitivity in vivo. AICAR increased AMPKα2 activity twofold; this did not suppress iNOS or improve whole-body or tissue-specific rates of glucose uptake during Lipid treatment. AICAR caused a marked increase in insulin-mediated glycogen synthesis in skeletal muscle. Consistent with this latter result, lean α2-KD mice exhibited impaired insulin-stimulated glycogen synthesis even though muscle glucose uptake was not affected. CONCLUSIONS/INTERPRETATION: Acute induction of insulin resistance via lipid infusion in healthy mice impairs AMPKα2, increases iNOS and causes insulin resistance in vivo. However, these changes do not appear to be interrelated. Rather, a functionally active AMPKα2 subunit is required for insulin-stimulated muscle glycogen synthesis.

Adherence to PEG/ribavirin treatment for chronic hepatitis C: prevalence, patterns, and predictors of missed doses and nonpersistence.

Adherence to treatment for hepatitis C virus (HCV) maximizes treatment efficacy. Missed doses and failing to persist on treatment are two patient-level processes that are rarely defined or analysed separately from other factors affecting treatment adherence. We evaluated the prevalence and patterns of missed doses and nonpersistence, and identified patient characteristics associated with these outcomes. Missed doses of ribavirin (RBV) and peginterferon (PEG), measured prospectively in Virahep-C using electronic monitoring technology, were analysed using generalized estimating equations. Cox proportional hazards models analysed time to nonpersistence from baseline to week 24 (N = 401) and from week 24 to 48 in Responders (N = 242). Average proportion of PEG- and RBV-missed doses increased over time from 5% to 15% and 7% to 27%, respectively. Patients who were younger, African-American, unemployed, or unmarried were at greater risk of missing PEG from week 0 to 24; higher baseline depression predicted missing PEG from weeks 24 to 48. Patients who were younger or African-American were more likely to miss daily RBV from weeks 0 to 24; and those without private insurance or employment were more likely to miss RBV from weeks 24 to 48. Fifty-two patients failed to persist on treatment for patient-driven deviations. Predictors of nonpersistence from weeks 0 to 24 included younger age, lower education, public or no insurance, or worse baseline headaches. In conclusion, electronic monitoring and the prospective Virahep-C design afforded a unique opportunity to evaluate missing doses and nonpersistence separately, and identify patients at risk of nonadherence. These processes will be important to investigate as the dosing schedules of antiviral regimens become increasingly complex.

Reliability and validity of a self-efficacy instrument for hepatitis C antiviral treatment regimens.

Self-efficacy or confidence in one's ability to successfully engage in goal-directed behaviour has been shown to influence medication adherence across many chronic illnesses. In the present study, we investigated the psychometric properties of a self-efficacy instrument used during treatment for chronic hepatitis C viral infection (HCV). Baseline (n = 394) and treatment week 24 (n = 254) data from the prospective, longitudinal Viral Resistance to Antiviral Therapy of Chronic Hepatitis C study were examined. Baseline participants were randomly split into two equal-sized subsamples (S(1) and S(2) ). Initial exploratory and confirmatory factor analyses (EFA/CFA) were performed on S(1), while S(2) was used to validate the factor structure of the S(1) results using CFA. An additional CFA was performed on the treatment week 24 participants. Convergent and discriminant validity were assessed by comparing the revised instrument with other psychosocial measures: depression, social support, quality of life and medication-taking behaviour. Our findings supported a reduced 17-item global measure of HCV treatment self-efficacy (HCV-TSE) with four underlying factors: patient communication self-efficacy, general physical coping self-efficacy, general psychological coping self-efficacy and adherence self-efficacy. The global score (0.92-0.94) and four factors (0.85-0.96) demonstrated good internal consistency. Correlations of convergent and discriminant validity yielded low to moderate associations with other measures of psychosocial functioning. The revised HCV-TSE instrument provides a reliable and valid global estimate of confidence in one's ability to engage in and adhere to HCV antiviral treatment. The four-factor structure suggests different types of efficacy beliefs may function during HCV treatment and should be explored further in relation to clinical outcomes.

Respiratory arrest in an obstetric patient using remifentanil patient-controlled analgesia.

Remifentanil patient-controlled analgesia is well established in many centres and provides satisfactory pain relief for many women in labour. We describe a patient using remifentanil patient-controlled analgesia who suffered a respiratory arrest requiring a brief period of ventilation. In our institution, remifentanil patient-controlled analgesia has been offered to women in labour since 2009. Up to this point, we had not observed any critical incidents in over 130 patients using this mode of analgesia in our labour suite.

Artificial intelligence for detection of microsatellite instability in colorectal cancer-a multicentric analysis of a pre-screening tool for clinical application.

BACKGROUND: Microsatellite instability (MSI)/mismatch repair deficiency (dMMR) is a key genetic feature which should be tested in every patient with colorectal cancer (CRC) according to medical guidelines. Artificial intelligence (AI) methods can detect MSI/dMMR directly in routine pathology slides, but the test performance has not been systematically investigated with predefined test thresholds. METHOD: We trained and validated AI-based MSI/dMMR detectors and evaluated predefined performance metrics using nine patient cohorts of 8343 patients across different countries and ethnicities. RESULTS: Classifiers achieved clinical-grade performance, yielding an area under the receiver operating curve (AUROC) of up to 0.96 without using any manual annotations. Subsequently, we show that the AI system can be applied as a rule-out test: by using cohort-specific thresholds, on average 52.73% of tumors in each surgical cohort [total number of MSI/dMMR = 1020, microsatellite stable (MSS)/ proficient mismatch repair (pMMR) = 7323 patients] could be identified as MSS/pMMR with a fixed sensitivity at 95%. In an additional cohort of N = 1530 (MSI/dMMR = 211, MSS/pMMR = 1319) endoscopy biopsy samples, the system achieved an AUROC of 0.89, and the cohort-specific threshold ruled out 44.12% of tumors with a fixed sensitivity at 95%. As a more robust alternative to cohort-specific thresholds, we showed that with a fixed threshold of 0.25 for all the cohorts, we can rule-out 25.51% in surgical specimens and 6.10% in biopsies. INTERPRETATION: When applied in a clinical setting, this means that the AI system can rule out MSI/dMMR in a quarter (with global thresholds) or half of all CRC patients (with local fine-tuning), thereby reducing cost and turnaround time for molecular profiling.

Wound healing following combined radiation and cetuximab therapy in head and neck cancer patients.

OBJECTIVE: This study set out to determine if cetuximab treatment increases the risk of wound healing complications when combined with radiation therapy. METHOD: We performed a retrospective chart review of head and neck cancer patients who received salvage neck dissections between 1999 and 2007, at two academic tertiary care centres. Complications from wound healing were compared between radiation and combined therapy groups. RESULTS: A total of 35 patients received radiation (n=20) or combined radiation and cetuximab therapy (n=15) prior to neck dissection. The treatment groups were similar in regard to demographic and primary tumour-related characteristics. The time between treatment and salvage neck dissection did not differ between the radiation (3.9 months) and combination treatment (3.0 months) groups (p=0.15). Wound healing complications occurred in 13% (2/15) of the patients treated with radiation and cetuximab and there were no complications in patients who received radiation alone (p=0.20). CONCLUSION: Cetuximab did not significantly increase the risk of post-surgical wound complications, although a higher absolute number of wound complications was observed in the group treated with cetuximab and radiation therapy, compared with the group treated with radiation alone. CONFLICT OF INTEREST: This work was supported by a grant from the National Institute of Health (2T32 CA091078-06). One of the authors, JAB, is an occasional consultant and honoraria for ImClone and Bristol-Meyers Squibb.

A novel primate model of delayed wound healing in diabetes: dysregulation of connective tissue growth factor.

AIMS/HYPOTHESIS: Chronic non-healing wounds are a common complication of diabetes. Prolonged inflammation and decreased matrix accumulation may contribute. Connective tissue growth factor (CTGF) is induced during normal wound healing, but its regulation in diabetic wounds is unknown. We developed a primate model for the study of in vivo wound healing in baboons with long diabetes duration. METHODS: Drum implants were placed subcutaneously into thighs of diabetic and non-diabetic control baboons. After 2 and 4 weeks the skin incision sites were removed for measurement of breaking strength and epithelial thickness. Drum implants were removed for analysis of granulation tissue and inflammatory cells, CTGF and tissue inhibitor of matrix metalloproteinase (TIMP-1). Degradation of added CTGF by wound fluid was also examined. RESULTS: Healed incision site skin was stiffer (less elastic) in diabetic baboons and epithelial remodelling was slower compared with controls. Granulation tissue from diabetic baboons was reduced at 2 and 4 weeks, with increased vessel lumen areas at 4 weeks. Macrophages were reduced while neutrophils persisted in diabetic tissue. In diabetic wound tissue at 4 weeks there was less CTGF induced, as shown by immunohistochemistry, compared with controls. In contrast, immunoreactive fragments of CTGF were significantly increased in whole tissue lysate in diabetic baboons, suggesting that CTGF is redistributed in diabetes from granulation tissue into wound fluid. When recombinant human CTGF was co-incubated with wound fluid, increased CTGF degradation products were observed in both control and diabetic samples. CONCLUSIONS/INTERPRETATION: This baboon model of wound healing reflects the abnormal microenvironment seen in human diabetic wounds and provides insights into the dysregulation of CTGF in diabetic wounds.

Ecology. The advantages of togetherness.

What would be the advantage of unicellular organisms becoming multicellular? For organisms that feed on organic food (heterotrophs), the most efficient way to produce energy is to metabolize the food by aerobic respiration, but the fastest way is to metabolize it by fermentation. In their Perspective, Cox and Bonner discuss a mathematical model (Pfeiffer et al.), which shows that when these two kinds of organisms (respirators and fermenters) compete for a limited food source, the respirators manage best when they are grouped in clusters rather than remaining as separate cells. In this way, multicellularity could have originated.

Localization of the globin gene in the template active fraction of chromatin of Friend leukemia cells.

Friend leukemia cell chromatin has been fractionated into template active and inactive components. The globin gene sequence is associated with the template active component both prior to and after the cells are induced with dimethyl sulfoxide to synthesize hemoglobin and therefore appears to be in an active configuration in uninduced as well as in induced Friend leukemia cells. In cells which have lost the ability to produce hemoglobin, the globin gene sequence is not associated with the template active fraction of chromatin. These results demonstrate the success of the fractionation procedure.

Chromosomal RNA: its properties.

We describe the properties of a special class of RNA associated with chromatin. We discuss why this RNA should be considered a distinct class of RNA and not an artifactual degradation product of either transfer or ribosomal RNA.

Fluorometric examination of a lunar sample.

We have been unable to detect porphyrins in 13 grams of the bulk fine lunar sample from the Sea of Tranquillity under conditions in which less than 10-(1J) mole per gram of lunar sample could have been detected. By appropriate extraction, however, the lunar sample yields a material which exhibits absorption maxima at 310 and 350 nanometers and a fluorescence maximum at 410 nanometers.

The Biology of Isolated Chromatin: Chromosomes, biologically active in the test tube, provide a powerful tool for the study of gene action.

The isolated chromatin of higher organisms possesses several properties characteristic of the same chromatin in life. These include the presence of histone bound to DNA, the state of repression of the genetic material, and the ability to serve as template for the readout of the derepressed portion of the genome by RNA polymerase. The important respect in which isolated chromatin differs from the material in vivo, fragmentation of DNA into pieces shorter (5 x 10(6) to 20 x 10(6) molecular weight) than the original, does not appear to importantly alter such transcription. The study of isolated chromatin has already revealed the material basis of the restriction of template activity; it is the formation of a complex between histone and DNA. Chromatin isolated by the methods now available, together with the basis provided by our present knowledge of chromatin biochemistry and biophysics, should make possible and indeed assure rapid increase in our knowledge of chromosomal structure and of all aspects of the control of gene activity and hence of developmental processes.

Consensus guidelines for the management of radiation dermatitis and coexisting acne-like rash in patients receiving radiotherapy plus EGFR inhibitors for the treatment of squamous cell carcinoma of the head and neck.

BACKGROUND: Radiation dermatitis occurs to some degree in most patients receiving radiotherapy, with or without chemotherapy. Patients with squamous cell carcinoma of the head and neck (SCCHN) who receive radiotherapy in combination with epidermal growth factor receptor (EGFR) inhibitors, such as cetuximab, may develop a characteristic acne-like rash in addition to dermatitis. DESIGN: An advisory board of 11 experienced radiation oncologists, medical oncologists and dermatologists discussed the management options for skin reactions in patients receiving EGFR inhibitors and radiotherapy for SCCHN. Skin toxicity was categorised according to the National Cancer Institute-Common Terminology Criteria for Adverse Events (version 3) grading. RESULTS: Both general and grade-specific approaches for the management of dermatitis in this patient group are presented. It was concluded that where EGFR inhibitor-related acne-like rash and dermatitis coexist within irradiated fields, management should be based on the grade of dermatitis: for grade 1 (or no dermatitis), treatment recommendations for EGFR-related acne-like rash outside irradiated fields should be followed; for grades 2 and above, treatment recommendations for dermatitis were proposed. CONCLUSIONS: This paper presents comprehensive consensus guidelines for the treatment of dermatitis in patients with SCCHN receiving EGFR inhibitors in combination with radiotherapy.

Development and applications of a physiologically-based model of paediatric oral drug absorption.

There is increasing interest in paediatric drug absorption and the development of biopharmaceutics tools to facilitate the development of oral formulations for neonates, infants and children. We describe the development and application of a physiologically-based model of paediatric drug absorption applicable from full term birth onwards. Paediatric age-specific parameters were included for salivary flow, gastric pH, gastric emptying (and associated food effects) and duodenal bile salt concentrations and the associated algorithms were integrated into a dissolution, absorption and metabolism model as part of a PBPK platform. For other parameters, there was either evidence for no age-related changes or a lack of data, so that adult values were applied. An initial assessment of the model was carried out by simulating the oral absorption of theophylline, paracetamol and ketoconazole over a range of paediatric ages. The absorption of the first two drugs, both BCS class 1 compounds, was predicted to be slower in early neonates compared to older age groups (median tmax values of 3 vs 2h, respectively), but with invariant fraction absorbed (fa). This is in agreement with clinical observations. The tmax of ketoconazole, a BCS class 2 compound, was predicted to be about 1h in both neonates and adults, but the fa value was higher in the former (0.87 vs 0.69). There is clearly a need to expand the components of the model as new information on the ontogeny of GI tract parameters becomes available, and to assess it against more in vivo data with evidence of specific age-related changes in oral drug absorption.

Chrysotile asbestos upregulates gene expression and production of alpha-receptors for platelet-derived growth factor (PDGF-AA) on rat lung fibroblasts.

PDGF isoforms have been postulated to serve as mediators of fibroblast proliferation and chemotaxis during lung fibrogenesis induced by asbestos inhalation. We have studied the interaction of chrysotile asbestos fibers with rat lung fibroblasts (RLF) in vitro and the consequent changes in PDGF receptor mRNA expression, PDGF binding, and mitogenic activity of PDGF isoforms. Northern blot analysis revealed that mRNA for the PDGF-receptor alpha subtype (PDGF-R alpha) on RLF was upregulated after a 24-h exposure to asbestos in culture (0.5-15 micrograms fibers/cm2). [125I]PDGF-BB receptor assays showed that normal RLF possess mainly PDGF-R beta and a paucity of PDGF-R alpha. In agreement with the Northern data, saturation binding of [125I]PDGF-BB to RLF exposed to asbestos demonstrated an approximately 40% increase in binding sites accompanied by a twofold decrease in receptor affinity. Treating asbestos-exposed RLF with PDGF-AA, which binds only PDGF-R alpha, blocked the PDGF binding sites that were upregulated by fiber exposure. PDGF-AA had increased mitogenic potency for fiber-exposed RLF, but PDGF-BB was a less potent mitogen for these RLF. Nonfibrogenic carbonyl iron spheres induced similar changes in PDGF growth responses. These data show that inorganic particulates alter the PDGF-R alpha population on RLF without significant change in PDGF-R beta.

Definition of immunoglobulin A receptors on eosinophils and their enhanced expression in allergic individuals.

Fc alpha receptors (Fc alpha R), detected by the binding of IgA and by anti-Fc alpha R antibodies, were found to be differentially expressed on eosinophils and neutrophils. Neutrophils were the major granulocyte population expressing Fc alpha R, and they expressed much higher levels of Fc alpha R than eosinophils. The expression of Fc alpha R by eosinophils could be upregulated approximately threefold by Ca2+ ionophore treatment in a dose- and time-dependent manner. This effect, which was blocked by a chelating agent, was not duplicated by other cellular stimuli. Eosinophils in allergic individuals displayed enhanced Fc alpha R expression, whereas neutrophils did not. The Fc alpha R on eosinophils had a higher molecular mass (70-100 kD) than those identified on neutrophils (55-75 kD). However, removal of N-linked carbohydrates from Fc alpha R of eosinophils and neutrophils revealed a major protein core of 32 kD for both cell types. The data indicate that expression of Fc alpha R molecules with a characteristic glycosylation pattern is upregulated on eosinophils in allergic individuals.

Genetic identification of the site of DNA contact in the yeast heat shock transcription factor.

The heat shock transcription factor (HSF), a trimeric transcription factor, activates the expression of heat shock genes in eukaryotes. We have isolated mutations in the HSF1 gene from Saccharomyces cerevisiae that severely compromise the ability of HSF to bind to its normal binding site, repeats of the module nGAAn. One of these mutations, Q229R, shows a "new specificity" phenotype, in which the protein prefers the mutant sequence nGACn. These results identify the region of HSF that contacts DNA, in complete agreement with the crystal structure of HSF of Kluyveromyces lactis and the nuclear magnetic resonance data from HSF of Drosophila melanogaster. To determine the orientation of the DNA-binding domain on the nGAAn motif, we performed site-specific cross-linking between cysteine residues of single-cysteine substitutions. Cysteines placed at the N terminus of the DNA contact helix formed cross-links readily, while cysteines placed at the C terminus of the helix did not.

Interactions between DNA-bound trimers of the yeast heat shock factor.

The heat shock transcription factor (HSF) is a trimer that binds to DNA containing inverted repeats of the sequence nGAAn. HSF can bind DNA with the sequence nGAAnnTTCn or with the sequence nTTCnnGAAn, with little preference for either sequence over the other. However, (nGAAnnTTCn)2 is considerably less active as a heat shock response element (HSE) than is (nTTCnnGAAn)2. The electrophoretic mobilities of DNA-protein complexes and chemical cross-linking between protein monomers indicate that the sequence (nGAAnnTTCn)2 is capable of binding a single HSF trimer. In contrast, the sequence with higher biological activity, (nTTCnnGAAn)2, is capable of binding two trimers. Thus, the ability of four-nGAAn-element HSEs to bind one or two trimers depends on the permutation with which the elements are presented. A survey of naturally occurring HSEs shows the sequence (nTTCnnGAAn)2 to be the more prevalent. We suggest that the greater ability of one permutation over the other to bind two HSF trimers accounts for the initial identification of the naturally occurring heat shock consensus sequence as a region of dyad symmetry.

Temperature-dependent regulation of a heterologous transcriptional activation domain fused to yeast heat shock transcription factor.

The heat shock transcription factor (HSF) of the yeast Saccharomyces cerevisiae is posttranslationally modified. At low growth temperatures, it activates transcription of heat shock genes only poorly; after shift to high temperatures, it activates transcription readily. In an effort to elucidate the mechanism of this regulation, we constructed a series of HSF-VP16 fusions that join the HSF DNA-binding domain to the strong transcriptional activation domain from the VP16 gene of herpes simplex virus. Replacement of the endogenous C-terminal transcriptional activation domain with that of VP16 generates an HSF derivative that exhibits behavior reminiscent of HSF itself: low transcriptional activation activity at normal growth temperature and high activity after heat shock. HSF can thus restrain the activity of the heterologous VP16 transcriptional activation domain. To determine what is required for repression of activity at low temperature, we deleted portions of HSF from this HSF-VP16 fusion to map the regulatory domain. We also isolated point mutations that convert the HSF-VP16 fusion into a constitutive transcriptional activator. We conclude that the central, evolutionarily conserved domain of HSF, encompassing the DNA-binding and multimerization domains, contains a major determinant of temperature-dependent regulation.