Combi-seq for multiplexed transcriptome-based profiling of drug combinations using deterministic barcoding in single-cell droplets.

Anti-cancer therapies often exhibit only short-term effects. Tumors typically develop drug resistance causing relapses that might be tackled with drug combinations. Identification of the right combination is challenging and would benefit from high-content, high-throughput combinatorial screens directly on patient biopsies. However, such screens require a large amount of material, normally not available from patients. To address these challenges, we present a scalable microfluidic workflow, called Combi-Seq, to screen hundreds of drug combinations in picoliter-size droplets using transcriptome changes as a readout for drug effects. We devise a deterministic combinatorial DNA barcoding approach to encode treatment conditions, enabling the gene expression-based readout of drug effects in a highly multiplexed fashion. We apply Combi-Seq to screen the effect of 420 drug combinations on the transcriptome of K562 cells using only ~250 single cell droplets per condition, to successfully predict synergistic and antagonistic drug pairs, as well as their pathway activities.

Validation of the Knowledge of Genome Sequencing (KOGS) scale in cancer patients.

INTRODUCTION: The Knowledge of Genome Sequencing (KOGS) questionnaire was recently developed to measure knowledge of genomic sequencing (GS), with preliminary psychometric data supporting its reliability and validity. The aim of this study was to test the reliability and validity of the KOGS in a larger sample, and to confirm its utility in a cancer setting. METHODS: The Genetic Cancer Risk in the Young (RisC) study recruits participants with a personal history of cancer, to investigate heritable cancer causes and future cancer risk using germline GS. Participants (n = 261) in a psychosocial substudy of RisC completed a questionnaire after consent to RisC but before GS, including the KOGS, the Intolerance of Uncertainty Scale, the Chew health literacy scale and items assessing demographic and disease variables. Confirmatory factor analysis (CFA), Cronbach alpha and correlational analyses were undertaken. RESULTS: The CFA testing a single-factor model yielded a good model fit, χ2/df = 2.43, comparative fit index (CFI) = 0.97, root mean square error of approximation (RMSEA) = 0.07 and weighted mean root square (WRMR) = 1.03. Factor loadings of all items were above 0.60 and ranged between.66 and.93. The single factor score demonstrated excellent internal consistency (α = 0.82). KOGS scores were significantly associated with health literacy (r = 0.23, p < .001), having a university education [t(258) = -4.53, p < .001] and having a medical or science background [t(259) = -3.52, p < .001] but not with speaking a language other than English at home, time since diagnosis, previous genetic counselling/testing or intolerance of uncertainty. DISCUSSION: This study confirmed a single-factor structure for the KOGS, and its reliability and validity in a cancer population. Associations with measures of health literacy and education were significant and positive as expected, supporting the KOG's construct validity. Previous genetic counselling may not be sufficient to provide specific knowledge of GS.

Long-term survival in patients with advanced non-small-cell lung cancer treated with atezolizumab versus docetaxel: Results from the randomised phase III OAK study.

BACKGROUND: Atezolizumab (anti-programmed death-ligand 1 [PD-L1]) received approval from the US Food and Drug Administration and European Medicines Agency for previously treated advanced non-small-cell lung cancer based on OAK-a randomised, phase III trial that showed significantly improved survival with atezolizumab versus docetaxel regardless of PD-L1 expression. With longer follow-up, we summarised the characteristics of long-term survivors (LTSs). METHODS: In OAK (NCT02008227), patients were randomised 1:1 to receive atezolizumab or docetaxel until loss of clinical benefit or disease progression, respectively. Overall survival was evaluated after a 26-month minimum follow-up, including in patient subgroups defined by best overall response (BOR). LTSs were defined as patients who lived ≥24 months since randomisation. Non-LTSs died within 24 months, and patients censored before 24 months were excluded from the analysis. The baseline characteristics, including biomarkers, BOR, subsequent non-protocol therapy (NPT) and safety, are reported. RESULTS: Survival benefit with atezolizumab was observed across all patient subgroups defined by BOR. More atezolizumab-treated patients were LTSs versus those treated with docetaxel (28% versus 18%). Most atezolizumab responders were LTSs (77%) versus only 48% of docetaxel responders. However, 21% of atezolizumab-arm LTSs had progressive disease (PD) as BOR, and more atezolizumab-arm LTSs than non-LTSs continued treatment post-PD. Fifty-two percent of docetaxel-arm LTSs received immunotherapy as subsequent NPT. Despite extended treatment duration in atezolizumab-arm LTSs (median, 18 months), atezolizumab was well tolerated. CONCLUSIONS: After >2 years of follow-up, atezolizumab continued to provide durable survival benefit versus docetaxel, with tolerable safety. Atezolizumab-arm LTSs were enriched for patients with high PD-L1 expression and included PD-L1-negative patients. Long-term survival was not limited to responders.

Sociodemographic, psychosocial and clinical factors associated with uptake of genetic counselling for hereditary cancer: a systematic review.

Evidence suggests that a significant proportion of individuals referred to cancer genetic counselling (GC) do not attend, and thus may not be engaged in adequate cancer risk management. We aimed to review the literature to better understand barriers to accessing GC and how they may be overcome. We conducted a systematic literature search for articles examining factors influencing cancer GC uptake as well as motivators and barriers to GC attendance. Factors were categorised as sociodemographic, psychosocial or clinical. The literature search identified 1413 citations, 35 of which met the inclusion criteria. GC uptake ranged from 19% to 88%. With the exceptions of education level, socioeconomic status, cancer-specific distress, personal cancer diagnosis and actual and perceived risk of cancer, support was lacking for most sociodemographic, clinical and psychosocial factors as predictors of GC uptake. Cost and logistical barriers, emotional concerns, family concerns and low perceived personal relevance were reported as important considerations for those declining GC. We conclude that there is poor understanding of GC and a lack of decision support among those referred to GC. Research into ways of providing education and support to referred individuals will be important as the scope and availability of GC and genetic testing broaden.

Early postnatal GABAA receptor modulation reverses deficits in neuronal maturation in a conditional neurodevelopmental mouse model of DISC1.

Exploring drug targets based on disease-associated molecular mechanisms during development is crucial for the generation of novel prevention and treatment strategies for neurodevelopmental psychiatric conditions. We report that prefrontal cortex (PFC)-specific postnatal knockdown of DISC1 via in utero electroporation combined with an inducible knockdown expression system drives deficits in synaptic GABAA function and dendritic development in pyramidal neurons, as well as abnormalities in sensorimotor gating, albeit without profound memory deficits. We show for the first time that DISC1 is specifically involved in regulating cell surface expression of α2 subunit-containing GABAA receptors in immature developing neurons, but not after full maturation. Notably, pharmacological intervention with α2/3 subtype-selective GABAA receptor positive allosteric modulators during the early postnatal period ameliorates dendritic deficits and behavioral abnormalities induced by knockdown of DISC1. These findings highlight a critical role of DISC1-mediated disruption of postnatal GABA signaling in aberrant PFC maturation and function.

Results of a self-absorption study on the Versapor 3000 47-mm filters for radioactive particulate air stack sampling.

Since the mid-1980's the Pacific Northwest National Laboratory (PNNL) has used a value of 0.85 as a correction factor for the self absorption of activity for particulate radioactive air samples collected from building exhaust for environmental monitoring. More recently, an effort was made to evaluate the current particulate radioactive air sample filters (Versapor 3000, 47-mm diameter) used at PNNL for self absorption effects. There were two methods used to characterize the samples. Sixty samples were selected from the archive for acid digestion to compare the radioactivity measured by direct gas-flow proportional counting of filters to the results obtained after acid digestion of the filter and counting again by gas-flow proportional detection. Thirty different sample filters were selected for visible light microscopy to evaluate filter loading and particulate characteristics. Mass-loading effects were also considered. Large error is associated with the sample filter analysis comparison and subsequently with the estimation of the absorption factor resulting in an inadequate method to estimate losses from self-absorption in the sample filter. The mass loading on the sample filter as determined after digestion and drying was approximately 0.08 mg cm; however, this value may not represent the total filter mass loading given that there may be undetermined losses associated with the digestion process. While it is difficult to determine how much material is imbedded in the filter, observations from the microscopy analysis indicate that the vast majority of the particles remain on the top of the filter. In comparing the results obtained, the continued use of 0.85 as a conservative correction factor is recommended.

Glycosaminoglycan synthesis and structure as targets for the prevention of calcific aortic valve disease.

Calcific aortic valve disease is frequently driven by ageing and the obesity-associated metabolic syndrome, and the increasing impact of these factors indicates that valve disease will become a cardiovascular disease of considerable significance. This disease is now thought to be an active cell-based disease process, which may therefore be amenable to therapeutic intervention. Some similarities are apparent with atherosclerosis. The accumulation of lipid, possibly by retention by proteoglycans and the attraction of inflammatory cells by hyaluronan, may be common to the early stages of both pathologies. The synthesis and structure of glycosaminoglycans, proteoglycans, and hyaluronan are exquisitely regulated, and the signalling pathways controlling these processes may provide tissue-specific opportunities for concomitant prevention of atherosclerosis and calcific aortic valve disease.

Fenofibrate modifies human vascular smooth muscle proteoglycans and reduces lipoprotein binding.

AIMS/HYPOTHESIS: Vascular disease in type 2 diabetes is associated with an up-regulation of atherogenic growth factors, which stimulate matrix synthesis including proteoglycans. We have examined the direct actions of fenofibrate on human vascular smooth muscle cells (VSMCs) and have specifically investigated proteoglycan synthesis and binding to LDL. METHODS: Proteoglycans synthesised by human VSMCs treated with fenofibrate (30 micromol/l) were assessed for binding to human LDL using a gel mobility shift assay, metabolically labelled with [(35)S]-sulphate and quantitated by cetylpyridinium chloride. They were then assessed for electrophoretic mobility by SDS-PAGE, for size by gel filtration, for sulphation pattern by fluorophore-assisted carbohydrate electrophoresis, and for glycosaminoglycan (GAG) composition by enzyme digestion. RESULTS: Proteoglycans synthesised in the presence of fenofibrate showed an increase in the half-maximum saturation concentration of LDL from 36.8+/-12.4 microg/ml to 77.7+/-17 microg/ml under basal conditions, from 24.9+/-4.6 microg/ml to 39.1+/-6.1 microg/ml in the presence of TGF-beta1, and from 9.5+/-4.4 microg/ml to 31.1+/-3.4 microg/ml in the presence of platelet-derived growth factor/insulin. Fenofibrate treatment in the presence of TGF-beta1 inhibited the incorporation of [(35)S]-sulphate into secreted and cell-associated proteoglycans synthesised by human VSMCs by 59.2% (p<0.01) and 39.8% (p<0.01) respectively. The changes in sulphate incorporation following treatment with fenofibrate were associated with a concentration-related increase in the electrophoretic mobility due to a reduction in GAG length. There was no change in the sulphation pattern; however, there was an alteration in the disaccharide composition of the GAGs. CONCLUSIONS/INTERPRETATION: Fenofibrate modifies the structure of vascular proteoglycans by reducing the length of the GAG chains and GAG composition, resulting in reduced binding to human LDL, a mechanism which may lead to a reduction of atherosclerosis and cardiovascular disease in people with diabetes treated with fenofibrate.

Regulation of glycosaminoglycan structure and atherogenesis.

Cardiovascular disease is the major cause of premature death in modern society, and its impact is increasing due to rising rates of obesity and type 2 diabetes. Clinical studies based on targeting metabolic abnormalities and biomarkers demonstrate significant benefits, but always an element of disease remains which is resistant to treatment. Recent evidence has strongly implicated an early interaction of atherogenic lipoproteins with vascular matrix proteoglycans as the initiating step in atherogenesis. Expert commentary has pointed to the need for vascular directed therapies to provide reductions in the residual disease component. We propose that the regulation of synthesis and thus structure of glycosaminoglycans on proteoglycans provides a potential pathway to this reduction. We review existing evidence that the vascular synthesis of glycosaminoglycan chains can be regulated in a manner which reduces lipoprotein binding and the potential application of this strategy to attenuation of the current cardiovascular disease pandemic.

Evaluation of sampling locations for two radionuclide air-sampling systems based on the requirements of ANSI/HPS N13.1-1999.

The Pacific Northwest National Laboratory characterized the performance of sampling locations for two radionuclide air-sampling systems that continuously monitor radioactive air emissions from research and development facilities. The testing was conducted to determine whether sampling system locations would meet the criteria for uniform air velocity and contaminant concentration in the American National Standard Institute standard, Sampling and Monitoring Releases of Airborne Radioactive Substances from the Stacks and Ducts of Nuclear Facilities (ANSI/HPS N13.1-1999). The standard is a revision of the 1969 version that the facilities have been required to meet. Whereas the 1969 standard provided prescriptive criteria for the selection of sampling locations, the 1999 standard is performance-based and requires well-mixed sampling locations that must be demonstrated through performance tests that are specified in the standard. Testing at the Life Sciences Laboratory I was performed on the existing stack at the current sampling location; a scale model was built and used in place of the Radiochemical Processing Laboratory. Although both facilities' sampling sites were compliant with the 1969 standard, only the Radiochemical Processing Laboratory met the criteria of the revised standard. In the future, the use of a computational fluid dynamics computer model may be useful in determining whether a sampling location is likely to test successfully.

Results of inspection and cleaning of two radionuclide air-sampling systems based on the requirements of ANSI/HPS N13.1-1999.

The Pacific Northwest National Laboratory inspected and cleaned two radionuclide air-sampling systems that continuously monitor radioactive air emissions from research and development facilities. The inspection and cleaning was performed to evaluate effective methods and potential cost impacts of maintenance requirements in the revised American National Standard Institute standard Sampling and Monitoring Releases of Airborne Radioactive Substances from the Stacks and Ducts of Nuclear Facilities. The standard requires at least annual inspections of sampling systems followed by cleaning if deposits are visible. During 2001 and 2002, inspections were performed leaving the sampling systems in place and inserting videoscope cables into different access points to allow viewing of the inside and outside of sampling manifolds and transport lines. Cleaning was performed on one of the systems by disconnecting and extracting the sampling manifold, then washing it with de-ionized water and scrub brushes. The wash water was analyzed for radioactivity and solids. Results of the inspection showed greater deposition in one of the systems than would be expected by a High Efficiency Particulate Air (HEPA) filtered exhaust stream, possibly due to accumulation of dust from a short period when unfiltered air was exhausted from construction areas. The second system was also downstream of HEPA filters and appeared much cleaner. The videoscope was a useful and cost-effective tool and provided a better view than could be obtained with the naked eye. However, because even small amounts of deposition were made visible with the videoscope, clarification is needed in defining when probe washing is merited, particularly in existing sampling systems whose design is not conducive to easy removal and cleaning.

Structural changes at cut ends of earthworm giant axons in the interval between dye barrier formation and neuritic outgrowth.

We describe structural changes at the cut ends of invertebrate myelinated earthworm giant axons beginning with the formation of a dye barrier (15 minutes posttransection or postcalcium addition) and ending with the formation of a neuritic outgrowth (2-10 days posttransection). The morphology of the cut end, and the location and morphological configuration of the dye barrier, were assessed by time-lapse confocal, fluorescence microscopy and by electron microscopy. During the interval from 15 to 35 minutes postcalcium addition, the dye barrier continuously migrated away from a cut axonal end; the dye barrier then remained stable for up to 5 hours. The size, packing density, and arrangement of membranous structures were correlated with changes in the dye barrier from 15 to 35 minutes postcalcium addition. During this interval, uptake of an externally placed hydrophilic dye by these membranous structures was also variable. After 35 minutes postcalcium addition, the membranous structures remained stable until they completely disappeared between 1 and 2 days posttransection. The disappearance of membranous structures always preceded neuritic outgrowth, which only arose from cut axonal ends. These results demonstrate that the dye barrier and associated membranous structures, which form after transection of earthworm giant axons, are very dynamic in the short term (35 minutes) with respect to their location and morphological configuration and suggest that axolemmal repair must be completed before neuritic outgrowth can occur.

Semirational design of a potent, artificial agonist of fibroblast growth factor receptors.

Fibroblast growth factors (FGFs) are being investigated in human clinical trials as treatments for angina, claudication, and stroke. We designed a molecule structurally unrelated to all FGFs, which potently mimicked basic FGF activity, by combining domains that (1) bind FGF receptors (2) bind heparin, and (3) mediate dimerization. A 26-residue peptide identified by phage display specifically bound FGF receptor (FGFR) 1c extracellular domain but had no homology with FGFs. When fused with the c-jun leucine zipper domain, which binds heparin and forms homodimers, the polypeptide specifically reproduced the mitogenic and morphogenic activities of basic FGF with similar potency (EC50 = 240 pM). The polypeptide required interaction with heparin for activity, demonstrating the importance of heparin for FGFR activation even with designed ligands structurally unrelated to FGF. Our results demonstrate the feasibility of engineering potent artificial agonists for the receptor tyrosine kinases, and have important implications for the design of nonpeptidic ligands for FGF receptors. Furthermore, artificial FGFR agonists may be useful alternatives to FGF in the treatment of ischemic vascular disease.

Calcium entry initiates processes that restore a barrier to dye entry in severed earthworm giant axons.

After severance, axons can restore structural barriers that are necessary for recovery of their electrical function. In earthworm myelinated axons, such a barrier to dye entry is mediated by many vesicles and myelin-derived membranous structures. From time-lapse confocal fluorescence and DIC images, we now report that Ca2+ entry and not axonal injury per se initiates the processes that form a dye barrier, as well as the subsequent structural changes in this barrier and associated membranous structures. The time required to restore a dye barrier after transection also depends only on the time of Ca2+ entry.

Rapid induction of functional and morphological continuity between severed ends of mammalian or earthworm myelinated axons.

The inability to rapidly restore the loss of function that results from severance (cutting or crushing) of PNS and CNS axons is a severe clinical problem. As a novel strategy to help alleviate this problem, we have developed in vitro procedures using Ca2+-free solutions of polyethylene glycol (PEG solutions), which within minutes induce functional and morphological continuity (PEG-induced fusion) between the cut or crushed ends of myelinated sciatic or spinal axons in rats. Using a PEG-based hydrogel that binds to connective tissue to provide mechanical strength at the lesion site and is nontoxic to nerve tissues in earthworms and mammals, we have also developed in vivo procedures that permanently maintain earthworm myelinated medial giant axons whose functional and morphological integrity has been restored by PEG-induced fusion after axonal severance. In all these in vitro or in vivo procedures, the success of PEG-induced fusion of sciatic or spinal axons and myelinated medial giant axons is measured by the restored conduction of action potentials through the lesion site, the presence of intact axonal profiles in electron micrographs taken at the lesion site, and/or the intra-axonal diffusion of fluorescent dyes across the lesion site. These and other data suggest that the application of polymeric fusiogens (such as our PEG solutions), possibly combined with a tissue adherent (such as our PEG hydrogels), could lead to in vivo treatments that rapidly and permanently repair cut or crushed axons in the PNS and CNS of adult mammals, including humans.

Heat-shock proteins in axoplasm: high constitutive levels and transfer of inducible isoforms from glia.

To characterize heat-shock proteins (HSPs) of the 70-kDa family in the crayfish medial giant axon (MGA), we analyzed axoplasmic proteins separately from proteins of the glial sheath. Several different molecular weight isoforms of constitutive HSP 70s that were detected on immunoblots were approximately 1-3% of the total protein in the axoplasm of MGAs. To investigate inducible HSPs, MGAs were heat shocked in vitro or in vivo, then the axon was bathed in radiolabeled amino acid for 4 hours. After either heat-shock treatment, protein synthesis in the glial sheath was decreased compared with that of control axons, and newly synthesized proteins of 72 kDa, 84 kDa, and 87 kDa appeared in both the axoplasm and the sheath. Because these radiolabeled proteins were present in MGAs only after heat-shock treatments, we interpreted the newly synthesized proteins of 72 kDa, 84 kDa, and 87 kDa to be inducible HSPs. Furthermore, the 72-kDa radiolabeled band in heat-shocked axoplasm and glial sheath samples comigrated with a band possessing HSP 70 immunoreactivity. The amount of heat-induced proteins in axoplasm samples was greater after a 2-hour heat shock than after a 1-hour heat shock. These data indicate that MGA axoplasm contains relatively high levels of constitutive HSP 70s and that, after heat shock, MGA axoplasm obtains inducible HSPs of 72 kDa, 84 kDa, and 87 kDa from the glial sheath. These constitutive and inducible HSPs may help MGAs maintain essential structures and functions following acute heat shock.

Binding interaction of the heregulinbeta egf domain with ErbB3 and ErbB4 receptors assessed by alanine scanning mutagenesis.

Individual residues of the heregulinbeta (HRG) egf domain were mutated to alanine and displayed monovalently on phagemid particles as gene III fusion proteins. Wild type HRGbeta egf domain displayed on phage was properly folded as evidenced by its ability to bind ErbB3 and ErbB4 receptor-IgG fusion proteins with affinities close to those measured for bacterially produced HRGbeta egf domain. Binding to ErbB3 and ErbB4 receptors was affected by mutation of residues throughout the egf domain; including the NH2 terminus (His2 and Leu3), the two beta-turns (Val15-Gly18 and Gly42-Gln46), and some discontinuous residues (including Leu3, Val4, Phe13, Val23, and Leu33) that form a patch on the major beta-sheet and the COOH-terminal region (Tyr48 and Met50-Phe53). Binding affinity was least changed by mutations throughout the Omega-loop and the second strand of the major beta-sheet. More mutants had greater affinity loss for ErbB3 compared with ErbB4 implying that it has more stringent binding requirements. Many residues important for HRG binding to its receptors correspond to critical residues for epidermal growth factor (EGF) and transforming growth factor alpha binding to the EGF receptor. Specificity may be determined in part by bulky groups that prevent binding to the unwanted receptor. All of the mutants tested were able to induce phosphorylation and mitogen-activated protein kinase activation through ErbB4 receptors and were able to modulate a transphosphorylation signal from ErbB3 to ErbB2 in MCF7 cells. An understanding of binding similarities and differences among the EGF family of ligands may facilitate the development of egf-like analogs with broad or narrow specificity.

Selection of heregulin variants having higher affinity for the ErbB3 receptor by monovalent phage display.

Heregulins (HRGs) are epidermal growth factor (egf) domain containing polypeptide growth factors that bind and activate several members of the ErbB receptor family. Although HRG can bind to ErbB3 and ErbB4 homodimers, the highest affinity and most intracellularly active receptor complexes are hetero-oligomers containing ErbB2. The HRGbeta egf domain was displayed on the surface of M13 phage to facilitate mutagenic analysis and optimize for binding to a homodimeric ErbB3-immunoglobulin (IgG) fusion. Nine libraries were constructed in which virtually the entire sequence was randomized in stretches of four to six amino acids. These were selected separately for binding to immobilized ErbB3-IgG. Analysis of the resulting sequences revealed some areas that diverged radically from the wild-type, whereas others showed strong conservation. The degree of wild-type conservation correlated strongly with the functional importance of the residues as determined by alanine scanning mutagenesis (Jones, J. T., Ballinger, M. D., Pisacane, P. I., Lofgren, J. A., Fitzpatrick, V. D., Fairbrother, W. J., Wells, J. A., and Sliwkowski, M. X. (1998) J. Biol. Chem. 273, 11667-11674). Some variants from several libraries showed significant improvements in binding affinity to the ErbB3-IgG. These optimized segments were combined in various ways in the same molecule to generate variants (containing up to 16 mutations) that had >50-fold higher affinity than wild-type HRGbeta. The optimized variants stimulated ErbB2 phophorylation on MCF7 cells at levels similar to wild-type. This indicates wild-type affinity is optimized for potency and that factors other than affinity for ErbB3 are limiting. These variants showed enhanced affinity toward the ErbB4 homodimer, suggesting these receptors use very similar binding determinants despite them having 65% sequence identity.