Primary Cilia Mediate Diverse Kinase Inhibitor Resistance Mechanisms in Cancer.

Primary cilia are microtubule-based organelles that detect mechanical and chemical stimuli. Although cilia house a number of oncogenic molecules (including Smoothened, KRAS, EGFR, and PDGFR), their precise role in cancer remains unclear. We have interrogated the role of cilia in acquired and de novo resistance to a variety of kinase inhibitors, and found that, in several examples, resistant cells are distinctly characterized by an increase in the number and/or length of cilia with altered structural features. Changes in ciliation seem to be linked to differences in the molecular composition of cilia and result in enhanced Hedgehog pathway activation. Notably, manipulating cilia length via Kif7 knockdown is sufficient to confer drug resistance in drug-sensitive cells. Conversely, targeting of cilia length or integrity through genetic and pharmacological approaches overcomes kinase inhibitor resistance. Our work establishes a role for ciliogenesis and cilia length in promoting cancer drug resistance and has significant translational implications.This research was partly funded by the Institute of Cancer Research and by grants from Sarcoma UK (to B.E.T. [14.2014] and P.H.H. [3.2014]), Kent Cancer Trust (to M.M.), Hilfe fuer Krebskranke Kinder Frankfurt e.V. and Frankfurter Stiftung fuer Krebskranke Kinder (to J.C.), CRUK-CI Core Grant (C14303/A17197), and S.H.D. Fellowship (Wellcome Trust/Royal Society [107609]) (to M.D.R.). B.E.T. was supported by an ICR fellowship

Three Novel Downstream Promoter Elements Regulate MHC Class I Promoter Activity in Mammalian Cells

BACKGROUND: MHC CLASS I TRANSCRIPTION IS REGULATED BY TWO DISTINCT TYPES OF REGULATORY PATHWAYS: 1) tissue-specific pathways that establish constitutive levels of expression within a given tissue and 2) dynamically modulated pathways that increase or decrease expression within that tissue in response to hormonal or cytokine mediated stimuli. These sets of pathways target distinct upstream regulatory elements, have distinct basal transcription factor requirements, and utilize discrete sets of transcription start sites within an extended core promoter. METHODOLOGY/PRINCIPAL FINDINGS: We studied regulatory elements within the MHC class I promoter by cellular transfection and in vitro transcription assays in HeLa, HeLa/CIITA, and tsBN462 of various promoter constructs. We have identified three novel MHC class I regulatory elements (GLE, DPE-L1 and DPE-L2), located downstream of the major transcription start sites, that contribute to the regulation of both constitutive and activated MHC class I expression. These elements located at the 3' end of the core promoter preferentially regulate the multiple transcription start sites clustered at the 5' end of the core promoter. CONCLUSIONS/SIGNIFICANCE: Three novel downstream elements (GLE, DPE-L1, DPE-L2), located between +1 and +32 bp, regulate both constitutive and activated MHC class I gene expression by selectively increasing usage of transcription start sites clustered at the 5' end of the core promoter upstream of +1 bp. Results indicate that the downstream elements preferentially regulate TAF1-dependent, relative to TAF1-independent, transcription

Using high-resolution contact networks to evaluate SARS-CoV-2 transmission and control in large-scale multi-day events

The emergence of highly transmissible SARS-CoV-2 variants has created a need to reassess the risk posed by increasing social contacts as countries resume pre-pandemic activities, particularly in the context of resuming large-scale events over multiple days. To examine how social contacts formed in different activity settings influences interventions required to control Delta variant outbreaks, we collected high-resolution data on contacts among passengers and crew on cruise ships and combined the data with network transmission models. We found passengers had a median of 20 (IQR 10–36) unique close contacts per day, and over 60% of their contact episodes were made in dining or sports areas where mask wearing is typically limited. In simulated outbreaks, we found that vaccination coverage and rapid antigen tests had a larger effect than mask mandates alone, indicating the importance of combined interventions against Delta to reduce event risk in the vaccine era

International genome-wide meta-analysis identifies new primary biliary cirrhosis risk loci and targetable pathogenic pathways.

Primary biliary cirrhosis (PBC) is a classical autoimmune liver disease for which effective immunomodulatory therapy is lacking. Here we perform meta-analyses of discovery data sets from genome-wide association studies of European subjects (n=2,764 cases and 10,475 controls) followed by validation genotyping in an independent cohort (n=3,716 cases and 4,261 controls). We discover and validate six previously unknown risk loci for PBC (Pcombined<5 × 10(-8)) and used pathway analysis to identify JAK-STAT/IL12/IL27 signalling and cytokine-cytokine pathways, for which relevant therapies exist

Flexible Cyclic Immunofluorescence (cyCIF) Using Oligonucleotide Barcoded Antibodies

Advances in our understanding of the complex, multifaceted interactions between tumor epithelia, immune infiltrate, and tumor microenvironmental cells have been driven by highly multiplexed imaging technologies. These techniques are capable of labeling many more biomarkers than conventional immunostaining methods. However, multiplexed imaging techniques suffer from low detection sensitivity, cell loss—particularly in fragile samples—, and challenges with antibody labeling. Herein, we developed and optimized an oligonucleotide antibody barcoding strategy for cyclic immunofluorescence (cyCIF) that can be amplified to increase the detection efficiency of low-abundance antigens. Stained fluorescence signals can be readily removed using ultraviolet light treatment, preserving tissue and fragile cell sample integrity. We also extended the oligonucleotide barcoding strategy to secondary antibodies to enable the inclusion of difficult-to-label primary antibodies in a cyCIF panel. Using both the amplification oligonucleotides to label DNA barcoded antibodies and in situ hybridization of multiple fluorescently labeled oligonucleotides resulted in signal amplification and increased signal-to-background ratios. This procedure was optimized through the examination of staining parameters including staining oligonucleotide concentration, staining temperature, and oligonucleotide sequence design, resulting in a robust amplification technique. As a proof-of-concept, we demonstrate the flexibility of our cyCIF strategy by simultaneously imaging with the original oligonucleotide conjugated antibody (Ab-oligo) cyCIF strategy, the novel Ab-oligo cyCIF amplification strategy, as well as direct and indirect immunofluorescence to generate highly multiplexed images

Flexible Cyclic Immunofluorescence (cyCIF) Using Oligonucleotide Barcoded Antibodies

Advances in our understanding of the complex, multifaceted interactions between tumor epithelia, immune infiltrate, and tumor microenvironmental cells have been driven by highly multiplexed imaging technologies. These techniques are capable of labeling many more biomarkers than conventional immunostaining methods. However, multiplexed imaging techniques suffer from low detection sensitivity, cell loss&mdash;particularly in fragile samples&mdash;, and challenges with antibody labeling. Herein, we developed and optimized an oligonucleotide antibody barcoding strategy for cyclic immunofluorescence (cyCIF) that can be amplified to increase the detection efficiency of low-abundance antigens. Stained fluorescence signals can be readily removed using ultraviolet light treatment, preserving tissue and fragile cell sample integrity. We also extended the oligonucleotide barcoding strategy to secondary antibodies to enable the inclusion of difficult-to-label primary antibodies in a cyCIF panel. Using both the amplification oligonucleotides to label DNA barcoded antibodies and in situ hybridization of multiple fluorescently labeled oligonucleotides resulted in signal amplification and increased signal-to-background ratios. This procedure was optimized through the examination of staining parameters including staining oligonucleotide concentration, staining temperature, and oligonucleotide sequence design, resulting in a robust amplification technique. As a proof-of-concept, we demonstrate the flexibility of our cyCIF strategy by simultaneously imaging with the original oligonucleotide conjugated antibody (Ab-oligo) cyCIF strategy, the novel Ab-oligo cyCIF amplification strategy, as well as direct and indirect immunofluorescence to generate highly multiplexed images

QUALICOPC (Quality and Costs of Primary Care) Canada : a focus on the aspects of primary care most highly rated by current patients of primary care practices

This Pan-Canadian report describes patient and physician perspectives regarding current performance of primary care in each province based on data collected as part of the Quality and Costs of Primary Care (QUALICOPC) study. The QUALICOPC study is an international study of quality and costs of primary care in 34 countries. This report describes results from the data collected in Canada. It includes only data collected from patients and physicians in primary care practices that provide comprehensive primary care. Two patient surveys were conducted as part of the study; one that asked patients about the importance of various aspects of primary care and the other about patients’ experiences with primary care. This report focuses on aspects of primary care that respondents to patient surveys (distributed during visits to primary care physicians) identified as very important aspects of their primary care experience. Primary care physicians from all Canadian provinces were asked to participate in the QUALICOPC study by a research team in each province. In general, one physician from each primary care practice was invited to participate (23 practices, primarily in Quebec, had more than one physician respondent). Physicians who agreed to participate were sent a package containing four different surveys regarding: the practice setting ; the services provided in the practice ; patient values and patient experience. In each practice, the physician was asked to complete the survey about the services provided and any staff member could complete the practice setting survey. One patient was expected to respond to the patient values survey and nine other patients were expected to respond to the patient experience survey. Physicians returned completed surveys to the research team in each province. A total of 8,332 patients of 810 primary care physicians in 785 practices across Canada responded to the surveys. A total of 1,160 completed the patient values survey and a different sample of 7,172 patients completed the patient experience survey. Two-thirds (67%) of participating patients were female, and three-quarters (74%) were in good to very good health . The median age of patient respondents was 53. The majority (59%) of participating patients had a post-secondary education, were fluent or native speaking in at least one of Canada’s official languages (80%) and were born in Canada (86%).To our knowledge, this is the largest study to date of patient values and patient experience regarding primary care in Canada in terms of the number of patients. The results reported provide important insight into the experience and values of primary care of the population sampled (as described above): patients who had access to a primary care physician, the majority of whom were in good to very good health. Whether the results reported herein and the associated reported policy implications would extend to other population groups, such those who do not have regular access to a primary care practice, requires further study

Seasonal Phenology of the Cerambycid Beetles of East Central Illinois

We summarize field data on the species composition and seasonal phenology of the community of cerambycid beetles of east-central Illinois. Data were drawn from field bioassays conducted during 2009 – 2012 that tested attraction of adult beetles of diverse species to a variety of synthetic pheromones and host plant volatiles. A total of 34,086 beetles of 114 species were captured, including 48 species in the subfamily Cerambycinae, 41 species in the Lamiinae, 19 species in the Lepturinae, two species in the Spondylidinae, and one species each in the Necydalinae, Parandrinae, Prioninae, and the Disteniidae. Most of the best-represented species were attracted to pheromones that were included in field experiments, particularly species that use (R)-3-hydroxyhexan-2-one as a pheromone component. The species captured, and their patterns of abundance and seasonal phenology were similar to those in an earlier study conducted in Pennsylvania. The most abundant species identified in both studies included the cerambycines Elaphidion mucronatum (Say), Neoclytus a. acuminatus (F.), Neoclytus m. mucronatus (F.), and Xylotrechus colonus (F.). Cerambycine species became active in an orderly progression from early spring through late fall, whereas most lamiine species were active in summer and fall, and lepturine species were limited to summer. Potential cross attraction between some cerambycine species that shared pheromone components may have been averted by differences in seasonal activity period, and by minor pheromone components that acted as synergists for conspecifics and/or antagonists for heterospecifics. These results provide quantitative data on the abundance and seasonal phenology of a large number of species

Seasonal Phenology of the Cerambycid Beetles of East Central Illinois

We summarize field data on the species composition and seasonal phenology of the community of cerambycid beetles of east-central Illinois. Data were drawn from field bioassays conducted during 2009 - 2012 that tested attraction of adult beetles of diverse species to a variety of synthetic pheromones and host plant volatiles. A total of 34,086 beetles of 114 species were captured, including 48 species in the subfamily Cerambycinae, 41 species in the Lamiinae, 19 species in the Lepturinae, two species in the Spondylidinae, and one species each in the Necydalinae, Parandrinae, Prioninae, and the Disteniidae. Most of the best-represented species were attracted to pheromones that were included in field experiments, particularly species that use (R)-3-hydroxyhexan-2-one as a pheromone component. The species captured, and their patterns of abundance and seasonal phenology were similar to those in an earlier study conducted in Pennsylvania. The most abundant species identified in both studies included the cerambycines Elaphidion mucronatum (Say), Neoclytus a. acuminatus (F.), Neoclytus m. mucronatus (F.), and Xylotrechus colonus (F.). Cerambycine species became active in an orderly progression from early spring through late fall, whereas most lamiine species were active in summer and fall, and lepturine species were limited to summer. Potential cross attraction between some cerambycine species that shared pheromone components may have been averted by differences in seasonal activity period, and by minor pheromone components that acted as synergists for conspecifics and/or antagonists for heterospecifics. These results provide quantitative data on the abundance and seasonal phenology of a large number of species