Most cancers preserve functional retinoblastoma (Rb) and may, therefore, respond to inhibition of D-cyclin-dependent Rb kinases, CDK4 and CDK6. To date, CDK4/6 inhibitors have shown promising ...clinical activity in breast cancer and lymphomas, but it is not clear which additional Rb-positive cancers might benefit from these agents. No systematic survey to compare relative sensitivities across tumor types and define molecular determinants of response has been described. We report a subset of cancers highly sensitive to CDK4/6 inhibition and characterized by various genomic aberrations known to elevate D-cyclin levels and describe a recurrent CCND1 3′UTR mutation associated with increased expression in endometrial cancer. The results suggest multiple additional classes of cancer that may benefit from CDK4/6-inhibiting drugs such as abemaciclib.
•A wide range of sensitivity to abemaciclib is observed among Rb+ tumor cells•CDKN2A mutant cancers show only intermediate sensitivity to CDK4/6 inhibition•D-cyclin activating features are associated with highly sensitive cells•About 5% of endometrial cancers bear a stabilizing mutation in the CCND1 3′UTR
Gong et al. identify a subset of cancers highly sensitive to CDK4/6 inhibition, which are characterized by various genomic aberrations known to elevate D-cyclin levels but not by CDKN2A mutations. They also identify a recurrent CCND1 3′UTR mutation associated with increased CCND1 expression in endometrial cancer.
We developed a novel approach for conducting multisample, multigene, ultradeep bisulfite sequencing analysis of DNA methylation patterns in clinical samples. A massively parallel ...sequencing-by-synthesis method (454 sequencing) was used to directly sequence >100 bisulfite PCR products in a single sequencing run without subcloning. We showed the utility, robustness, and superiority of this approach by analyzing methylation in 25 gene-related CpG rich regions from >40 cases of primary cells, including normal peripheral blood lymphocytes, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), and mantle cell lymphoma (MCL). A total of 294,631 sequences was generated with an average read length of 131 bp. On average, >1,600 individual sequences were generated for each PCR amplicon far beyond the few clones (<20) typically analyzed by traditional bisulfite sequencing. Comprehensive analysis of CpG methylation patterns at a single DNA molecule level using clustering algorithms revealed differential methylation patterns between diseases. A significant increase in methylation was detected in ALL and FL samples compared with CLL and MCL. Furthermore, a progressive spreading of methylation was detected from the periphery toward the center of select CpG islands in the ALL and FL samples. The ultradeep sequencing also allowed simultaneous analysis of genetic and epigenetic data and revealed an association between a single nucleotide polymorphism and the methylation present in the LRP1B promoter. This new generation of methylome sequencing will provide digital profiles of aberrant DNA methylation for individual human cancers and offers a robust method for the epigenetic classification of tumor subtypes.
Sensitive and specific detection of breast cancer biomarker CA15-3 in human serum is an important step toward successful evaluation of clinical treatment and prediction of breast cancer recurrence. ...In this work, we developed an optofluidic ring resonator (OFRR) sensor and the corresponding sensing protocols for label-free CA15-3 detection without any additional signal amplification steps. Nonspecific serum protein adsorption was minimized with effective surface blocking methods. The sensor performance for CA15-3 detection was first characterized in phosphate-buffered saline (PBS) buffer and in fetal calf serum. Then the potential use of the OFRR as a simple clinical laboratory testing device for breast cancer diagnostics was tested by measuring the CA15-3 level in clinical human serum samples, and the results were compared with those of standard clinical lab tests. It was found that the OFRR was capable of detecting approximately 1 unit/mL CA15-3 in both PBS buffer and diluted serum within approximately 30 min. Our work marks the first demonstration of the optical ring resonator biosensor in real clinical applications that features low cost, simple detection procedures, rapid response time, low sample consumption, and high specificity.
Identifying perturbed or dysregulated pathways is critical to understanding the biological processes that change within an experiment. Previous methods identified important pathways that are ...significantly enriched among differentially expressed genes; however, these methods cannot account for small, coordinated changes in gene expression that amass across a whole pathway. In order to overcome this limitation, we use microarray gene expression data to identify pathway perturbation based on pathway correlation profiles. By identifying the distribution of gene-gene pair correlations within a pathway, we can rank the pathways based on the level of perturbation and dysregulation. We have shown this successfully for differences between two experimental conditions in Escherichia coli and changes within time series data in Saccharomyces cerevisiae, as well as two estrogen receptor response classes of breast cancer. Overall, our method made significant predictions as to the pathway perturbations that are involved in the experimental conditions.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Knowledge of epigenetic alterations in cancer is rapidly increasing due to the development of genome-wide techniques for their identification. DNA methylation is the best understood epigenetic ...adaptation and disease-specific aberrant DNA methylation is a well-recognized hallmark of cancer. Recently, novel modifications, including 5-hydroxymethylation have been described, adding a new layer of complexity to understanding the epigenetic machinery and their role in cancer. There have been significant advances in techniques for the discovery and validation of DNA methylation- and hydroxymethylation-based biomarkers, each with its own advantages and limitations. With the advent of new profiling technologies, the ever-growing list of genes that show epigenetic alterations, particularly DNA methylation, emphasizes the role of these changes for early detection, diagnosis, prognosis, and prediction of response to therapies. While there are yet many challenges to the effective implementation of DNA-methylation/hydroxymethylation-based biomarkers and epigenetic therapeutics, the field is moving closer to the goal of defining personalized medicine.
Follicular lymphoma (FL) is a form of non-Hodgkin's lymphoma (NHL) that arises from germinal center (GC) B-cells. Despite the significant advances in immunotherapy, FL is still not curable. Beyond ...transcriptional profiling and genomics datasets, there currently is no epigenome-scale dataset or integrative biology approach that can adequately model this disease and therefore identify novel mechanisms and targets for successful prevention and treatment of FL.
We performed methylation-enriched genome-wide bisulfite sequencing of FL cells and normal CD19(+) B-cells using 454 sequencing technology. The methylated DNA fragments were enriched with methyl-binding proteins, treated with bisulfite, and sequenced using the Roche-454 GS FLX sequencer. The total number of bases covered in the human genome was 18.2 and 49.3 million including 726,003 and 1.3 million CpGs in FL and CD19(+) B-cells, respectively. 11,971 and 7,882 methylated regions of interest (MRIs) were identified respectively. The genome-wide distribution of these MRIs displayed significant differences between FL and normal B-cells. A reverse trend in the distribution of MRIs between the promoter and the gene body was observed in FL and CD19(+) B-cells. The MRIs identified in FL cells also correlated well with transcriptomic data and ChIP-on-Chip analyses of genome-wide histone modifications such as tri-methyl-H3K27, and tri-methyl-H3K4, indicating a concerted epigenetic alteration in FL cells.
This study is the first to provide a large scale and comprehensive analysis of the DNA methylation sequence composition and distribution in the FL epigenome. These integrated approaches have led to the discovery of novel and frequent targets of aberrant epigenetic alterations. The genome-wide bisulfite sequencing approach developed here can be a useful tool for profiling DNA methylation in clinical samples.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
We demonstrated quantitative real-time label-free detection of DNA sequences using the liquid core optical ring resonator (LCORR) sensor. The LCORR is a recently developed sensing platform that ...integrates microfluidics and photonic sensing technology with low detection limit and sub-nanoliter detection volume. We analyzed experimentally and theoretically the LCORR response to a variety of DNA samples that had different strand lengths (25–100 bases), number of base- mismatches (1–5), and concentrations (10
pM to 10
μM) to evaluate the LCORR sequence detection capability. In particular, we established the linear correlation between the LCORR sensing signal and the molecule density, which allows us to accurately calculate the molecule density on the surface. It is found that the probe surface coverage was 26–51% and the extent of hybridization was 40–50%. The titration curve for 25-base probe and 25-base target DNA yields a dissociation constant of 2.9
nM. With a 37.1
nm/RIU LCORR, detection of 10
pM bulk DNA concentration was demonstrated. The mass detection limit was estimated to be 4
pg/mm
2, corresponding to a density of 10
10
molecules/cm
2 on the surface. We also showed that the LCORR was sensitive enough to differentiate DNA with only a few base-mismatches based on the raw sensing signal and kinetic analysis. Our work will provide important insight into the light-DNA interaction at the ring resonator surface and lay a foundation for future LCORR-based DNA label-free microarray development.
Introduction
Hospital pharmacists' have traditionally focused on the manufacture and supply of medicines. However, the increasing complexity and range of medicines and a greater awareness of ...medication errors has facilitated a change towards a patient‐centred role. Given this movement, it is surprising that a search of the published literature shows very little research that evaluated patients' views of hospital‐based pharmacy services.
Objective
To explore inpatients' expectations and experiences of hospital‐based pharmacy services.
Study setting and design
Face‐to‐face semi‐structured interviews with inpatients admitted to acute medical wards of three NHS general hospitals.
Principal findings
Seventy‐four inpatients were interviewed: 37 were male with average age 73 years (age range of 19 –86 years). The predominate number of participants (62/74, 84%) being in the 65–80 years of age group. Thematic analysis of the data was driven by three themes; patients' expectations of the pharmacist's involvement in their treatment and care, the patients' experiences of any interaction that may have taken place and the patients' evaluation of their interaction with the pharmacist.
Conclusions
There was a dichotomy of expectations and opinions from patients about the role of hospital pharmacists and the services being provided. As pharmacists' roles are developing towards a patient‐orientated model in which pharmacists have direct contact with patients and their care, it is important to ensure that patients are aware of these developments to help them maximize the benefit they derive from their country's health‐care system.
The opto-fluidic ring resonator (OFRR) is a sensitive label-free optical biosensor that is uniquely well suited for photonic and fluidic integration. For the first time we have explored the utility ...of this novel instrument for the analysis of methylation in oligonucleotides using the MBD-2 (methyl binding) protein as the capture molecule. This application has strong relevance to cancer research and future clinical tools through the study of methylation patterns in important gene promoters. In this work we quantitatively characterized the OFRR's response to artificially methylated ssDNA and dsDNA as a function of the number of methylated cytosines and DNA concentration. The effect of hemi- versus fully methylated oligonucleotides was also investigated. Additionally, anti 5-methylcytidine antibody was also used as the capture molecule and compared with MBD-2. It is found that the antibody has stronger affinity for ssDNA, whereas MBD-2 is much better at binding dsDNA.
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