Proteomic characterization of blood plasma is of central importance to clinical proteomics and particularly to biomarker discovery studies. The vast dynamic range and high complexity of the plasma ...proteome have, however, proven to be serious challenges and have often led to unacceptable tradeoffs between depth of coverage and sample throughput. We present an optimized sample-processing pipeline for analysis of the human plasma proteome that provides greatly increased depth of detection, improved quantitative precision and much higher sample analysis throughput as compared with prior methods. The process includes abundant protein depletion, isobaric labeling at the peptide level for multiplexed relative quantification and ultra-high-performance liquid chromatography coupled to accurate-mass, high-resolution tandem mass spectrometry analysis of peptides fractionated off-line by basic pH reversed-phase (bRP) chromatography. The overall reproducibility of the process, including immunoaffinity depletion, is high, with a process replicate coefficient of variation (CV) of <12%. Using isobaric tags for relative and absolute quantitation (iTRAQ) 4-plex, >4,500 proteins are detected and quantified per patient sample on average, with two or more peptides per protein and starting from as little as 200 μl of plasma. The approach can be multiplexed up to 10-plex using tandem mass tags (TMT) reagents, further increasing throughput, albeit with some decrease in the number of proteins quantified. In addition, we provide a rapid protocol for analysis of nonfractionated depleted plasma samples analyzed in 10-plex. This provides ∼600 quantified proteins for each of the ten samples in ∼5 h of instrument time.
A fast, noninvasive test with high sensitivity (SN) and a negative predictive value (NPV), which is able to detect recurrences in bladder cancer (BC) patients, is needed. A newly developed urine ...assay, Xpert Bladder Cancer Monitor (Xpert), measures five mRNA targets (ABL1, CRH, IGF2, UPK1B, and ANXA10) that are frequently overexpressed in BC.
To validate Xpert characteristics in patients previously diagnosed with non-muscle-invasive BC.
Voided precystoscopy urine samples were prospectively collected at 22 sites. Xpert, cytology, and UroVysion were performed. If cystoscopy was suspicious for BC, a histologic examination was performed. Additionally, technical validation was performed and specificity was determined in patients without a history or clinical evidence of BC.
Test characteristics were calculated based on cystoscopy and histology results, and compared between Xpert, cytology, and UroVysion.
Of the eligible patients, 239 with a history of BC had results for all assays. The mean age was 71 yr; 190 patients were male, 53 never smoked, and 64% had previous intravesical immunotherapy (35%) or chemotherapy (29%). Forty-three cases of recurrences occurred. Xpert had overall SN of 74% (95% confidence interval CI: 60–85) and 83% (95% CI: 64–93) for high-grade (HG) tumors. The NPV was 93% (95% CI: 89–96) overall and 98% (95% CI: 94–99) for HG tumors. Specificity was 80% (95% CI: 73–85). Xpert SN and NPV were superior to those of cytology and UroVysion. Specificity in non-BC individuals (n=508) was 95% (95% CI: 93–97).
Xpert has an improved NPV compared with UroVysion and cytology in patients under follow-up for BC. It represents a promising tool for excluding BC in these patients, reducing the need for cystoscopy.
Xpert is an easy-to-perform urine test with good performance compared with standard urine tests. It should help optimize the follow-up of recurrent bladder cancer patients.
The Xpert test is a mRNA-based urine assay for bladder cancer detection. In recurrent bladder cancer patients, its test characteristics compare favorably with those of UroVysion and cytology. It represents a promising tool for excluding cancer, thus reducing the need for cystoscopy.
In patients with haematuria, a fast, noninvasive test with high sensitivity (SN) and negative predictive value (NPV), which is able to detect or exclude bladder cancer (BC), is needed. A newly ...developed urine assay, Xpert Bladder Cancer Detection (Xpert), measures five mRNA targets (ABL1, CRH, IGF2, UPK1B, and ANXA10) that are frequently overexpressed in BC.
To validate the performance of Xpert in patients with haematuria.
Voided precystoscopy urine specimens were prospectively collected at 22 sites from patients without prior BC undergoing cystoscopy for haematuria. Xpert, cytology, and UroVysion procedures were performed. Technical validation was performed and specificity (SP) was determined in patients without BC.
Test characteristics were calculated based on cystoscopy and histology results, and compared between Xpert, cytology, and UroVysion.
We included 828 patients (mean age 64.5 yr, 467 males, 401 never smoked). Xpert had an SN of 78% (95% confidence interval CI: 66–87) overall and 90% (95% CI: 76–96) for high-grade (HG) tumours. The NPV was 98% (95% CI: 97–99) overall. The SP was 84% (95% CI: 81–86). In patients with microhaematuria, only one HG patient was missed (NPV 99%). Xpert had higher SN and NPV than cytology and UroVysion. Cytology had the highest SP (97%). In a separate SP study, Xpert had an SP of 89% in patients with benign prostate hypertrophy and 92% in prostate cancer patients.
Xpert is an easy-to-use, noninvasive test with improved SN and NPV compared with cytology and UroVysion, representing a promising tool for identifying haematuric patients with a low likelihood of BC who might not need to undergo cystoscopy.
Xpert is an easy-to-perform urine test with good performance compared with standard urine tests. It should help identify (micro)haematuria patients with a very low likelihood to have bladder cancer.
In haematuric patients, the characteristics of the mRNA-based Xpert urine assay for bladder cancer detection compare favourably with UroVysion and cytology. It represents a promising tool for selecting patients at a low risk of cancer, reducing the need for cystoscopy.
Stereocontrolled syntheses of the C16-C28 CD-spiroacetal subunit of altohyrtin A/spongistatin 1 , relying on kinetic and thermodynamic control of the spiroacetal formation, are described. The kinetic ...control approach resulted in a slight preference (60 : 40) for the desired spiroacetal isomer. The thermodynamic approach allowed ready access to the desired spiroacetal by acid-promoted equilibration, chromatographic separation of the C23 epimers and resubjection of the undesired isomer to the equilibration conditions. This scalable synthetic sequence provided multi-gram quantities of , thus enabling the successful completion of the total synthesis of altohyrtin A/spongistatin 1, as reported in Part 4 of this series.
As an exceptionally potent antimitotic macrolide, altohyrtin A/spongistatin 1 shows great promise in cancer chemotherapy but its extreme scarcity in the natural sponges has halted its further ...preclinical development. A highly stereocontrolled total synthesis, which exploits boron‐mediated aldol bond constructions, has been realized to provide, for the first time, a useful amount of synthetic material.
Cancers avoid immune surveillance through an array of mechanisms, including perturbation of HLA class I antigen presentation. Merkel cell carcinoma (MCC) is an aggressive, HLA-I-low, neuroendocrine ...carcinoma of the skin often caused by the Merkel cell polyomavirus (MCPyV). Through the characterization of 11 newly generated MCC patient-derived cell lines, we identified transcriptional suppression of several class I antigen presentation genes. To systematically identify regulators of HLA-I loss in MCC, we performed parallel, genome-scale, gain- and loss-of-function screens in a patient-derived MCPyV-positive cell line and identified MYCL and the non-canonical Polycomb repressive complex 1.1 (PRC1.1) as HLA-I repressors. We observed physical interaction of MYCL with the MCPyV small T viral antigen, supporting a mechanism of virally mediated HLA-I suppression. We further identify the PRC1.1 component USP7 as a pharmacologic target to restore HLA-I expression in MCC.
Abstract
Intravenous (IV) anti-CD20 monoclonal antibody (mAb) therapy for chronic lymphocytic leukemia (CLL) patients often produces a first dose infusion reaction (FDIR) within the first 2 h that ...requires careful monitoring to avoid serious complications and fatalities. To better understand FDIR, we studied blood samples collected from 37 treatment-naïve CLL patients in our clinical trial NCT03788291 undergoing IV treatment with 50 mg of anti-CD20 mAb (rituximab). We analyzed four time points: baseline (prior to infusion), 1 h later (during infusion), at the end of infusion (~2.5 h) and at 48 h. Infusion reactions (CTCAE (v5) grade ≥2 events) were managed by interrupting the infusion and with symptomatic treatment. 24 (65%) patients had a FDIR and all patients completed the infusion.
Patient and CLL characteristics including measures of disease severity (Rai stage, IGHV mutation status, cytogenetic defects) did not correlate with FDIR. High levels of CLL cells and/or CD20 and their subsequent depletion resulting in decreased serum mAb and/or complement are thought to be risks for FDIR. However, measurements of these parameters did not correlate. Induction of cytokine release syndrome (CRS) may correlate with FDIR. However, CRS was observed in all samples after mAb infusion. The only significant associations with FDIR were higher levels of IP-10, IL-6, and IL-8, with IP-10 most significant. Anti-CD20 mAb activated cytotoxic effector cells, such as tissue-resident macrophages that ingest CLL cells via antibody-dependent cellular phagocytosis (ADCP), may be responsible for these events leading to FDIR. A mouse model of anti-CD20 mAb FDIR and in vitro ADCP cell systems will be useful to further understand the biology of FDIR.
Supported by funding from Acerta/AstraZeneca, the Cadregari Foundation, a generous donation by Elizabeth Aaron, and the NIH NCI grant number R21CA267040.
Cancers avoid immune surveillance through an array of mechanisms, including perturbation of HLA class I antigen presentation. Merkel cell carcinoma (MCC) is an aggressive, HLA-I-low, neuroendocrine ...carcinoma of the skin often caused by the Merkel cell polyomavirus (MCPyV). Through the characterization of 11 newly generated MCC patient-derived cell lines, we identified transcriptional suppression of several class I antigen presentation genes. To systematically identify regulators of HLA-I loss in MCC, we performed parallel, genome-scale, gain- and loss-of-function screens in a patient-derived MCPyV-positive cell line and identified MYCL and the non-canonical Polycomb repressive complex 1.1 (PRC1.1) as HLA-I repressors. We observed physical interaction of MYCL with the MCPyV small T viral antigen, supporting a mechanism of virally mediated HLA-I suppression. We further identify the PRC1.1 component USP7 as a pharmacologic target to restore HLA-I expression in MCC.
None of these is true.5 Policymaking is not an entirely rational process of identifying problems and choosing optimal solutions, especially when scientists must make value-laden assumptions and ...extrapolations in the face of highly uncertain data to answer questions posed by policymakers.6 What is needed is a way to ensure, politics aside, that our understanding of the workings of complex ecological systems informs public policy choices about where and how development should proceed, how natural resources are managed to ensure sustainable supplies, and whether and how to regulate economic activities that pose a threat to human health and safety as well as environmental protection. In recognition that science is needed now more than ever to inform societal decisions, politicians, government and nongovernmental agencies, and citizens have been asking with a mounting sense of urgency for scientists and science organizations to make their research more relevant to society's needs and to become involved in policymaking.39 Yet even as scientists heed this call, more often than not, they still find themselves and their work ignored, marginalized, or misrepresented in deeply contentious environmental policy debates.
The present study examined recidivism risk factors in a sample of 320 male batterers attending community treatment. Recidivism was assessed by new charges for violence or for any offence during a ...5-year follow-up period. The variables associated with recidivism among male batterers were similar to those found for other criminal populations (e.g. young, unstable lifestyle, substance abuse, criminal history). There was no evidence that potential offenders were deterred by expectations of negative consequences, either social (e.g. friends would disapprove) or official (e.g. being arrested, losing job). Maintaining positive relationships with treatment providers was associated with reduced recidivism.
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