Acyl-coenzyme A synthetases in metabolic control

The 11 long-chain (ACSL) and very long chain acyl-coenzyme A (acyl-CoA) synthetases [(ACSVL)/fatty acid transport protein] are receiving considerable attention because it has become apparent that their individual functions are not redundant

Multiple Recurrent De Novo CNVs, Including Duplications of the 7q11.23 Williams Syndrome Region, Are Strongly Associated with Autism

SummaryWe have undertaken a genome-wide analysis of rare copy-number variation (CNV) in 1124 autism spectrum disorder (ASD) families, each comprised of a single proband, unaffected parents, and, in most kindreds, an unaffected sibling. We find significant association of ASD with de novo duplications of 7q11.23, where the reciprocal deletion causes Williams-Beuren syndrome, characterized by a highly social personality. We identify rare recurrent de novo CNVs at five additional regions, including 16p13.2 (encompassing genes USP7 and C16orf72) and Cadherin 13, and implement a rigorous approach to evaluating the statistical significance of these observations. Overall, large de novo CNVs, particularly those encompassing multiple genes, confer substantial risks (OR = 5.6; CI = 2.6–12.0, p = 2.4 × 10-7). We estimate there are 130–234 ASD-related CNV regions in the human genome and present compelling evidence, based on cumulative data, for association of rare de novo events at 7q11.23, 15q11.2-13.1, 16p11.2, and Neurexin 1

Report on computational assessment of Tumor Infiltrating Lymphocytes from the International Immuno-Oncology Biomarker Working Group

Funder: U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)Funder: National Center for Research Resources under award number 1 C06 RR12463-01, VA Merit Review Award IBX004121A from the United States Department of Veterans Affairs Biomedical Laboratory Research and Development Service, the DOD Prostate Cancer Idea Development Award (W81XWH-15-1-0558), the DOD Lung Cancer Investigator-Initiated Translational Research Award (W81XWH-18-1-0440), the DOD Peer Reviewed Cancer Research Program (W81XWH-16-1-0329), the Ohio Third Frontier Technology Validation Fund, the Wallace H. Coulter Foundation Program in the Department of Biomedical Engineering and the Clinical and Translational Science Award Program (CTSA) at Case Western Reserve University.Funder: Susan G Komen Foundation (CCR CCR18547966) and a Young Investigator Grant from the Breast Cancer Alliance.Funder: The Canadian Cancer SocietyFunder: Breast Cancer Research Foundation (BCRF), Grant No. 17-194Abstract: Assessment of tumor-infiltrating lymphocytes (TILs) is increasingly recognized as an integral part of the prognostic workflow in triple-negative (TNBC) and HER2-positive breast cancer, as well as many other solid tumors. This recognition has come about thanks to standardized visual reporting guidelines, which helped to reduce inter-reader variability. Now, there are ripe opportunities to employ computational methods that extract spatio-morphologic predictive features, enabling computer-aided diagnostics. We detail the benefits of computational TILs assessment, the readiness of TILs scoring for computational assessment, and outline considerations for overcoming key barriers to clinical translation in this arena. Specifically, we discuss: 1. ensuring computational workflows closely capture visual guidelines and standards; 2. challenges and thoughts standards for assessment of algorithms including training, preanalytical, analytical, and clinical validation; 3. perspectives on how to realize the potential of machine learning models and to overcome the perceptual and practical limits of visual scoring

Application of a risk-management framework for integration of stromal tumor-infiltrating lymphocytes in clinical trials

Funder: Breast Cancer Research Foundation (BCRF); doi: https://doi.org/10.13039/100001006Abstract: Stromal tumor-infiltrating lymphocytes (sTILs) are a potential predictive biomarker for immunotherapy response in metastatic triple-negative breast cancer (TNBC). To incorporate sTILs into clinical trials and diagnostics, reliable assessment is essential. In this review, we propose a new concept, namely the implementation of a risk-management framework that enables the use of sTILs as a stratification factor in clinical trials. We present the design of a biomarker risk-mitigation workflow that can be applied to any biomarker incorporation in clinical trials. We demonstrate the implementation of this concept using sTILs as an integral biomarker in a single-center phase II immunotherapy trial for metastatic TNBC (TONIC trial, NCT02499367), using this workflow to mitigate risks of suboptimal inclusion of sTILs in this specific trial. In this review, we demonstrate that a web-based scoring platform can mitigate potential risk factors when including sTILs in clinical trials, and we argue that this framework can be applied for any future biomarker-driven clinical trial setting

Pitfalls in assessing stromal tumor infiltrating lymphocytes (sTILs) in breast cancer

Abstract: Stromal tumor-infiltrating lymphocytes (sTILs) are important prognostic and predictive biomarkers in triple-negative (TNBC) and HER2-positive breast cancer. Incorporating sTILs into clinical practice necessitates reproducible assessment. Previously developed standardized scoring guidelines have been widely embraced by the clinical and research communities. We evaluated sources of variability in sTIL assessment by pathologists in three previous sTIL ring studies. We identify common challenges and evaluate impact of discrepancies on outcome estimates in early TNBC using a newly-developed prognostic tool. Discordant sTIL assessment is driven by heterogeneity in lymphocyte distribution. Additional factors include: technical slide-related issues; scoring outside the tumor boundary; tumors with minimal assessable stroma; including lymphocytes associated with other structures; and including other inflammatory cells. Small variations in sTIL assessment modestly alter risk estimation in early TNBC but have the potential to affect treatment selection if cutpoints are employed. Scoring and averaging multiple areas, as well as use of reference images, improve consistency of sTIL evaluation. Moreover, to assist in avoiding the pitfalls identified in this analysis, we developed an educational resource available at www.tilsinbreastcancer.org/pitfalls

Phosphorylation and Acetylation of Acyl-CoA Synthetase- I

Long chain acyl-CoA synthetase 1 (ACSL1) contributes 50 to 90% of total ACSL activity in liver, adipose tissue, and heart and appears to direct the use of long chain fatty acids for energy. Although the functional importance of ACSL1 is becoming clear, little is understood about its post-translational regulation. In order to investigate the post-translational modifications of ACSL1 under different physiological conditions, we overexpressed ACSL1 in hepatocytes, brown adipocytes, and 3T3-L1 differentiated adipocytes, treated these cells with different hormones, and analyzed the resulting phosphorylated and acetylated amino acids by mass spectrometry. We then compared these results to the post-translational modifications observed in vivo in liver and brown adipose tissue after mice were fasted or exposed to a cold environment. We identified universal N-terminal acetylation, 15 acetylated lysines, and 25 phosphorylation sites on ACSL1. Several unique acetylation and phosphorylation sites occurred under conditions in which fatty acid β-oxidation is normally enhanced. Thirteen of the acetylated lysines had not previously been identified, and none of the phosphorylation sites had been previously identified. Site-directed mutagenesis was used to introduce mutations at three potential acetylation and phosphorylation sites believed to be important for ACSL1 function. At the ATP/AMP binding site and at a highly conserved site near the C terminus, modifications of Ser278 or Lys676, respectively, totally inhibited ACSL1 activity. In contrast, mutations of Lys285 that mimicked acetylation (Lys285Ala and Lys285Gln) reduced ACSL activity, whereas full activity was retained by Lys285Arg, suggesting that acetylation of Lys285 would be likely to decrease ACSL1 activity. These results indicate that ACSL1 is highly modified post-translationally. Several of these modifications would be expected to alter enzymatic function, but others may affect protein stability or protein-protein interactions

A Modified Database Search Strategy Leads to Improved Identification of in Vitro Brominated Peptides Spiked into a Complex Proteomic Sample

Inflammation leads to activation of immune cells, resulting in production of hypobromous acid. Few investigations have been performed on protein bromination on a proteomic scale, even though bromination is a relatively abundant protein modification in endogenously brominated proteomes. Such studies have been hampered by the lack of an optimized database search strategy. In order to address this issue, we performed nano-LC–MS/MS analysis of an in vitro generated, trypsin-digested brominated human serum albumin standard, spiked into a complex trypsin-digested proteomic background, in an LTQ-Orbitrap instrument. We found that brominated peptides spiked in at a 1–10% ratio (mass:mass) were easily identified by manual inspection when higher-energy collisional dissociation (HCD) and collision induced dissociation (CID) were employed as the dissociation mode; however, confident assignment of brominated peptides from protein database searches required a novel approach. By addition of a custom modification, corresponding to the substitution of a single bromine with ⁸¹Br rather than ⁷⁹Br for dibromotyrosine (⁷⁹Br⁸¹BrY), the number of validated assignments for peptides containing dibromotyrosine increased significantly when analyzing both high resolution and low resolution MS/MS data. This new approach will facilitate the identification of proteins derived from endogenously brominated proteomes, providing further knowledge about the role of protein bromination in various pathological states

A Modified Database Search Strategy Leads to Improved Identification of in Vitro Brominated Peptides Spiked into a Complex Proteomic Sample

Inflammation leads to activation of immune cells, resulting in production of hypobromous acid. Few investigations have been performed on protein bromination on a proteomic scale, even though bromination is a relatively abundant protein modification in endogenously brominated proteomes. Such studies have been hampered by the lack of an optimized database search strategy. In order to address this issue, we performed nano-LC–MS/MS analysis of an in vitro generated, trypsin-digested brominated human serum albumin standard, spiked into a complex trypsin-digested proteomic background, in an LTQ-Orbitrap instrument. We found that brominated peptides spiked in at a 1–10% ratio (mass:mass) were easily identified by manual inspection when higher-energy collisional dissociation (HCD) and collision induced dissociation (CID) were employed as the dissociation mode; however, confident assignment of brominated peptides from protein database searches required a novel approach. By addition of a custom modification, corresponding to the substitution of a single bromine with ⁸¹Br rather than ⁷⁹Br for dibromotyrosine (⁷⁹Br⁸¹BrY), the number of validated assignments for peptides containing dibromotyrosine increased significantly when analyzing both high resolution and low resolution MS/MS data. This new approach will facilitate the identification of proteins derived from endogenously brominated proteomes, providing further knowledge about the role of protein bromination in various pathological states