Digital Commons Network^™

Pan-Cancer Analysis Reveals Recurrent Bcar4 Gene Fusions Across Solid Tumors, Andrew Nickless, Jin Zhang, Ghofran Othoum, Jace Webster, Matthew J Inkman, Emily Coonrod, Sherron Fontes, Emily B Rozycki, Christopher A Maher, Nicole M White

2020-Current year OA Pubs

UNLABELLED: Chromosomal rearrangements often result in active regulatory regions juxtaposed upstream of an oncogene to generate an expressed gene fusion. Repeated activation of a common downstream partner-with differing upstream regions across a patient cohort-suggests a conserved oncogenic role. Analysis of 9,638 patients across 32 solid tumor types revealed an annotated long noncoding RNA (lncRNA), Breast Cancer Anti-Estrogen Resistance 4 (BCAR4), was the most prevalent, uncharacterized, downstream gene fusion partner occurring in 11 cancers. Its oncogenic role was confirmed using multiple cell lines with endogenous BCAR4 gene fusions. Furthermore, overexpressing clinically prevalent BCAR4 gene fusions in untransformed cell lines was sufficient …

Shared Brain And Genetic Architectures Between Mental Health And Physical Activity, Wei Zhang, Sarah E Paul, Anderson Winkler, Ryan Bogdan, Janine D Bijsterbosch

2020-Current year OA Pubs

Physical activity is correlated with, and effectively treats various forms of psychopathology. However, whether biological correlates of physical activity and psychopathology are shared remains unclear. Here, we examined the extent to which the neural and genetic architecture of physical activity and mental health are shared. Using data from the UK Biobank (N = 6389), we applied canonical correlation analysis to estimate associations between the amplitude and connectivity strength of subnetworks of three major neurocognitive networks (default mode, DMN; salience, SN; central executive networks, CEN) with accelerometer-derived measures of physical activity and self-reported mental health measures (primarily of depression, anxiety disorders, …

The Transcription Factor Bach2 Negatively Regulates Murine Natural Killer Cell Maturation And Function, Shasha Li, Michael D Bern, Benpeng Miao, Changxu Fan, Xiaoyun Xing, Takeshi Inoue, Sytse J Piersma, Ting Wang, Marco Colonna, Tomohiro Kurosaki, Wayne M Yokoyama

2020-Current year OA Pubs

BTB domain And CNC Homolog 2 (Bach2) is a transcription repressor that actively participates in T and B lymphocyte development, but it is unknown if Bach2 is also involved in the development of innate immune cells, such as natural killer (NK) cells. Here, we followed the expression of Bach2 during murine NK cell development, finding that it peaked in immature CD27

Effectiveness Of The Ad26.Cov2.S (Johnson & Johnson) Coronavirus Disease 2019 (Covid-19) Vaccine For Preventing Covid-19 Hospitalizations And Progression To High Disease Severity In The United States, Nathaniel M Lewis, Hilary M Babcock, Jennie H Kwon, Et Al.

2020-Current year OA Pubs

Background . Adults in the United States (US) began receiving the adenovirus vector coronavirus disease 2019 (COVID-19) vaccine, Ad26.COV2.S (Johnson & Johnson [Janssen]), in February 2021. We evaluated Ad26.COV2.S vaccine effectiveness (VE) against COVID-19 hospitalization and high disease severity during the first 10 months of its use. Methods . In a multicenter case-control analysis of US adults (≥18 years) hospitalized 11 March to 15 December 2021, we estimated VE against susceptibility to COVID-19 hospitalization (VEs), comparing odds of prior vaccination with a single dose Ad26.COV2.S vaccine between hospitalized cases with COVID-19 and controls without COVID-19. Among hospitalized patients with COVID-19, …

Peristaltic Pumps Adapted For Laminar Flow Experiments Enhance In Vitro Modeling Of Vascular Cell Behavior, Javier Abello, Shreya Raghavan, Yvette Y. Yien, Amber N. Stratman

2020-Current year OA Pubs

Endothelial cells (ECs) are the primary cellular constituent of blood vessels that are in direct contact with hemodynamic forces over their lifetime. Throughout the body, vessels experience different blood flow patterns and rates that alter vascular architecture and cellular behavior. Because of the complexities of studying blood flow in an intact organism, particularly during development, the field has increasingly relied on in vitro modeling of blood flow as a powerful technique for studying hemodynamic-dependent signaling mechanisms in ECs. While commercial flow systems that recirculate fluids exist, many commercially available pumps are peristaltic and best model pulsatile flow conditions. However, there …

The Gut Microbial Metabolic Capacity Of Microbiome-Humanized Vs. Wild Type Rodents Reveals A Likely Dual Role Of Intestinal Bacteria In Hepato-Intestinal Schistosomiasis, Alba Cortés, John Martin, Bruce A. Rosa, Klara A. Stark, Simon Clare, Catherine Mccarthy, Katherine Harcourt, Cordelia Brandt, Charlotte Tolley, Trevor D. Lawley, Makedonka Mitreva, Matthew Berriman, Gabriel Rinaldi, Cinzia Cantacessi

2020-Current year OA Pubs

Increasing evidence shows that the host gut microbiota might be involved in the immunological cascade that culminates with the formation of tissue granulomas underlying the pathophysiology of hepato-intestinal schistosomiasis. In this study, we investigated the impact of Schistosoma mansoni infection on the gut microbial composition and functional potential of both wild type and microbiome-humanized mice. In spite of substantial differences in microbiome composition at baseline, selected pathways were consistently affected by parasite infection. The gut microbiomes of infected mice of both lines displayed, amongst other features, enhanced capacity for tryptophan and butyrate production, which might be linked to the activation …

Telomere Biology Disorders: Time For Moving Towards The Clinic?, Luis F Z Batista, Inderjeet Dokal, Roy Parker

2020-Current year OA Pubs

Telomere biology disorders (TBDs) are a group of rare diseases caused by mutations that impair telomere maintenance. Mutations that cause reduced levels of TERC/hTR, the telomerase RNA component, are found in most TBD patients and include loss-of-function mutations in hTR itself, in hTR-binding proteins [NOP10, NHP2, NAF1, ZCCHC8, and dyskerin (DKC1)], and in proteins required for hTR processing (PARN). These patients show diverse clinical presentations that most commonly include bone marrow failure (BMF)/aplastic anemia (AA), pulmonary fibrosis, and liver cirrhosis. There are no curative therapies for TBD patients. An understanding of hTR biogenesis, maturation, and degradation has identified pathways and …

Comprehensive Characterization Of The Multiple Myeloma Immune Microenvironment Using Integrated Scrna-Seq, Cytof, And Cite-Seq Analysis, Lijun Yao, Reyka G Jayasinghe, Mark A Fiala, Michael C Wendl, Stephen T Oh, John F Dipersio, Ravi Vij, Li Ding

2020-Current year OA Pubs

UNLABELLED: As part of the Multiple Myeloma Research Foundation (MMRF) immune atlas pilot project, we compared immune cells of multiple myeloma bone marrow samples from 18 patients assessed by single-cell RNA sequencing (scRNA-seq), mass cytometry (CyTOF), and cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) to understand the concordance of measurements among single-cell techniques. Cell type abundances are relatively consistent across the three approaches, while variations are observed in T cells, macrophages, and monocytes. Concordance and correlation analysis of cell type marker gene expression across different modalities highlighted the importance of choosing cell type marker genes best suited to …

Radiotherapy For Elderly Patients With Glioblastoma: An Assessment Of Hypofractionation And Modern Treatment Techniques, Jennifer K. Matsui, Jiayi Huang, Et Al.

2020-Current year OA Pubs

Glioblastoma (GBM) is a disease with a poor prognosis. For decades, radiotherapy has played a critical role in the management of GBM. The standard of care radiation prescription is 60 Gy in 30 fractions, but landmark trials have historically excluded patients older than 70 years. Currently, there is considerable variation in the management of elderly patients with GBM. Shortened radiation treatment (hypofractionated) regimens have been explored since conventional treatment schedules are lengthy and many elderly patients have functional, cognitive, and social limitations. Clinical trials have demonstrated the effectiveness of hypofractionated radiotherapy (40 Gy in 15 fractions) to treat elderly or …

Targeting Pim2 By Jp11646 Results In Significant Antitumor Effects In Solid Tumors, Eriko Katsuta, Malgorzata Gil-Moore, Justine Moore, Mohamed Yousif, Alex A Adjei, Yi Ding, Justin Caserta, Carmen M Baldino, Kelvin P Lee, Irwin H Gelman, Kazuaki Takabe, Mateusz Opyrchal

2020-Current year OA Pubs

Proviral integration of Moloney virus 2 (PIM2) is a pro‑survival factor of cancer cells and a possible therapeutic target in hematological malignancies. However, the attempts at inhibiting PIM2 have yielded underwhelming results in early clinical trials on hematological malignancies. Recently, a novel pan‑PIM inhibitor, JP11646, was developed. The present study examined the utility of targeting PIM2 in multiple solid cancers and investigated the antitumor efficacy and the mechanisms of action of JP11646. When PIM2 expression was compared between normal and cancer tissues in publicly available datasets, PIM2 was found to be overexpressed in several types of solid cancers. PIM2 ectopic …

Functional Characterization Of Enhancer Activity During A Long Terminal Repeat's Evolution, Alan Y Du, Xiaoyu Zhuo, Vasavi Sundaram, Nicholas O Jensen, Hemangi G Chaudhari, Nancy L Saccone, Barak A Cohen, Ting Wang

2020-Current year OA Pubs

Many transposable elements (TEs) contain transcription factor binding sites and are implicated as potential regulatory elements. However, TEs are rarely functionally tested for regulatory activity, which in turn limits our understanding of how TE regulatory activity has evolved. We systematically tested the human LTR18A subfamily for regulatory activity using massively parallel reporter assay (MPRA) and found AP-1- and CEBP-related binding motifs as drivers of enhancer activity. Functional analysis of evolutionarily reconstructed ancestral sequences revealed that LTR18A elements have generally lost regulatory activity over time through sequence changes, with the largest effects occurring owing to mutations in the AP-1 and CEBP …

A Saturated Map Of Common Genetic Variants Associated With Human Height, Loïc Yengo, E Warwick Daw, Lihua Wang, Mary F. Feitosa, Michael A. Province, Dabeeru C. Rao, Adam E. Locke, Et Al.

2020-Current year OA Pubs

No abstract provided.

Quantitative Single-Cell Analysis Of Leishmania Major Amastigote Differentiation Demonstrates Variably Extended Expression Of The Lipophosphoglycan (Lpg) Virulence Factor In Different Host Cell Types, Michael A. Mandell, Wandy L. Beatty, Stephen M. Beverley

2020-Current year OA Pubs

Immediately following their deposition into the mammalian host by an infected sand fly vector, Leishmania parasites encounter and are engulfed by a variety of cell types. From there, parasites may transit to other cell types, primarily macrophages or dendritic cells, where they replicate and induce pathology. During this time, Leishmania cells undergo a dramatic transformation from the motile non-replicating metacyclic stage to the non-motile replicative amastigote stage, a differentiative process that can be termed amastigogenesis. To follow this at the single cell level, we identified a suite of experimental 'landmarks' delineating different stages of amastigogenesis qualitatively or quantitatively, including new …

The Inner Nuclear Membrane Protein Nemp1 Supports Nuclear Envelope Openings And Enucleation Of Erythroblasts, Didier Hodzic, Jun Wu, Karen Krchma, Andrea Jurisicova, Yonit Tsatskis, Yijie Liu, Peng Ji, Kyunghee Choi, Helen Mcneill

2020-Current year OA Pubs

Nuclear envelope membrane proteins (NEMPs) are a conserved family of nuclear envelope (NE) proteins that reside within the inner nuclear membrane (INM). Even though Nemp1 knockout (KO) mice are overtly normal, they display a pronounced splenomegaly. This phenotype and recent reports describing a requirement for NE openings during erythroblasts terminal maturation led us to examine a potential role for Nemp1 in erythropoiesis. Here, we report that Nemp1 KO mice show peripheral blood defects, anemia in neonates, ineffective erythropoiesis, splenomegaly, and stress erythropoiesis. The erythroid lineage of Nemp1 KO mice is overrepresented until the pronounced apoptosis of polychromatophilic erythroblasts. We show …

Neoadjuvant Atezolizumab For Resectable Non-Small Cell Lung Cancer: An Open-Label, Single-Arm Phase Ii Trial, Jamie E Chaft, G Alexander Patterson, Saiama N Waqar, Et Al.

2020-Current year OA Pubs

In an ongoing, open-label, single-arm phase II study ( NCT02927301 ), 181 patients with untreated, resectable, stage IB-IIIB non-small cell lung cancer received two doses of neoadjuvant atezolizumab monotherapy. The primary end point was major pathological response (MPR; ≤10% viable malignant cells) in resected tumors without EGFR or ALK alterations. Of the 143 patients in the primary end point analysis, the MPR was 20% (95% confidence interval, 14-28%). With a minimum duration of follow-up of 3 years, the 3-year survival rate of 80% was encouraging. The most common adverse events during the neoadjuvant phase were fatigue (39%, 71 of 181) …

Multi-Modulated Frequency Domain High Density Diffuse Optical Tomography, Guy A. Perkins, Adam T. Eggebrecht, Hamid Dehghani

2020-Current year OA Pubs

No abstract provided.

High Polygenic Risk Scores Are Associated With Early Age Of Onset Of Alcohol Use Disorder In Adolescents And Young Adults At Risk, John I Nurnberger Jr, Kathleen Bucholz, Laura Bierut, Vivia Mccutcheon, Andrey P Anokhin, Et Al.

2020-Current year OA Pubs

BACKGROUND: Genome-wide association studies have been conducted in alcohol use disorder (AUD), and they permit the use of polygenic risk scores (PRSs), in combination with clinical variables, to predict the onset of AUD in vulnerable populations.

METHODS: A total of 2794 adolescent/young adult subjects from the Collaborative Study on the Genetics of Alcoholism were followed, with clinical assessments every 2 years. Subjects were genotyped using a genome-wide chip. Separate PRS analyses were performed for subjects of European ancestry and African ancestry. Age of onset of DSM-5 AUD was evaluated using the Cox proportional hazard model. Predictive power was assessed using …

Semiautomated Analysis Of An Optical Atp Indicator In Neurons, Taher Dehkharghanian, Arsalan Hashemiaghdam, Ghazaleh Ashrafi

2020-Current year OA Pubs

No abstract provided.

Response To Brentuximab Vedotin By Cd30 Expression In Non-Hodgkin Lymphoma, Deepa Jagadeesh, Steve Horwitz, Nancy L Bartlett, Youn Kim, Eric Jacobsen, Madeleine Duvic, Meredith Little, William Trepicchio, Keenan Fenton, Matthew Onsum, Julie Lisano, Ranjana Advani

2020-Current year OA Pubs

BACKGROUND: The safety and efficacy of brentuximab vedotin (BV), an antibody-drug conjugate directed to the CD30 antigen, has been assessed in several trials in patients with peripheral T-cell lymphoma (PTCL), cutaneous T-cell lymphoma (CTCL), or B-cell non-Hodgkin lymphoma (NHL). The objective of this research was to examine the relationship between CD30 expression level and clinical response to BV.

PATIENTS AND METHODS: We analyzed response in patients treated with BV monotherapy in 5 prospective clinical studies in relapsed or refractory PTCL, CTCL, or B-cell NHL. CD30 expression was assessed by immunohistochemistry (IHC) using the Ber H2 antibody for 275 patients.

RESULTS: …

Stroke Genetics Informs Drug Discovery And Risk Prediction Across Ancestries, Aniket Mishra, Carlos Cruchaga, Et Al.

2020-Current year OA Pubs

Previous genome-wide association studies (GWASs) of stroke - the second leading cause of death worldwide - were conducted predominantly in populations of European ancestry

A Novel Piggyback Strategy For Mrna Delivery Exploiting Adenovirus Entry Biology, Myungeun Lee, Paul J Rice-Boucher, Logan Thrasher Collins, Ernst Wagner, Lorenzo Aulisa, Jeffrey Hughes, David T Curiel

2020-Current year OA Pubs

Molecular therapies exploiting mRNA vectors embody enormous potential, as evidenced by the utility of this technology for the context of the COVID-19 pandemic. Nonetheless, broad implementation of these promising strategies has been restricted by the limited repertoires of delivery vehicles capable of mRNA transport. On this basis, we explored a strategy based on exploiting the well characterized entry biology of adenovirus. To this end, we studied an adenovirus-polylysine (AdpL) that embodied "piggyback" transport of the mRNA on the capsid exterior of adenovirus. We hypothesized that the efficient steps of Ad binding, receptor-mediated entry, and capsid-mediated endosome escape could provide an …

Transcriptional And Immune Landscape Of Cardiac Sarcoidosis, Jing Liu, Pan Ma, Lulu Lai, Ana Villanueva, Andrew Koenig, Gregory R. Bean, Dawn E. Bowles, Carolyn Glass, Michael Watson, Kory J. Lavine, Chieh-Yu Lin

2020-Current year OA Pubs

BACKGROUND: Cardiac involvement is an important determinant of mortality among sarcoidosis patients. Although granulomatous inflammation is a hallmark finding in cardiac sarcoidosis, the precise immune cell populations that comprise the granuloma remain unresolved. Furthermore, it is unclear how the cellular and transcriptomic landscape of cardiac sarcoidosis differs from other inflammatory heart diseases.

METHODS: We leveraged spatial transcriptomics (GeoMx digital spatial profiler) and single-nucleus RNA sequencing to elucidate the cellular and transcriptional landscape of cardiac sarcoidosis. Using GeoMX digital spatial profiler technology, we compared the transcriptomal profile of CD68

RESULTS: Despite overlapping histological features, spatial transcriptomics identified transcriptional signatures and associated …

Proteomic And Phosphoproteomic Landscapes Of Acute Myeloid Leukemia, Michael H Kramer, Qiang Zhang, Robert Sprung, Ryan B Day, Petra Erdmann-Gilmore, Yang Li, Ziheng Xu, Nichole M Helton, Daniel R George, Yiling Mi, Peter Westervelt, Jacqueline E Payton, Sai M Ramakrishnan, Christopher A Miller, Daniel C Link, John F Dipersio, Matthew J Walter, R Reid Townsend, Timothy J Ley

2020-Current year OA Pubs

We have developed a deep-scale proteome and phosphoproteome database from 44 representative acute myeloid leukemia (AML) patients from the LAML TCGA dataset and 6 healthy bone marrow-derived controls. After confirming data quality, we orthogonally validated several previously undescribed features of AML revealed by the proteomic data. We identified examples of posttranscriptionally regulated proteins both globally (ie, in all AML samples) and also in patients with recurrent AML driver mutations. For example, samples with IDH1/2 mutations displayed elevated levels of the 2-oxoglutarate-dependent histone demethylases KDM4A/B/C, despite no changes in messenger RNA levels for these genes; we confirmed this finding in vitro. …

A Case Of Persistent Human Pegivirus Infection In Two Separate Pregnancies Of A Woman, Mathieu Garand, Susie S Y Huang, Lisa S Goessling, Donna A Santillan, Mark K Santillan, Anoop Brar, Todd N Wylie, Kristine M Wylie, Pirooz Eghtesady

2020-Current year OA Pubs

Human pegivirus (HPgV) is best known for persistent, presumably non-pathogenic, infection and a propensity to co-infect with human immunodeficiency virus or hepatitis C virus. However, unique attributes, such as the increased risk of malignancy or immune modulation, have been recently recognized for HPgV. We have identified a unique case of a woman with high levels HPgV infection in two pregnancies, which occurred 4 years apart and without evidence of human immunodeficiency virus or hepatitis C virus infection. The second pregnancy was complicated by congenital heart disease. A high level of HPgV infection was detected in the maternal blood from different …

Meeting The Challenges Of Myocarditis: New Opportunities For Prevention, Detection, And Intervention-A Report From The 2021 National Heart, Lung, And Blood Institute Workshop, Daniela Čiháková, Yang Shi, Bishow Adhikari, W Patricia Bandettini, Madeleine W Cunningham, Narasimhan Danthi, Matthias G Friedrich, Peter Liu, Lisa Schwartz Longacre, Douglas L Mann, Filip K Swirski, W H Wilson Tang, Guofei Zhou, Leslie T Cooper Jr., Workshop Speakers

2020-Current year OA Pubs

The National Heart, Lung, and Blood Institute (NHLBI) convened a workshop of international experts to discuss new research opportunities for the prevention, detection, and intervention of myocarditis in May 2021. These experts reviewed the current state of science and identified key gaps and opportunities in basic, diagnostic, translational, and therapeutic frontiers to guide future research in myocarditis. In addition to addressing community-acquired myocarditis, the workshop also focused on emerging causes of myocarditis including immune checkpoint inhibitors and SARS-CoV-2 related myocardial injuries and considered the use of systems biology and artificial intelligence methodologies to define workflows to identify novel mechanisms of …

Trending Anti-E7 Serology Predicts Mortality And Recurrence Of Hpv-Associated Cancers Of The Oropharynx, Luke Johnson, Dat T Ha, Melissa B Hall, Gregory Shoemaker, Paul A Bevins, John Strickley, Shadmehr Demehri, Rebecca A Redman, Joongho Joh

2020-Current year OA Pubs

High-risk human papillomavirus (HPV) is among the most common causes of head and neck cancer (HNC) with increasing incidence. HPV-associated HNC patients' clinical response to treatment varies drastically, which has made treatment de-escalation clinical trials challenging. To address the need for noninvasive biomarkers that differentiate patient outcomes, serum antibodies to E7 oncoprotein levels were evaluated in serial serum specimens from HPV-positive HNC patients (

The Material Properties Of A Bacterial-Derived Biomolecular Condensate Tune Biological Function In Natural And Synthetic Systems, Keren Lasker, Steven Boeynaems, Vinson Lam, Daniel Scholl, Emma Stainton, Adam Briner, Maarten Jacquemyn, Dirk Daelemans, Ashok Deniz, Elizabeth Villa, Alex S Holehouse, Aaron D Gitler, Lucy Shapiro

2020-Current year OA Pubs

Intracellular phase separation is emerging as a universal principle for organizing biochemical reactions in time and space. It remains incompletely resolved how biological function is encoded in these assemblies and whether this depends on their material state. The conserved intrinsically disordered protein PopZ forms condensates at the poles of the bacterium Caulobacter crescentus, which in turn orchestrate cell-cycle regulating signaling cascades. Here we show that the material properties of these condensates are determined by a balance between attractive and repulsive forces mediated by a helical oligomerization domain and an expanded disordered region, respectively. A series of PopZ mutants disrupting this …

Non-Sedative Cortical Eeg Signatures Of Allopregnanolone And Functional Comparators, Peter M Lambert, Richard Ni, Ann Benz, Nicholas R Rensing, Michael Wong, Charles F Zorumski, Steven Mennerick

2020-Current year OA Pubs

Neurosteroids that positively modulate GABA

Islet Primary Cilia Motility Controls Insulin Secretion, Jung Hoon Cho, Zipeng A Li, Lifei Zhu, Brian D Muegge, Henry F Roseman, Eun Young Lee, Toby Utterback, Louis G Woodhams, Philip V Bayly, Jing W Hughes

2020-Current year OA Pubs

Primary cilia are specialized cell-surface organelles that mediate sensory perception and, in contrast to motile cilia and flagella, are thought to lack motility function. Here, we show that primary cilia in human and mouse pancreatic islets exhibit movement that is required for glucose-dependent insulin secretion. Islet primary cilia contain motor proteins conserved from those found in classic motile cilia, and their three-dimensional motion is dynein-driven and dependent on adenosine 5'-triphosphate and glucose metabolism. Inhibition of cilia motion blocks beta cell calcium influx and insulin secretion. Human beta cells have enriched ciliary gene expression, and motile cilia genes are altered in …

Multidimensional Analysis And Therapeutic Development Using Patient Ipsc-Derived Disease Models Of Wolfram Syndrome, Rie Asada Kitamura, Kristina G. Maxwell, Kelly Kries, Cris M. Brown, Punn Augsornworawat, Jeffrey R Millman, Fumihiko Urano, Et Al

2020-Current year OA Pubs

Wolfram syndrome is a rare genetic disorder largely caused by pathogenic variants in the WFS1 gene and manifested by diabetes mellitus, optic nerve atrophy, and progressive neurodegeneration. Recent genetic and clinical findings have revealed Wolfram syndrome as a spectrum disorder. Therefore, a genotype-phenotype correlation analysis is needed for diagnosis and therapeutic development. Here, we focus on the WFS1 c.1672C>T, p.R558C variant, which is highly prevalent in the Ashkenazi Jewish population. Clinical investigation indicated that patients carrying the homozygous WFS1 c.1672C>T, p.R558C variant showed mild forms of Wolfram syndrome phenotypes. Expression of WFS1 p.R558C was more stable compared with …