bluuebunny/crossref_metadata_2025_split huggingface dataset:
Protein Structure Prediction and Bioinfamatic Analysis of Novel Fusion Gene IKZF1/Pfas in BPDCN
Authors: Le Yin , Yu Qian , Zhu Hongkai , Jiang Yi , Wang Zhihua , Li Ruijuan , Li Heng , Sheng Yue , Cheng Zhao , Peng Hongling 
Published: November 2021

Abstract
A novel fusion gene, IKAROS Family Zinc Finger 1 (IKZF1)/Phosphoribosylformylglycinamidine Synthase (PFAS), was identified in a patient diagnosed with Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN). Secondary protein structure analysis, 3D protein structure determination and domain analysis revealed loss of the ZnF-C2H2 domains of IKZF1 caused by premature translation termination at amino acid (AA) residue 55, which might cause dysfunction of IKZF1.
Bioinformatic analysis was conducted using RNA-seq data for 37 BPDCN samples from the GSE database (GSE62014 and GSE89565) to elucidate the role of IKZF1 in BPDCN. WGCNA showed that IKZF1 expression could be used to categorize BPDCN samples into two distinct clusters (IKZF1 high and IKZF1 low). Differentially expressed genes (DEGs) were identified in these two subgroups and subjected to GO and KEGG analyses. Mitochondrial function-related pathways and ubiquitin-mediated proteolysis were the most enriched pathways in the GO and KEGG analyses, respectively. A PPI network of the DEGs was constructed, and 10 key genes were identified (CSTF2, SF3B1, U2AF2, HNRNPH1, SF3B3, SNRNP200, LSM2, SLU7, CPSF4 and UPF3B). Most of these genes are related to tumorigenesis, cancer metastasis, and hematopoietic malignancies.
In conclusion, the novel IKZF1/PFAS fusion gene could cause dysfunction of the IKZF1. The bioinformatic analysis results emphasized the role of IKZF1 in BPDCN and identified 10 key genes closely related to IKZF1, most of which are tumorigenesis-related.

Disclosures
No relevant conflicts of interest to declare.
Next-Generation Sequencing Reveals Molecular Heterogeneity in Primary Testicular DLBCL
Authors: Liu Shuozi , Yang Ping , Wang Jing , Zhang Weilong , Jing Hongmei 
Published: November 2021

Abstract
Background:
Primary testicular lymphomas (PTLs) are extra-nodal large B-cell lymphomas (LBCLs) that respond poorly to current empirical treatment options. To date, no PTL whole-exome sequencing analysis based on the Chinese population has been reported. The main objective of this study was to explore the genomic landscape of PTDLBCL, correlating the results with clinical, histological, and immunogenetic features.
Methods:
We retrospectively analyzed the disease characteristics in a series of 20 patients (13 paired data and 7 unpaired data). In all cases, the diagnosis of PTL was made using appropriate diagnostic criteria for the 2008 WHO classification of lymphoid tumors with combinations of histologic, immunohistochemical, flow cytometric, and genetic evaluation. Medical records were reviewed for demographic and clinical data. We performed whole-exome next-generation sequencing (NGS) and mapped the mutational landscape. Based on the differential gene results, we performed KEGG and GO enrichment analysis, and five potential new pathogenic genes for DLBCL were obtained based on the Polyphen score.
Results:
A total of 20 DLBCL samples were collected. All samples were subjected to whole-exome sequencing (WES). A standard bioinformatics analysis process was used to identify somatic mutations in each sample, and an average of 1200 somatic mutations was identified, involving an average of 420 mutated genes. (Figure 1.) More than 85% of the somatic mutations were found in only 1 sample, indicating that somatic mutations are highly heterogeneous. In the matched 13 cases, we validated common gene mutations that have been reported, including PIM1 53.84% (7/13), CD79B 38.46% (5/13), CDKN2A 15.38% (2/13), and MYD88 15.38% (2/13). Venn diagram shows that 25% of the mutated genes were specific to the relapsed sample. GO analysis (Figure 2) shows the functional enrichment and differences between relapsed and non-relapsed samples. Non-relapsed samples are enriched in the herpes simplex virus 1 infection pathway. (Figure 3). Focusing on relapsed/non-relapsed samples, we identified 12 mutated genes specific to relapsed samples. Mutation Polyphen scores were calculated to assess mutation deleteriousness. Five potential new pathogenic genes for DLBCL were obtained.
Conclusions:
Based on whole-sequencing data, we validated the previously reported common genes for PTDLBCL. In addition, we performed GO and KEGG analysis according to the relapsed and non-relapsed groups. In addition, based on the difference in enrichment results, 12 significantly enriched mutated genes in relapsed DLBCL samples are obtained, and mutation Polyphen scores are calculated to assess mutation deleteriousness. And 5 potential new pathogenic genes of DLBCL are identified.
Figure 1 Figure 1.

Disclosures
No relevant conflicts of interest to declare.
Hematopoietic Reconstitution of Human Umbilical Cord Mesenchymal Stem Cells after Leukemia Chemotherapy: Effectiveness and Safety
Authors: Tan Yuxin , Ding Lu , Can Can , Zhou Fuling 
Published: November 2021

Abstract
Objective: As the incidence of tumors increases, more patients need chemotherapy. Patients receiving chemotherapy also inevitably suffer side effects from treatment. Damage to the immune and hematopoietic system causes the failure of therapies. Mesenchymal stem cells have excellent capabilities in immune regulation and hematopoietic support. This study preliminarily explored the safety and effectiveness and of human umbilical cord mesenchymal stem cells (UCMSC) in myelosuppressive patient-derived tumor xenograft (PDX) models of acute myeloid leukemia (AML).
Methods: B-NDG mice were used to establish the PDX models. The mice were randomly divided into three groups: AML group, AML+Ara-C group and AML+Ara-C+MSC group. Mice in the AML+Ara-C group and AML+Ara-C+MSC group were injected intraperitoneally with cytarabine (Ara-C) 60mg/kg on day1-day3 to induce myelosuppression. Mice in the AML+Ara-C+MSC group were injected 3×10 6 UCMSC through the tail vein on day4. We observed the changes in peripheral blood, bone marrow signaling pathways, and AML progression in mice.
Results: The experiment found that UCMSC rescued the body weight and peripheral blood. We also found that UCMSC could increase the number of bone marrow CD117 + hematopoietic stem/progenitor cells and CD41 + megakaryocytes through flow cytometry. We verified at protein level that the hematopoiesis-related signaling pathway JAK2/STAT3 was up-regulated in AML+Ara-C+MSC group compared with AML+Ara-C group through flow cytometry and immunohistochemistry. At the same time, the infusion of UCMSC after Ara-C had no influence on disease progression. On day15, the proportion of human CD45 + cells in the bone marrow of between AML+Ara-C+MSC group and AML+Ara-C group was similar, and there was no statistical difference (21.96±3.10 vs. 23.04±1.51; P=0.6792).
Conclusion: Infusion of exogenous UCMSC after chemotherapy in PDX models could promote the recovery of hematopoiesis without affecting the efficacy of Ara-C. A reasonable UCMSC infusion scheme is potential to be used in the treatment of AML patients and it requires a lot of preclinical exploration in the future.

Disclosures
No relevant conflicts of interest to declare.

OffLabel Disclosure:
Cytarabine (Ara-C) was administrated to mice models to induce myelosuppression.
Human Genetic Diversity Alters Therapeutic Gene Editing Off-Target Outcomes
Authors: Lin Linda Yingqi , Cancellieri Samuele , Zeng Jing , Masillo Francesco , Nguyen My Anh , Bombieri Nicola , Maitland Stacy A. , Ciuculescu Marioara-Felicia , Katta Varun , Tsai Shengdar Q. , Armant Myriam , Wolfe Scot A. , Giugno Rosalba , Pinello Luca , Bauer Daniel E. 
Published: November 2021

Abstract
CRISPR gene editing holds great promise to modify somatic genomes to ameliorate disease. In silico prediction of homologous sites coupled with biochemical evaluation of possible genomic off-targets may predict genotoxicity risk of individual gene editing reagents. However, standard computational and biochemical methods focus on reference genomes and do not consider the impact of genetic diversity on off-target potential. Here we developed a web application called CRISPRme that explicitly and efficiently integrates human genetic variant datasets with orthogonal genomic annotations to predict and prioritize off-target sites at scale. The method considers both single-nucleotide variants (SNVs) and indels, accounts for bona fide haplotypes, accepts spacer:protospacer mismatches and bulges, and is suitable for personal genome analyses. We tested the tool with a guide RNA (gRNA) targeting the BCL11A erythroid enhancer that has shown therapeutic promise in clinical trials for sickle cell disease (SCD) and β-thalassemia (Frangoul et al. NEJM 2021). We find that the top predicted off-target site is produced by a non-reference allele common in African-ancestry populations (rs114518452, minor allele frequency (MAF) = 4.5%) that introduces a protospacer adjacent motif (PAM) for SpCas9. We validate that SpCas9 generates indels (~9.6% frequency) and chr2 pericentric inversions in a strictly allele-specific manner in edited CD34+ hematopoietic stem/progenitor cells (HSPCs), although a high-fidelity Cas9 variant mitigates this off-target. This report illustrates how population and private genetic variants should be considered as modifiers of genome editing outcomes. We expect that variant-aware off-target assessment will be required for therapeutic genome editing efforts going forward, including both ongoing and future clinical trials, and we provide a powerful approach for comprehensive off-target prediction. CRISPRme is available at crisprme.di.univr.it.

Disclosures
No relevant conflicts of interest to declare.
Prognosis and Therapeutic Response Transfer Learning Model for Multiple Myeloma
Authors: Voegeli Jerome , Moreno Dana , Kryukov Maxim , Mathez Gregory , Petremand Remy , Pere Arthur , Ekstrom Leeland , Jaun Andre 
Published: November 2021

Abstract
The combination of clinical knowledge (applicable for a majority of patients, published in the form of review articles), real world evidence (describing more nuanced outcomes for small cohorts) and innovative artificial intelligence algorithms opens potent avenues to re-examine clinical findings and uncover new biomarkers for the prognosis / prediction of therapeutic responses--in a manner that can directly be incorporated in clinical decision support tools.
In this work, neural networks are first developed to reproduce clinical guidelines with &gt;95% accuracy. After mastering the complex knowledge that is generally expected from human doctors, a transfer learning technique was used to sift through de-identified longitudinal data of 9267 patients with multiple myeloma-related conditions at the Vanderbilt University Medical Center using progression free survival (PFS) to quantify therapeutic outcomes. The "precision medicine neural networks" obtained as a result can be compared with conventional and less portable survival model algorithms, using Shapley values to explain prediction differences.
Testing a first hypothesis that "lytic bone lesions are a prognostic factor for poor PFS", a study involving 1530 patients confirmed that the median PFS of 54 ± 6 months (684 censored patients showing progression) in the presence of bone lesions is significantly lower than the 107 ± 40 months (90 censored patients) in the absence of lesions.
Keeping only 179 patients for which a full range of cytogenetic factors are available and using a Cox regression & Random Survival Forests that provide the best fit of the data, we confirmed previous findings for high-risk trisomies 1 and 7, monosomy 13, deletion 12p and translocation t(11;14)(q13;q32). We furthermore uncovered new additional adverse factors del6q, 2+, 21-, 16- to formulate a model that achieves a statistically significant concordance of 0.72 ± 0.07.
Comparing therapeutic effects for patients in a real-world hospital setting with clinical trials, we found that lenalidomide + bortezomib + dexamethasone used in a first-line therapy resulted in lower median PFS of 17-47 months than the 39-52 months published in the SWOG S0777 trial, most likely because comorbidities contributed to shortening the PFS in real-world settings.
We conclude with an analysis of concrete examples where therapeutic recommendations differ from guidelines, explaining the reason with statistically significant cohorts observed in the data.

Disclosures
No relevant conflicts of interest to declare.
Factor VIII Epitope Analysis Using a Random Peptide Phage-Display Library Approach in the Sippet Cohort
Authors: Hassan Shermarke , Baselli Guido , Palm Kaia , Rosendaal Frits Richard , Palla Roberta , Peyvandi Flora 
Published: November 2021

Abstract
Background Inhibitor development is the most severe complication of hemophilia A care, and is associated with increased morbidity and mortality.
Aims The aim of this study was to use a novel epitope mapping method to explore the factor VIII (FVIII)-specific epitope profile in the SIPPET cohort population.
Methods The population consisted of 122 previously untreated patients with severe hemophilia A that were followed-up for 50 days of exposure to FVIII. Sampling was performed before FVIII treatment and at the end of the follow-up. The outcome was inhibitor development. The FVIII-specific IgG epitope repertoire was assessed by means of a novel high-throughput epitope mapping technique using a random peptide phage-display library. Using this assay, a set of affinity-selected 12-mer peptide sequences (also called mimotopes) that were strongly bound by FVIII-specific antibodies were identified. These mimotopes were clustered on the basis of sequence similarity and a consensus motif was generated for each mimotope cluster. Discriminative performance of these mimotope clusters was assessed by ROC analysis. Mimotope clusters were mapped onto the 3D structure of a B-domain deleted FVIII model using a B-cell epitope prediction algorithm (Mapitope).
Results The FVIII-specific antibody response is polyclonal with several mimotope clusters. The most predominant mimotope clusters in inhibitor patients were mapped to the heavy chain of the FVIII molecule. Using plasma samples taken before exposure to FVIII, three mimotopes (with the consensus motifs "QM", "PSLxWK" and "SWPHxxxxK") were identified that predicted inhibitor development (with an AUC of 0.76, 0.80 and 0.76 respectively).
Conclusion Information on immunodominant epitope clusters can be used to generate novel, less immunogenic FVIII proteins and set up diagnostic tests that predict the risk of inhibitor development before starting treatment with FVIII.
Figure 1 Figure 1.

Disclosures
Palm: Protobios LLC: Current Employment, Patents & Royalties: Inventor of the patent application (PCT Application No. US/14079626) filed by Protobios that covers the use of phage display method to manipulate and monitor humoral immunity.. Palla: Pfizer: Other: Travel support; Kedrion: Other: Travel support; Novonordisk: Speakers Bureau. Peyvandi: Roche: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Sobi: Consultancy, Honoraria; Takeda: Consultancy, Honoraria.
The Role of Endothelial Superoxide Dismutase 2 in Modulating Sickle Pathology
Authors: Dosunmu-Ogunbi Atinuke , Yuan Shuai , Straub Adam C 
Published: November 2021

Abstract
Sickle cell disease (SCD) patients suffer from hemolysis in microcirculation. On the one hand, hemoglobin and heme released from sickled red blood cells catalyze reactive oxygen species (ROS) generation by Fenton chemistry. On the other hand, sickled red blood cells occlude capillaries, creating a hypoxic condition that exacerbates ROS production. As an essential antioxidant mechanism, superoxide dismutase 2 (SOD2) detoxifies superoxide by converting it to hydrogen peroxide (H 2O 2) in the mitochondria. In SCD patients, despite the elevated ROS production, we found that SOD2 expression is suppressed in the pulmonary endothelium (Figure 1A,B). Therefore, we hypothesize the depletion of endothelial SOD2 compromises endothelial function and exacerbates the progression of SCD.
To examine the role of endothelial SOD2, we silenced SOD2 gene expression (SOD2 KD) with siRNA in primary human pulmonary microvascular endothelial cells (hPMVECs). Knocking down SOD2 in hPMVECs accelerated mitochondrial superoxide production and compromised mitochondrial potential. However, mitochondrial respiration, the activity of respiratory complexes, and the cellular ATP content were not affected by SOD2 KD. An important function of endothelial cells is to form a barrier and sequester cellular and molecular contents in the blood. SOD2 KD hPMVECs exhibited increased albumin leakage and decreased transendothelial resistance, indicating a disrupted endothelial barrier(Figure 1C). The defect in the endothelial barrier was rescued by adding 4 mM H 2O 2(Figure 1D), suggesting SOD2-derived H 2O 2 may serve as a critical signaling molecule. Moreover, cell migration or proliferation was inhibited in SOD2 KD hPMVECs, which was examined by a scratch assay.
Since both cell migration and barrier maintenance require focal adhesion assembly, we next investigated the role of SOD2 in focal adhesion dynamics. In an attachment assay, SOD2 KD reduced cell attachment rate on uncoated plates, which was blunted by fibronectin precoating, indicating a fibronectin-dependent defect in cell adhesion(Figure 1E,F). Notably, although fibronectin protein expression in hPMVECs was not altered, SOD2 KD decreased the dimer/monomer ratio. Furthermore, confocal images showed that fibronectin was retained in SOD2 KD cells(Figure 1G), highlighting the importance of SOD2 in fibronectin assembly.
In conclusion, we demonstrated for the first time that SOD2 expression is diminished in the pulmonary endothelium of SCD patients and that endothelial SOD2 is crucial for endothelial function by facilitating fibronectin assembly. The importance of SOD2 in endothelial function may prove therapeutic value in SCD patients, which requires further investigation.
Figure 1 Figure 1.

Disclosures
No relevant conflicts of interest to declare.
The Small Gtpase Rap1 in Platelets Is Critical for Arterial but Not Venous Thrombosis in Mice
Authors: Mwiza Jean , Lee Robert H , Paul David , Holle Lori A , Cooley Brian C , Wolberg Alisa S. , Bergmeier Wolfgang 
Published: November 2021

Abstract
Background: Platelets and their main adhesion receptors, integrins, are critical in hemostasis and arterial thrombosis, i.e., in situations involving severe insult to the vasculature and elevated shear stress, respectively. We recently demonstrated that integrin activation under both of these conditions depends on the small GTPase Rap1 directly activating the integrin adapter protein, Talin1. Our studies further suggested that the Rap1-talin1 axis is less important for platelet function at sites of inflammation, i.e., in situations of mild endothelial insult and low shear stress.
Aim: To investigate the contribution of the platelet Rap1-Talin1-integrin signaling axis in the pathogenesis of venous thrombosis (VT).
Methods: The following mice with documented deficiencies in platelets were subjected to the inferior vena cava (IVC) stenosis VT model: Rap1amKO (Rap1a fl/fl pf4-Cre+), Rap1bmKO (Rap1b fl/fl pf4-Cre+), or both isoforms (Rap1a fl/flRap1b fl/fl pf4-Cre+ [Rap1dKO]), and Talin1mKO (Tln1 fl/flpf4-Cre+). Thrombus weight was determined 48 hours after flow restriction. Clot contraction and tissue plasminogen activator (tPA)-mediated lysis of whole blood clots were studied ex vivo to characterize effects on clot consolidation and stability .
Results: Compared to control mice, thrombus weight was markedly reduced in Talin1mKO mice (13.1±2 and 2.2±1.2 mg, p&lt;0.05), but not Rap1dKO or single knockout mice. Ex vivo clot contraction was significantly impaired in whole blood from Talin1mKO (clot weight: control 13.35±3.4 mg vs. 34.63±4.2 mg), Rap1amKO (control 18.0±3.9 mg vs. 25.6±6.0 mg), Rap1bmKO (control 18.0±3.9 vs 23.0±6.6 mg) and in Rap1dKO (control 18.03±3.9 mg vs. 30.02±4.6 mg). Clot weights were not significantly different between Rap1dKO and Talin1mKO mice. However, ex vivo clot lysis assays revealed that compared to controls, clots formed in whole blood from Talin1mKO mice lysed faster, whereas clots from Rap1dKO did not.
Conclusions: Platelet Talin1 is critical for VT in mice, and this role is partially independent of its best-known upstream regulator, Rap1. Future studies will identify the alternative mechanism allowing for Rap1-independent Talin1 signaling and platelet function in VT and how these signaling affect various phases of VT.

Disclosures
Wolberg: Takeda: Research Funding; Bristol Myers Squibb: Research Funding; CSL Behring: Consultancy.
Histogram analysis based on multi-parameter MR imaging as a biomarker to predict lymph node metastasis in T3 stage rectal cancer
Authors: Zhou Yang , Yang Rui , Wang Yuan , Zhou Meng , Zhou Xueyan , Xing JiQing , Wang Xinxin , Zhang Chunhui 
Published: November 2021

Abstract
Background
Preoperative identification of rectal cancer lymph node status is crucial for patient prognosis and treatment decisions. Rectal magnetic resonance imaging (MRI) plays an essential role in the preoperative staging of rectal cancer, but its ability to predict lymph node metastasis (LNM) is insufficient. This study explored the value of histogram features of primary lesions on multi-parametric MRI for predicting LNM of stage T3 rectal carcinoma.

Methods
We retrospectively analyzed 175 patients with stage T3 rectal cancer who underwent preoperative MRI, including diffusion-weighted imaging (DWI) before surgery. 62 patients were included in the LNM group, and 113 patients were included in the non-LNM group. Texture features were calculated from histograms derived from T2 weighted imaging (T2WI), DWI, ADC, and T2 maps. Stepwise logistic regression analysis was used to screen independent predictors of LNM from clinical features, imaging features, and histogram features. Predictive performance was evaluated by receiver operating characteristic (ROC) curve analysis. Finally, a nomogram was established for predicting the risk of LNM.

Results
The clinical, imaging and histogram features were analyzed by stepwise logistic regression. Preoperative carbohydrate antigen 199 level (p = 0.009), MRN stage (p < 0.001), T2WIKurtosis (p = 0.010), DWIMode (p = 0.038), DWICV (p = 0.038), and T2-mapP5 (p = 0.007) were independent predictors of LNM. These factors were combined to form the best predictive model. The model reached an area under the ROC curve (AUC) of 0.860, with a sensitivity of 72.8% and a specificity of 85.5%.

Conclusion
The histogram features on multi-parametric MRI of the primary tumor in rectal cancer were related to LN status, which is helpful for improving the ability to predict LNM of stage T3 rectal cancer.