The identification of effective tumor-suppressing neoantigens using a tumor-reactive TIL TCR-pMHC ternary complex.

Neoantigens are ideal targets for cancer immunotherapy because they are expressed de novo in tumor tissue but not in healthy tissue and are therefore recognized as foreign by the immune system. Advances in next-generation sequencing and bioinformatics technologies have enabled the quick identification and prediction of tumor-specific neoantigens; however, only a small fraction of predicted neoantigens are immunogenic. To improve the predictability of immunogenic neoantigens, we developed the in silico neoantigen prediction workflows VACINUSpMHC and VACINUSTCR: VACINUSpMHC incorporates physical binding between peptides and MHCs (pMHCs), and VACINUSTCR integrates T cell reactivity to the pMHC complex through deep learning-based pairing with T cell receptors (TCRs) of putative tumor-reactive CD8 tumor-infiltrating lymphocytes (TILs). We then validated our neoantigen prediction workflows both in vitro and in vivo in patients with hepatocellular carcinoma (HCC) and in a B16F10 mouse melanoma model. The predictive abilities of VACINUSpMHC and VACINUSTCR were confirmed in a validation cohort of 8 patients with HCC. Of a total of 118 neoantigen candidates predicted by VACINUSpMHC, 48 peptides were ultimately selected using VACINUSTCR. In vitro validation revealed that among the 48 predicted neoantigen candidates, 13 peptides were immunogenic. Assessment of the antitumor efficacy of the candidate neoepitopes using a VACINUSTCR in vivo mouse model suggested that vaccination with the predicted neoepitopes induced neoantigen-specific T cell responses and enabled the trafficking of neoantigen-specific CD8 + T cell clones into the tumor tissue, leading to tumor suppression. This study showed that the prediction of immunogenic neoantigens can be improved by integrating a tumor-reactive TIL TCR-pMHC ternary complex.

Barcoded multiple displacement amplification for high coverage sequencing in spatial genomics.

Determining mutational landscapes in a spatial context is essential for understanding genetically heterogeneous cell microniches. Current approaches, such as Multiple Displacement Amplification (MDA), offer high genome coverage but limited multiplexing, which hinders large-scale spatial genomic studies. Here, we introduce barcoded MDA (bMDA), a technique that achieves high-coverage genomic analysis of low-input DNA while enhancing the multiplexing capabilities. By incorporating cell barcodes during MDA, bMDA streamlines library preparation in one pot, thereby overcoming a key bottleneck in spatial genomics. We apply bMDA to the integrative spatial analysis of triple-negative breast cancer tissues by examining copy number alterations, single nucleotide variations, structural variations, and kataegis signatures for each spatial microniche. This enables the assessment of subclonal evolutionary relationships within a spatial context. Therefore, bMDA has emerged as a scalable technology with the potential to advance the field of spatial genomics significantly.

G-RANK: an equivariant graph neural network for the scoring of protein-protein docking models.

Motivation: Protein complex structure prediction is important for many applications in bioengineering. A widely used method for predicting the structure of protein complexes is computational docking. Although many tools for scoring protein-protein docking models have been developed, it is still a challenge to accurately identify near-native models for unknown protein complexes. A recently proposed model called the geometric vector perceptron-graph neural network (GVP-GNN), a subtype of equivariant graph neural networks, has demonstrated success in various 3D molecular structure modeling tasks. Results: Herein, we present G-RANK, a GVP-GNN-based method for the scoring of protein-protein docking models. When evaluated on two different test datasets, G-RANK achieved a performance competitive with or better than the state-of-the-art scoring functions. We expect G-RANK to be a useful tool for various applications in biological engineering. Availability and implementation: Source code is available at https://github.com/ha01994/grank. Contact: kds@kaist.ac.kr. Supplementary information: Supplementary data are available at Bioinformatics Advances online.

A Sensitive Immunodetection Assay Using Antibodies Specific to Staphylococcal Enterotoxin B Produced by Baculovirus Expression.

Staphylococcal enterotoxin B (SEB) is a potent bacterial toxin that causes inflammatory stimulation and toxic shock, thus it is necessary to detect SEB in food and environmental samples. Here, we developed a sensitive immunodetection system using monoclonal antibodies (mAbs). Our study is the first to employ a baculovirus expression vector system (BEVS) to produce recombinant wild-type SEB. BEVS facilitated high-quantity and pure SEB production from suspension-cultured insect cells, and the SEB produced was characterized by mass spectrometry analysis. The SEB was stable at 4 °C for at least 2 years, maintaining its purity, and was further utilized for mouse immunization to generate mAbs. An optimal pair of mAbs non-competitive to SEB was selected for sandwich enzyme-linked immunosorbent assay-based immunodetection. The limit of detection of the immunodetection method was 0.38 ng/mL. Moreover, it displayed higher sensitivity in detecting SEB than commercially available immunodetection kits and retained detectability in various matrices and S. aureus culture supernatants. Thus, the results indicate that BEVS is useful for producing pure recombinant SEB with its natural immunogenic property in high yield, and that the developed immunodetection assay is reliable and sensitive for routine identification of SEB in various samples, including foods.

Phenotype-based single cell sequencing identifies diverse genetic subclones in CD133 positive cancer stem cells.

Tumor heterogeneity is one of the ongoing huddles in the field of colon cancer therapy. It is evident that there are countless clones which exhibit different phenotypes and therefore, single cell analysis is inevitable. Cancer stem cells (CSCs) are rare cell population within tumor which is known to function in cancer metastasis and recurrence. Although there have been trials to prove intra-tumoral heterogeneity using single cell sequencing, that of CSCs has not been clearly elucidated. Here, we articulate the presence of heterogeneous subclones within CD133 positive cancer stem cells through single cell sequencing. As a proof of principle, we performed phenotype-based high-throughput laser isolation and single cell sequencing (PHLI-seq) of CD133 positive cells in a frozen tumor tissue obtained from a patient with colorectal cancer. The result proved that CD133 positive cells were shown to be heterogeneous both in copy number and mutational profiles. Single cancer stem cell specific mutations such as RNF144A, PAK2, PARP4, ADAM21, HYDIN, KRT38 and CELSR1 could be also detected in liver metastatic tumor of the same patient. Collectively, these data suggest that single cell analysis used to spot subclones with genetic variation within rare population, will lead to new strategies to tackle colon cancer metastasis.

Targeted sequencing aids in identifying clonality in chronic myelomonocytic leukemia.

Chronic myelomonocytic leukemia (CMML) typically shows monocytosis in the peripheral blood (PB), which must be differentiated from reactive monocytosis. To determine the clonality of CMML, we performed molecular and cytogenetic analysis in Korean patients. To investigate whether monocytes in the PB harbored clonal mutational changes, we performed single-cell sequencing after selecting monocytes, neutrophils, and lymphocytes by morphology-aided laser microdissection. Targeted sequencing was performed in 35 patients with CMML with 41 bone marrow samples. Single-cell analysis was performed in two cases. Most (94.3%) patients harbored at least one variant, in genes considered as potential therapeutic targets, while cytogenetic aberrations occurred in only 28.6% of cases. ASXL1 (54.3%), SRSF2 (37.1%), NRAS (31.4%), and TET2 (25.7%) were frequently mutated, with lower frequencies of TET2 mutation and higher frequencies of NRAS, DNMT3A (17.1%), and NPM1 (11.4%) mutations compared to in previous studies of Caucasians. Patients with SETBP1 mutation and those with more than two variants showed poorer survival than those without mutation (P < 0.001 and P = 0.007, respectively). Most (70.8%) variants were detected at diagnosis and follow-up with no significant differences in variant allele frequency, warranting sequencing during follow-up if diagnostic samples were unavailable. Single-cell analysis revealed clonal monocytes with mutations, and the same mutations were also identified in lymphocytes and neutrophils. Targeted sequencing aided in clonality detection in most patients with CMML and single-cell sequencing facilitated identification of clonal monocytes and the co-existence of mutations in non-myeloid cells, suggesting that certain mutations are acquired by pluripotent stem cells.

Characteristics of Waldenström Macroglobulinemia in Korean Patients According to Mutational Status of MYD88 and CXCR4: Analysis Using Ultra-Deep Sequencing.

BACKGROUND: Little is known about the mutational frequency of myeloid differentiation factor 88 (MYD88) and C-X-C chemokine receptor type 4 (CXCR4) and the corresponding characteristics in Asian individuals afflicted with Waldenström macroglobulinemia (WM). We investigated the characteristics of WM according to mutational status of MYD88/CXCR4, and attempted to determine the lineage commitment among hematopoietic cells by MYD88L265P single-cell sequencing on bone marrow (BM) smear slides. MATERIALS AND METHODS: CXCR4 mutations (muts) were detected using ultra-deep sequencing using target capture. Mutational burden of MYD88 was assessed using real-time polymerase chain reaction. Single-cell sequencing for MYD88 was performed on lymphocytes, plasmacytoid lymphocytes, plasma cells, and neutrophils using laser microdissection. RESULTS: Among 31 patients, the frequencies of MYD88/CXCR4 muts were as follows: MYD88 wild type (WT) CXCR4WT (6 patients, 19.4%), MYD88L265PCXCR4WT (19 patients, 61.4%), MYD88L265PCXCR4mut (6 patients, 19.4%; 1 frameshift and 5 nonsense muts). Immunoglobulin M levels of MYD88L265CXCR4WT patients were significantly higher than those of MYD88WTCXCR4WT patients (P = .024). Tumor burden in BM was highest in patients with MYD88L265PCXCR4mut (82.0%), followed by MYD88L265PCXCR4WT (52.8%) and MYD88WTCXCR4WT (14.2%) (P < .001). The quantity of MYD88-mutated DNA tended to correlate with tumor burden in BM (correlation coefficient 0.647; P = .009). MYD88L265P was detected in plasma cells, plasmacytoid lymphocytes, and lymphocytes but not neutrophils. CONCLUSION: The frequency of MYD88/CXCR4 muts in Korean and Caucasian patients with WM was similar, however 5 of the 6 CXCR4 muts were nonsense-a proportion higher than reported frequencies in Caucasian individuals. Ultra-deep sequencing was capable of detecting CXCR4 muts not detectable using Sanger sequencing, suggesting a possible replacement of the B-cell sorting.

Ginkgetin, a biflavone from Ginkgo biloba leaves, prevents adipogenesis through STAT5-mediated PPARγ and C/EBPα regulation.

Adipogenesis involved in hypertrophy and hyperplasia of adipocytes is responsible for expanding the mass of adipose tissues in obese individuals. Peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein α (C/EBPα) are two principal transcription factors induced by delicate signaling pathways, including signal transducer and activator of transcription 5 (STAT5), in adipogenesis. Here, we demonstrated a novel role of ginkgetin, a biflavone from Ginkgo biloba leaves, as a STAT5 inhibitor that blocks the differentiation of preadipocytes into adipocytes. During the differentiation of 3T3-L1 cells, ginkgetin treatment during the first 2 days markedly inhibited the formation of lipid-bearing adipocytes. PPARγ and C/EBPα expression was decreased in 3T3-L1 cells during adipogenesis following ginkgetin treatment, whereas no change was observed in C/EBPß or C/EBPδ expression. Inhibition of PPARγ and C/EBPα expression by ginkgetin occurred through the prevention of STAT5 activation during the initiation phase of adipogenesis. In addition, ginkgetin-mediated the inhibition of adipogenesis was recapitulated in the differentiation of primary preadipocytes. Lastly, we confirmed the inhibitory effects of ginkgetin on the hypertrophy of white adipose tissues from high-fat diet-fed mice. These results indicate that ginkgetin is a potential anti-adipogenesis and anti-obesity drug.

Author Correction: Evaluating Tumor Evolution via Genomic Profiling of Individual Tumor Spheroids in a Malignant Ascites.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

PHLI-seq: constructing and visualizing cancer genomic maps in 3D by phenotype-based high-throughput laser-aided isolation and sequencing.

Spatial mapping of genomic data to tissue context in a high-throughput and high-resolution manner has been challenging due to technical limitations. Here, we describe PHLI-seq, a novel approach that enables high-throughput isolation and genome-wide sequence analysis of single cells or small numbers of cells to construct genomic maps within cancer tissue in relation to the images or phenotypes of the cells. By applying PHLI-seq, we reveal the heterogeneity of breast cancer tissues at a high resolution and map the genomic landscape of the cells to their corresponding spatial locations and phenotypes in the 3D tumor mass.

Evaluating Tumor Evolution via Genomic Profiling of Individual Tumor Spheroids in a Malignant Ascites.

Epithelial ovarian cancer (EOC) is a silent but mostly lethal gynecologic malignancy. Most patients present with malignant ascites and peritoneal seeding at diagnosis. In the present study, we used a laser-aided isolation technique to investigate the clonal relationship between the primary tumor and tumor spheroids found in the malignant ascites of an EOC patient. Somatic alteration profiles of ovarian cancer-related genes were determined for eight spatially separated samples from primary ovarian tumor tissues and ten tumor spheroids from the malignant ascites using next-generation sequencing. We observed high levels of intra-tumor heterogeneity (ITH) in copy number alterations (CNAs) and single-nucleotide variants (SNVs) in the primary tumor and the tumor spheroids. As a result, we discovered that tumor cells in the primary tissues and the ascites were genetically different lineages. We categorized the CNAs and SNVs into clonal and subclonal alterations according to their distribution among the samples. Also, we identified focal amplifications and deletions in the analyzed samples. For SNVs, a total of 171 somatic mutations were observed, among which 66 were clonal mutations present in both the primary tumor and the ascites, and 61 and 44 of the SNVs were subclonal mutations present in only the primary tumor or the ascites, respectively. Based on the somatic alteration profiles, we constructed phylogenetic trees and inferred the evolutionary history of tumor cells in the patient. The phylogenetic trees constructed using the CNAs and SNVs showed that two branches of the tumor cells diverged early from an ancestral tumor clone during an early metastasis step in the peritoneal cavity. Our data support the monophyletic spread of tumor spheroids in malignant ascites.

Preferential Impairment of the Contralesional Posterior Semicircular Canal in Internuclear Ophthalmoplegia.

BACKGROUND: The vertical vestibulo-ocular reflex (VOR) may be impaired in internuclear ophthalmoplegia (INO) as the medial longitudinal fasciculus (MLF) conveys VOR-signals from the vertical semicircular canals. It has been proposed that signals from the contralesional posterior semicircular canal (PSC) are exclusively transmitted through the MLF, while for the contralesional anterior canal other pathways exist. OBJECTIVE: Here, we aimed to characterize dysfunction in individual canals in INO-patients using the video-head-impulse test (vHIT) and to test the hypothesis of dissociated vertical canal impairment in INO. METHODS: Video-head-impulse testing and magnetic resonance imaging were obtained in 21 consecutive patients with unilateral (n = 16) or bilateral (n = 5) INO and 42 controls. VOR-gains and compensatory catch-up saccades were analyzed and the overall function (normal vs. impaired) of each semicircular canal was rated. RESULTS: In unilateral INO, largest VOR-gain reductions were noted in the contralesional PSC (0.55 ± 0.11 vs. 0.89 ± 0.08, p < 0.001), while in bilateral INO both posterior (0.43 ± 0.11 vs. 0.89 ± 0.08, p < 0.001) and anterior (0.58 ± 0.19 vs. 0.88 ± 0.09, p < 0.001) canals showed marked drops. Small, but significant VOR-gain reductions were also found in the other canals in unilateral and bilateral INO-patients. Impairment of overall canal function was restricted to the contralesional posterior canal in 60% of unilateral INO-patients, while isolated involvement of the posterior canal was rare in bilateral INO-patients (20%). Reviewers correctly identified the INO-pattern in 15/21 (71%) patients and in all controls (sensitivity = 84.2% [95%-CI = 0.59.5-95.8]; specificity = 95.5% [95%-CI = 83.3-99.2]). CONCLUSION: Using a vHIT based overall rating of canal function, the correct INO-pattern could be identified with high accuracy. The predominant and often selective impairment of the contralesional posterior canal in unilateral INO further supports the role of the MLF in transmitting posterior canal signals. In patients with acute dizziness and abnormal vHIT-results, central pathologies such as INO should be considered as well, especially when the posterior canal is involved.

Gene Expression Profiling of Hepatocellular Carcinoma Derived Cancer Stem Like Cell under Hypoxia.

PURPOSE: Cancer stem like cells (CSCs), with unlimited self-renewal potential and other stem cell characteristics, occur in several cancers including hepatocellular carcinoma (HCC). Although CSCs can initiate tumors, malignant proliferation, relapse and multi-drug resistance, the ways how to activate them still remain unknown. This study aims to evaluate whether CSC acquire tumorigenic characters under tumor hypoxia, analyzed by microarray analysis. MATERIALS AND METHODS: CSCs were purified from HCC patients and Affymetrix microarray was used to investigate their gene expression profiles. The results were validated by real-time polymerase chain reaction (PCR). RESULTS: The results of the microarray indicated that 18 genes were up-regulated and 10 genes were down-regulated in CSCs. Several genes were identified to be significantly involved in the regulation of CSCs such as HCC. Furthermore, the up-regulated genes were related with metabolism, angiogenesis and hypoxia, whereas the down-regulated genes were related with apoptosis and inflammation. CONCLUSION: The results may help to understand the mechanisms of tumor development through CSCs which acquired their distinctive tumorogenic properties by hypoxic stimulation.

Central positional nystagmus associated with cerebellar tumors: Clinical and topographical analysis.

PURPOSE: Positional nystagmus is usually caused by peripheral vestibular disorder, mostly benign paroxysmal positional vertigo (BPPV). However, positional nystagmus is also encountered in central lesions. We aimed to determine clinical characteristics of the structures responsible for central positional nystagmus (CPN) associated with brain tumors. METHODS: All four patients (3 men; range=19-77years) had an evaluation of spontaneous and positional nystagmus using video-oculography. Brain MRIs were performed in all patients. RESULTS: All patients showed apogeotropic positional nystagmus during supine roll tests, and had an initial diagnosis of BPPV. Except for the positional nystagmus, findings of neurological examination were normal. Because all subjects were initially diagnosed with BPPV, canalith repositioning maneuvers were applied repeatedly but without a success. Brain MRI finally disclosed brain tumors involving the midline cerebellar structures around the fourth ventricle and the nodulus. The pathological diagnosis was hemangioblastoma in two and metastatic tumor in the others. CONCLUSIONS: An apogeotropic type of CPN may be an isolated finding in patients with a cerebellar tumor. Even in patients with isolated apogeotropic positional nystagmus, a central lesion should be sought especially when refractory to repeated canalith repositioning maneuvers.

Programmable gradational micropatterning of functional materials using maskless lithography controlling absorption.

The demand for patterning functional materials precisely on surfaces of stimuli-responsive devices has increased in many research fields. In situ polymerization technology is one of the most convenient ways to place the functional materials on a desired location with micron-scale accuracy. To fabricate stimuli-responsive surfaces, controlling concentration of the functional material is much as important as micropatterning them. However, patterning and controlling concentration of the functional materials simultaneously requires an additional process, such as preparing multiple co-flow microfluidic structures and numbers of solutions with various concentrations. Despite applying these processes, fabricating heterogeneous patterns in large scale (millimeter scale) is still impossible. In this study, we propose an advanced in situ polymerization technique to pattern the surface in micron scale in a concentration-controlled manner. Because the concentration of the functional materials is manipulated by self-assembly on the surface, a complex pattern could be easily fabricated without any additional procedure. The complex pattern is pre-designed with absorption amount of the functional material, which is pre-determined by the duration of UV exposure. We show that the resolution reaches up to 2.5 µm and demonstrate mm-scale objects, maintaining the same resolution. We also fabricated Multi-bit barcoded micro particles verify the flexibility of our system.

A high-throughput optomechanical retrieval method for sequence-verified clonal DNA from the NGS platform.

Writing DNA plays a significant role in the fields of synthetic biology, functional genomics and bioengineering. DNA clones on next-generation sequencing (NGS) platforms have the potential to be a rich and cost-effective source of sequence-verified DNAs as a precursor for DNA writing. However, it is still very challenging to retrieve target clonal DNA from high-density NGS platforms. Here we propose an enabling technology called 'Sniper Cloning' that enables the precise mapping of target clone features on NGS platforms and non-contact rapid retrieval of targets for the full utilization of DNA clones. By merging the three cutting-edge technologies of NGS, DNA microarray and our pulse laser retrieval system, Sniper Cloning is a week-long process that produces 5,188 error-free synthetic DNAs in a single run of NGS with a single microarray DNA pool. We believe that this technology has potential as a universal tool for DNA writing in biological sciences.

Maxillary yaw as the primary predictor of maxillary dental midline deviation: 3D analysis using cone-beam computed tomography.

PURPOSE: The purpose of this study was to investigate which factors affect upper dental midline deviation in dentofacial deformity patients using cone-beam computed tomography analysis. PATIENTS AND METHODS: Twenty-eight patients were selected for this study. Subjects were divided into 2 groups according to the amount of upper incisor (U1) midline deviation from the clinical facial midline: group 1 (U1 deviation <2 mm) and group 2 (U1 deviation >2 mm). Linear measurements, angles, and reference planes on 3-dimensional (3D) computed tomograms were obtained. The predictor variables were maxillary yaw, palatal plane angle, differences of maxillary point to the coronal and sagittal planes, and maxillary canting. The outcome variable was U1 deviation. The variables between the 2 groups and 2 sides were analyzed with a t test. Pearson correlation coefficient and multiple regression analysis were also calculated within each group for each measurement against the U1 deviation to determine which variables affect U1 deviation. P < .05 was considered statistically significant. RESULTS: The patients were evenly distributed between each group (n = 14 in each group). There was significant deviation of U1 from the sagittal plane in group 2 compared with group 1 (0.99 mm in group 1 vs 1.73 mm in group 2, P < .05). When we compared yaw with the sagittal plane, group 2 was more rotated than group 1 (1.16° in group 1 vs 2.28° in group 2, P < .01). Through multiple regression analysis, the primary predictor variable for U1 deviation was maxillary yaw (P < .05). CONCLUSIONS: This study suggests that maxillary yaw is the primary contributing factor for upper dental midline deviation. The use of maxillary yaw should be considered when one is performing orthognathic surgery in patients with U1 deviation to achieve optimum esthetics.

Evaluation of Cardiac Function by Transthoracic Echocardiography in Subjects with ST-Segment Elevation Myocardial Infarction Following Primary Percutaneous Coronary Intervention according to Valsartan Dose: The Valsartan One Center Trial.

BACKGROUND: The aim of this study was to evaluate the mid-term changes in cardiac function by transthoracic echocardiogram (TTE) in patients with ST-segment elevation myocardial infarction (STEMI) undergoing primary percutaneous coronary intervention (PCI) according to valsartan dose. METHODS: Between April 2006 and February 2009, 78 subjects (mean age: 57 ± 12 years, M : F = 74 : 4) with STEMI who underwent primary PCI were enrolled. Fifty three patients received low dose valsartan (40 or 80 mg) and 25 patients received high dose valsartan (160 or 320 mg). Follow-up TTE was done approximately 2 years later. We evaluated the changes in left ventricular (LV) function between initial and final TTE after primary PCI and compared the changes between low and high dose valsartan group. RESULTS: The mean follow-up TTE duration was 24 ± 8 months. Deceleration time (188.6 ± 56.3 msec vs. 221.5 ± 71.3 msec, p = 0.01), E/e' (12.24 ± 5.2 vs. 10.1 ± 4.9, p = 0.002), ejection fraction (52.7 ± 8% vs. 55.2 ± 8.4%, p < 0.01), and wall motion score index (1.45 ± 0.30 vs. 1.33 ± 0.32, p < 0.01) showed significant changes during the follow-up period. Wall motion improvement in injured myocardial segments was more frequently observed in the high-dose valsartan group compared to the low-dose group [18/25 (72%) vs. 24/53 (43.7%), p = 0.03]. There was no significant difference in the changes in cardiac dimensions and function between the low and high dose valsartan group. CONCLUSION: In patients with STEMI who undergoing primary PCI, high-dose valsartan treatment may be more helpful than low-dose in improving wall motion in the injured myocardium.