Automated estimation of cancer cell deformability with machine learning and acoustic trapping.

Cell deformability is a useful feature for diagnosing various diseases (e.g., the invasiveness of cancer cells). Existing methods commonly inflict pressure on cells and observe changes in cell areas, diameters, or thickness according to the degree of pressure. Then, the Young's moduli (i.e., a measure of deformability) of cells are estimated based on the assumption that the degrees of the changes are inversely proportional to Young's moduli. However, manual measurements of the physical changes in cells are labor-intensive, and the subjectivity of the operators can intervene during this step, thereby causing considerable uncertainty. Further, because the shapes of cells are nonuniform, we cannot ensure the assumption for linear correlations of physical changes in cells with their deformability. Therefore, this study aims at measuring non-linear elastic moduli of live cells (degrees of cell deformability) automatically by employing conventional neural networks (CNN) and multilayer perceptrons (MLP) while preserving (or enhancing) the accuracy of the manual methods. First, we obtain photomicrographs of cells on multiple pressure levels using single-beam acoustic tweezers, and then, we suggest an image preprocessing method for emphasizing changes in cell areas on the photomicrographs. The CNN model is trained to measure the ratios of the cell area change at each pressure level. Then, we apply the multilayer perceptron (MLP) to learn the correlations of the cell area change ratios according to the pressure levels with cell deformability. The accuracy of the CNN was evaluated using two types of breast cancer cells: MDA-MB-231 (invasive) and MCF-7 (noninvasive). The MLP was assessed using five different beads (Young's moduli from 0.214 to 9.235 kPa), which provides standardized reference data of the non-linear elastic moduli of live cells. Finally, we validated the practicality of the proposed system by examining whether the non-linear elastic moduli estimated by the proposed system can distinguish invasive breast cancer cells from noninvasive ones.

Classification of Breast Cancer Cells Using the Integration of High-Frequency Single-Beam Acoustic Tweezers and Convolutional Neural Networks.

Single-beam acoustic tweezers (SBAT) is a widely used trapping technique to manipulate microscopic particles or cells. Recently, the characterization of a single cancer cell using high-frequency (>30 MHz) SBAT has been reported to determine its invasiveness and metastatic potential. Investigation of cell elasticity and invasiveness is based on the deformability of cells under SBAT's radiation forces, and in general, more physically deformed cells exhibit higher levels of invasiveness and therefore higher metastatic potential. However, previous imaging analysis to determine substantial differences in cell deformation, where the SBAT is turned ON or OFF, relies on the subjective observation that may vary and requires follow-up evaluations from experts. In this study, we propose an automatic and reliable cancer cell classification method based on SBAT and a convolutional neural network (CNN), which provides objective and accurate quantitative measurement results. We used a custom-designed 50 MHz SBAT transducer to obtain a series of images of deformed human breast cancer cells. CNN-based classification methods with data augmentation applied to collected images determined and validated the metastatic potential of cancer cells. As a result, with the selected optimizers, precision, and recall of the model were found to be greater than 0.95, which highly validates the classification performance of our integrated method. CNN-guided cancer cell deformation analysis using SBAT may be a promising alternative to current histological image analysis, and this pretrained model will significantly reduce the evaluation time for a larger population of cells.

Learning Hierarchical Representations of Stories by Using Multi-layered Structures in Narrative Multimedia.

Narrative works (e.g., novels and movies) consist of various utterances (e.g., scenes and episodes) with multi-layered structures. However, the existing studies aimed to embed only stories in a narrative work. By covering other granularity levels, we can easily compare narrative utterances that are coarser (e.g., movie series) or finer (e.g., scenes) than a narrative work. We apply the multi-layered structures on learning hierarchical representations of the narrative utterances. To represent coarser utterances, we consider adjacency and appearance of finer utterances in the coarser ones. For the movies, we suppose a four-layered structure (character roles ∈ characters ∈ scenes ∈ movies) and propose three learning methods bridging the layers: Char2Vec, Scene2Vec, and Hierarchical Story2Vec. Char2Vec represents a character by using dynamic changes in the character's roles. To find the character roles, we use substructures of character networks (i.e., dynamic social networks of characters). A scene describes an event. Interactions between characters in the scene are designed to describe the event. Scene2Vec learns representations of a scene from interactions between characters in the scene. A story is a series of events. Meanings of the story are affected by order of the events as well as their content. Hierarchical Story2Vec uses sequential order of scenes to represent stories. The proposed model has been evaluated by estimating the similarity between narrative utterances in real movies.

An 8-week scapular stabilization exercise program in an elite archer with scapular dyskinesis presenting joint noise: A case report with one-year follow-up.

This case report demonstrates the effects of a scapular stabilization exercise program for managing the clinical symptoms of scapular dyskinesis in an archer presenting joint noise during shoulder movement. A 31-year-old man with a 20-year career in archery who complained of scapular dyskinesis and joint noise during shoulder movement was referred for proper management. The player performed the scapular stabilization exercise program, with an emphasis on strengthening scapular stabilizers and neutralization of scapular position, for 40 min, three times per week for 8 weeks (a total of 24 sessions). Measurements included the extent of scapular deviation, strength of scapular stabilizers, emotional burden related to joint noise, and upper limb disability. These measurements were taken before and after the intervention and during 1-year follow-up. After the 8-week intervention, scapular position improved by a range of 3.75-12.88% and muscle strength improved by a range of 8.69-28.60%. Further, emotional burden and upper limb disability improved by 56.86% and 91.67%, respectively. A 1-year follow-up showed that these improvements had been favorably maintained. These findings indicate that shoulder stabilization exercise may be helpful for alleviating scapular and shoulder problems related to archery performance.

A cloning and expression system to probe T-cell receptor specificity and assess functional avidity to neoantigens.

Recent studies have highlighted the promise of targeting tumor neoantigens to generate potent antitumor immune responses and provide strong motivation for improving our understanding of antigen-T-cell receptor (TCR) interactions. Advances in single-cell sequencing technologies have opened the door for detailed investigation of the TCR repertoire, providing paired information from TCRα and TCRß, which together determine specificity. However, a need remains for efficient methods to assess the specificity of discovered TCRs. We developed a streamlined approach for matching TCR sequences with cognate antigen through on-demand cloning and expression of TCRs and screening against candidate antigens. Here, we first demonstrate the system's capacity to identify viral-antigen-specific TCRs and compare the functional avidity of TCRs specific for a given antigen target. We then apply this system to identify neoantigen-specific TCR sequences from patients with melanoma treated with personalized neoantigen vaccines and characterize functional avidity of neoantigen-specific TCRs. Furthermore, we use a neoantigen-prediction pipeline to show that an insertion-deletion mutation in a putative chronic lymphocytic leukemia (CLL) driver gives rise to an immunogenic neoantigen mut-MGA, and use this approach to identify the mut-MGA-specific TCR sequence. This approach provides a means to identify and express TCRs, and then rapidly assess antigen specificity and functional avidity of a reconstructed TCR, which can be applied for monitoring antigen-specific T-cell responses, and potentially for guiding the design of effective T-cell-based immunotherapies.

Alteration of immunoproteome profile of Echinococcus granulosus hydatid fluid with progression of cystic echinococcosis.

BACKGROUND: Cystic echinococcosis (CE), caused by Echinococcus granulosus metacestode, invokes a serious public health concern. Early diagnosis has great impacts on reduction of disability-adjusted life years. Several antigen B-related molecules (EgAgB; EgAgB1-5) are known to be immunopotent, but detection of EgAgB is variable in many patients and may not allow reliable interpretation of its immunological relevance. More importantly, the immunoproteome profile of hydatid fluid (HF) has not been addressed. METHODS: We conducted a proteome analysis of the HF of a single fertile cyst of CE1 and CE2 stages through two-dimensional electrophoresis (2-DE). Each protein spot was analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). We subsequently determined the immunoproteome profile employing patient sera of entire disease spectrum from CE1 to CE5 stages. RESULTS: We identified 40 parasite proteins, of which EgAgB (28 spots) and antigen 5 (EgAg5; 5 molecules) were abundant. EgAgB proteoforms constituted the majority, mostly EgAgB1 (24 spots), followed by EgAgB2 and EgAgB4 (2 spots each). EgAgB3 was detected only by liquid chromatography-MS/MS. EgAgB5 was not recognized. We also detected 38 host proteins, which were largely composed of serum components, antioxidant/xenobiotic enzymes, and enzymes involved in carbohydrate metabolism. CE1 and CE2 HF exhibited comparable spotting patterns, but CE2 HF harbored greater amounts of EgAgB and EgAg5 complexes. CE sera demonstrated complicated immune recognition patterns according to the disease progression; CE2 and CE3 stages exhibited strong antibody responses against diverse EgAgB and EgAg5 proteoforms, while CE1, CE4, and CE5 stages mainly reacted to EgAg5 and cathepsin B. Patient sera of alveolar echinococcosis (AE) cross-reacted with diverse EgAgB isoforms (36%). EgAg5 and cathepsin B also demonstrated cross-reactions with sera from neurocysticercosis and sparganosis. CONCLUSIONS: Our results demonstrated that detection of a single defined molecule may not properly diagnose CE, since specific immunodominant epitopes changed as the disease progresses. Immunoproteome analysis combined with imaging studies may be practical in the differential diagnosis of CE from AE and other cystic lesions, as well as for staging CE, which are pertinent to establish appropriate patient management.

Expression characteristics and specific antibody reactivity of diverse cathepsin F members of Paragonimus westermani.

Paragonimiasis, caused by the lung fluke Paragonimus, is a major food-borne helminthic disease. Differential diagnosis of paragonimiasis from tuberculosis and other infectious granulomas in the lung is a prerequisite to proper management of patients. Cysteine proteases of Paragonimus westermani (PwCPs) invoke specific antibody responses against patient sera, while antibody capturing activity of different PwCPs has not been comparatively analyzed. In this study, we observed the expressional regulation of 11 species of different PwCPs (PwCP1-11). We expressed recombinant PwCPs and assessed diagnostic reliability employing sera from patients with P. westermani (n=138), other trematodiases (n=80), cestodiases (n=60) and pulmonary tuberculosis (n=20), and those of normal controls (n=20). PwCPs formed a monophyletic clade into cathepsin F and showed differential expression patterns along with developmental stages of worm. Bacterially expressed recombinant PwCPs (rPwCPs) exhibited variable sensitivity of 38.4-84.5% and specificity of 87.2-100% in diagnosing homologous infection. rPwCPs recognized specific antibodies of experimental cat sera as early as 3 or 6weeks after infection. Patient sera of fascioliasis, Schistosomiasis japonicum and clonorchiasis demonstrated weak cross-reactions. Our results demonstrate that diverse PwCPs of the cathepsin F family participate in inducing specific antibody responses. Most P. westermani cathepsin F, except for PwCP2 (AAF21461), which showed negligible antibody responses, might be applicable for paragonimiasis serodiagnosis.

Taenia solium metacestode fasciclin-like protein is reactive with sera of chronic neurocysticercosis.

OBJECTIVES: Neurocysticercosis (NC), an infection of the central nervous system with Taenia solium metacestodes (TsM), invokes a formidable neurological disease. A bundle of antigens is applicable for serodiagnosis of active cases, while they demonstrate fairly low reactivity against sera of chronic NC. Identification of sensitive biomarkers for chronic NC is critical for appropriate management of patients. METHODS: Proteome analysis revealed several isoforms of 65- and 83-kDa TsM fasciclin-like proteins (TsMFas) to be highly reactive with sera of chronic NC. A cDNA encoding one of the 83-kDa TsMFas (TsMFas1) was isolated from a cDNA library. We expressed a recombinant protein (rTsMFas1) and evaluated its diagnostic potential employing sera from chronic NC (n = 80), tissue-invasive cestodiases (n = 169) and trematodiases (n = 80) and those of normal controls (n = 50). RESULTS: Secretory TsMFas1 was composed of 766 amino acid polypeptide and harboured fasciclin and fasciclin-superfamily domains. The protein was constitutively expressed in metacestode and adult stages, with preferential locality in the scolex. Bacterially expressed rTsMFas1 exhibited 78.8% sensitivity (63/80 cases) and 93% specificity (278/299 samples) in diagnosing chronic NC. Some cross-reactivity was observed with sera of cystic echinococcosis (10/56, 17.8%) and sparganosis (4/50, 8%). Positive and negative predictive values were 75% and 95.5%, respectively. CONCLUSION: TsM fasciclin-like protein may be useful for differential diagnosis of chronic NC in clinical settings, especially where both NC and other infectious cerebral granulomatoses are prevalent.

EGFR T790M mutation as a possible target for immunotherapy; identification of HLA-A*0201-restricted T cell epitopes derived from the EGFR T790M mutation.

Treatment with epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), such as gefitinib and erlotinib, has achieved high clinical response rates in patients with non-small cell lung cancers (NSCLCs). However, over time, most tumors develop acquired resistance to EGFR-TKIs, which is associated with the secondary EGFR T790M resistance mutation in about half the cases. Currently there are no effective treatment options for patients with this resistance mutation. Here we identified two novel HLA-A*0201 (A2)-restricted T cell epitopes containing the mutated methionine residue of the EGFR T790M mutation, T790M-5 (MQLMPFGCLL) and T790M-7 (LIMQLMPFGCL), as potential targets for EGFR-TKI-resistant patients. When peripheral blood cells were repeatedly stimulated in vitro with these two peptides and assessed by antigen-specific IFN-γ secretion, T cell lines responsive to T790M-5 and T790M-7 were established in 5 of 6 (83%) and 3 of 6 (50%) healthy donors, respectively. Additionally, the T790M-5- and T790M-7-specific T cell lines displayed an MHC class I-restricted reactivity against NSCLC cell lines expressing both HLA-A2 and the T790M mutation. Interestingly, the NSCLC patients with antigen-specific T cell responses to these epitopes showed a significantly less frequency of EGFR-T790M mutation than those without them [1 of 7 (14%) vs 9 of 15 (60%); chi-squared test, p  =  0.0449], indicating the negative correlation between the immune responses to the EGFR-T790M-derived epitopes and the presence of EGFR-T790M mutation in NSCLC patients. This finding could possibly be explained by the hypothesis that immune responses to the mutated neo-antigens derived from T790M might prevent the emergence of tumor cell variants with the T790M resistance mutation in NSCLC patients during EGFR-TKI treatment. Together, our results suggest that the identified T cell epitopes might provide a novel immunotherapeutic approach for prevention and/or treatment of EGFR-TKI resistance with the secondary EGFR T790M resistance mutation in NSCLC patients.

5-O-(4-[125 I]Iodobenzyl)-L-ascorbic acid: electrophilic radioiodination and biodistribution in mice.

As a part of our efforts to develop potential imaging agents for ascorbate bioactivity, 5-O-(4-[(125)I]iodobenzyl)-L-ascorbic acid ([(125)I]1) was prepared through a two-step sequence which involved radioiodo-destannylation of a protected tributylstannyl precursor 6, followed by hydrolysis in acidic methanol of the protecting groups in 61% overall radiochemical yield, with a radiochemical purity of over 98% and a specific activity of more than 15.4 GBq/µmol. Tissue distribution of [(125)I]1 in tumor-bearing mice showed signs of distribution profiles similar to the reported results for 6-deoxy-6-[(18)F]fluoro-L-ascorbic (6-(18)FAsA) acid and 6-deoxy-6-[(131)I]iodo-L-ascorbic acid (6-(131)IAsA) but with notable differences in the adrenal glands, in which considerably lower uptake of radioactivity and rapid clearance with time were observed. Pretreatment of mice with a known inhibitor of ascorbate transport, sulfinpyrazone, did not produce any significant change in the adrenal uptake of radioactivity after injection of [(125)I]1 compared to the control, suggesting that uptake in the adrenal glands is independent of the sodium-dependent vitamin C transporter 2 transport mechanism. Introduction of a bulky substituent at C-5 on AsA, such as an iodobenzyloxy group, may not be suitable for the design of analogs that may still be able to maintain characteristic distribution properties in vivo seen with AsA itself.

6-Deoxy-6-[131I]iodo-L-ascorbic acid for the in vivo study of ascorbate: autoradiography, biodistribution in normal and hypolipidemic rats, and in tumor-bearing nude mice.

Normal female rat distribution studies showed high and specific uptake of 6-deoxy-6-[(131)I]iodo-L-ascorbic acid (6-(131)IAsA) into the adrenal glands, known to highly express the ascorbate sodium-dependent vitamin C transporter-2 (SVCT-2), and the adrenal gland was clearly visualized by whole-body autoradiography. Preinjection of sulfinpyrazone, a known blocker of ascorbate transport, with 6-(131)IAsA resulted in decreased uptake of radioactivity in rat adrenal glands compared to the control group, seemingly illustrating the participation of the SVCT transporter (probably the SVCT-2 subtype) in the uptake process in vivo. 4-Aminopyrazolo[3,4-d]pyrimidine-induced hypolipidemic rats showed a 1.7-fold increase in adrenal uptake of radioactivity at 30 min postinjection of 6-(131)IAsA, compared to the control, with increased adrenal-to-liver and adrenal-to-kidney ratios. To further characterize 6-(131)IAsA for its tumor uptake properties, biodistribution studies were also performed using male nude mice implanted with either Y-1 adrenocortical tumor cells or adrenal medulla-derived PC12 cells. None of these tumors exhibited relevant uptake of 6-(131)IAsA while normal adrenal glands showed high uptake of radioactivity, suggesting that these tumors in this model have only a poor transport capacity for this agent. The present study demonstrates that the use of radioiodinated 6-IAsA may help to obtain information about functional alterations in diseased adrenal glands, but it does not exhibit desirable properties as a tumor-seeking agent for ascorbic acid bioactivity.

The potent protective effect of wild ginseng (Panax ginseng C.A. Meyer) against benzo[alpha]pyrene-induced toxicity through metabolic regulation of CYP1A1 and GSTs.

Wild Panax ginseng C.A. Meyer (WG) is a well-known medicinal herb. In this study, the protective effects of a water extract from the root of WG on benzo[alpha]pyrene (BP)-induced hepatotoxicity and the mechanism of these effects were investigated for the first time. The effects of WG on liver toxicities induced by BP were assessed by blood biochemical and histopathological analyses. BP caused severe liver injury in rats, as indicated by elevated plasma ALT, AST and LPO levels. Pretreatment with WG for 4 weeks completely abrogated increases in the ALT, AST and LPO levels when challenged with BP. Reductions in GSH content and GST activity by BP were reversed by WG. These protective effects of WG against BP-induced toxicity were consistent with the results of histopathological examinations. We next examined the effects of WG on the gene expression of the enzymes that metabolize BP in H4IIE cells. CYP1A1 mRNA and protein expression were increased by BP. WG moderately inhibited BP-induced CYP1A1 gene expression. Moreover, GSTA2, GSTA3 and GSTM2 gene expressions were significantly increased by WG through the Nrf2/antioxidant responsive element pathway for enzyme induction. In summary, WG is efficacious in protecting against BP-induced hepatotoxicity as results of metabolic regulations through both the inhibition of metabolic enzyme activation and the enhancement of electrophilic detoxification, implying that WG should be considered a potential chemopreventive agent.