Extracellular vesicle proteome unveils cathepsin B connection to Alzheimer's disease pathogenesis.

Extracellular vesicles (EVs) are membrane vesicles that are released extracellularly and considered to be implicated in the pathogenesis of neurodegenerative diseases including Alzheimer's disease. Here, CSF EVs of 16 ATN-classified cases were subjected to quantitative proteome analysis. In these CSF EVs, levels of 11 proteins were significantly altered during the ATN stage transitions (P < 0.05 and fold-change > 2.0). These proteins were thought to be associated with Alzheimer's disease pathogenesis and represent candidate biomarkers for pathogenic stage classification. Enzyme-linked immunosorbent assay analysis of CSF and plasma EVs revealed altered levels of cathepsin B (CatB) during the ATN transition (seven ATN groups in validation set, n = 136). The CSF and plasma EV CatB levels showed a negative correlation with CSF amyloid-ß42 concentrations. This proteomic landscape of CSF EVs in ATN classifications can depict the molecular framework of Alzheimer's disease progression, and CatB may be considered a promising candidate biomarker and therapeutic target in Alzheimer's disease amyloid pathology.

Succinyl-CoA-based energy metabolism dysfunction in chronic heart failure.

Heart failure (HF) is a leading cause of death and repeated hospitalizations and often involves cardiac mitochondrial dysfunction. However, the underlying mechanisms largely remain elusive. Here, using a mouse model in which myocardial infarction (MI) was induced by coronary artery ligation, we show the metabolic basis of mitochondrial dysfunction in chronic HF. Four weeks after ligation, MI mice showed a significant decrease in myocardial succinyl-CoA levels, and this decrease impaired the mitochondrial oxidative phosphorylation (OXPHOS) capacity. Heme synthesis and ketolysis, and protein levels of several enzymes consuming succinyl-CoA in these events, were increased in MI mice, while enzymes synthesizing succinyl-CoA from α-ketoglutarate and glutamate were also increased. Furthermore, the ADP-specific subunit of succinyl-CoA synthase was reduced, while its GDP-specific subunit was almost unchanged. Administration of 5-aminolevulinic acid, an intermediate in the pathway from succinyl-CoA to heme synthesis, appreciably restored succinyl-CoA levels and OXPHOS capacity and prevented HF progression in MI mice. Previous reports also suggested the presence of succinyl-CoA metabolism abnormalities in cardiac muscles of HF patients. Our results identified that changes in succinyl-CoA usage in different metabolisms of the mitochondrial energy production system is characteristic to chronic HF, and although similar alterations are known to occur in healthy conditions, such as during strenuous exercise, they may often occur irreversibly in chronic HF leading to a decrease in succinyl-CoA. Consequently, nutritional interventions compensating the succinyl-CoA consumption are expected to be promising strategies to treat HF.

Brain-specific glycosylation enzyme GnT-IX maintains levels of protein tyrosine phosphatase receptor PTPRZ, thereby mediating glioma growth.

Gliomas are the most prevalent primary tumor of the central nervous system. Despite advances in imaging technologies, neurosurgical techniques, and radiotherapy, a cure for high-grade glioma remains elusive. Several groups have reported that protein tyrosine phosphatase receptor type Z (PTPRZ) is highly expressed in glioblastoma, and that targeting PTPRZ attenuates tumor growth in mice. PTPRZ is modified with diverse glycan, including the PTPRZ-unique human natural killer-1 capped O-mannosyl core M2 glycans. However, the regulation and function of these unique glycans are unclear. Using CRISPR genome-editing technology, we first demonstrated that disruption of the PTPRZ gene in human glioma LN-229 cells resulted in profoundly reduced tumor growth in xenografted mice, confirming the potential of PTPRZ as a therapeutic target for glioma. Furthermore, multiple glycan analyses revealed that PTPRZ derived from glioma patients and from xenografted glioma expressed abundant levels of human natural killer-1-capped O-Man glycans via extrinsic signals. Finally, since deficiency of O-Man core M2 branching enzyme N-acetylglucosaminyltransferase IX (GnT-IX) was reported to reduce PTPRZ protein levels, we disrupted the GnT-IX gene in LN-229 cells and found a significant reduction of glioma growth both in vitro and in the xenograft model. These results suggest that the PTPR glycosylation enzyme GnT-IX may represent a promising therapeutic target for glioma.

Emerging potential of immunopeptidomics by mass spectrometry in cancer immunotherapy.

With significant advances in analytical technologies, research in the field of cancer immunotherapy, such as adoptive T cell therapy, cancer vaccine, and immune checkpoint blockade (ICB), is currently gaining tremendous momentum. Since the efficacy of cancer immunotherapy is recognized only by a minority of patients, more potent tumor-specific antigens (TSAs, also known as neoantigens) and predictive markers for treatment response are of great interest. In cancer immunity, immunopeptides, presented by human leukocyte antigen (HLA) class I, play a role as initiating mediators of immunogenicity. The latest advancement in the interdisciplinary multiomics approach has rapidly enlightened us about the identity of the "dark matter" of cancer and the associated immunopeptides. In this field, mass spectrometry (MS) is a viable option to select because of the naturally processed and actually presented TSA candidates in order to grasp the whole picture of the immunopeptidome. In the past few years the search space has been enlarged by the multiomics approach, the sensitivity of mass spectrometers has been improved, and deep/machine-learning-supported peptide search algorithms have taken immunopeptidomics to the next level. In this review, along with the introduction of key technical advancements in immunopeptidomics, the potential and further directions of immunopeptidomics will be reviewed from the perspective of cancer immunotherapy.

Pks-positive Escherichia coli in tumor tissue and surrounding normal mucosal tissue of colorectal cancer patients.

A significant association exists between the gut microbiome and colorectal carcinogenesis, as well as cancer progression. It has been reported that Escherichia coli (E. coli) containing polyketide synthetase (pks) island contribute to colorectal carcinogenesis by producing colibactin, a polyketide-peptide genotoxin. However, the functions of pks+ E. coli in initiation, proliferation, and metastasis of colorectal cancer (CRC) remain unclear. We investigated the clinical significance of pks+ E. coli to clarify its functions in CRC. This study included 413 patients with CRC. Pks+ E. coli of tumor tissue and normal mucosal tissue were quantified using droplet digital PCR. Pks+ E. coli was more abundant in Stages 0-I tumor tissue than in normal mucosal tissue or in Stages II-IV tumor tissue. High abundance of pks+ E. coli in tumor tissue was significantly associated with shallower tumor depth (hazard ratio [HR] = 5.0, 95% confidence interval [CI] = 2.3-11.3, p < 0.001) and absence of lymph node metastasis (HR = 3.0, 95% CI = 1.8-5.1, p < 0.001) in multivariable logistic analyses. Pks+ E. coli-low and -negative groups were significantly associated with shorter CRC-specific survival (HR = 6.4, 95% CI = 1.7-25.6, p = 0.005) and shorter relapse-free survival (HR = 3.1, 95% CI = 1.3-7.3, p = 0.01) compared to the pks+ E. coli-high group. Pks+ E. coli was abundant in Stages 0-I CRC and associated with CRC prognosis. These results suggest that pks+ E. coli might contribute to carcinogenesis of CRC but might not be associated with tumor progression.

Pericentromeric noncoding RNA changes DNA binding of CTCF and inflammatory gene expression in senescence and cancer.

Cellular senescence causes a dramatic alteration of chromatin organization and changes the gene expression profile of proinflammatory factors, thereby contributing to various age-related pathologies through the senescence-associated secretory phenotype (SASP). Chromatin organization and global gene expression are maintained by the CCCTC-binding factor (CTCF); however, the molecular mechanism underlying CTCF regulation and its association with SASP gene expression remains unclear. We discovered that noncoding RNA (ncRNA) derived from normally silenced pericentromeric repetitive sequences directly impairs the DNA binding of CTCF. This CTCF disturbance increases the accessibility of chromatin and activates the transcription of SASP-like inflammatory genes, promoting malignant transformation. Notably, pericentromeric ncRNA was transferred into surrounding cells via small extracellular vesicles acting as a tumorigenic SASP factor. Because CTCF blocks the expression of pericentromeric ncRNA in young cells, the down-regulation of CTCF during cellular senescence triggers the up-regulation of this ncRNA and SASP-related inflammatory gene expression. In this study, we show that pericentromeric ncRNA provokes chromosomal alteration by inhibiting CTCF, leading to a SASP-like inflammatory response in a cell-autonomous and non-cell-autonomous manner and thus may contribute to the risk of tumorigenesis during aging.

Development of a phantom for assessing the precision of setup in skin mark-less surface-guided radiotherapy.

BACKGROUND: Surface-guided radiotherapy (SGRT) is adopted by several institutions; however, reports on the phantoms used to assess the precision of the SGRT setup are limited. PURPOSE: The purpose of this study was to develop a phantom to verify the accuracy of the irradiation position during skin mark-less SGRT. METHODS: An acrylonitrile butadiene styrene (ABS) plastic cube phantom with a diameter of 150 mm on each side containing a dummy target of 15 mm and two types of body surface-shaped phantoms (breast/face shape) that could be attached to the cube phantom were fabricated. Films can be inserted on four sides of the cubic phantom (left, right, anterior and posterior), and the center of radiation can be calculated by irradiating the dummy target with orthogonal MV beams. Three types of SGRT using a VOXELAN-HEV600M (Electronics Research&Development Corporation, Okayama, Japan) were evaluated using this phantom: (i) SGRTCT-a SGRT set-up based solely on a computed tomography (CT)-reference image. (ii) SGRTCT + CBCT-a method where cone beam computed tomography (CBCT) matching was performed after SGRTCT. (iii) SGRTScan-a resetup technique using a scan reference image obtained after completing the (ii) step. RESULTS: Both the breast and face phantoms were recognized in the SGRT system without problems. SGRTScan ensure precision within 1 mm/1° for breast and face verification, respectively. All SGRT methods showed comparable rotational accuracies with no significant disparities. CONCLUSIONS: The developed phantom was useful for verifying the accuracy of skin mark-less SGRT position matching. The SGRTScan demonstrated the feasibility of achieving skin-mark less SGRT with high accuracy, with deviations of less than 1 mm. Additional research is necessary to evaluate the suitability of the developed phantoms for use in various facilities and systems. This phantom could be used for postal surveys in the future.

Frontiers in mass spectrometry-based clinical proteomics for cancer diagnosis and treatment.

Numerous omics studies, primarily genomics analyses, have been conducted to fully understand the molecular biological characteristics of cancer. In recent years, the depth of proteomic analysis, which comprehensively analyzes proteins and molecules that function directly in vivo, has increased dramatically. Proteomics using mass spectrometry (MS) is a promising technology to directly examine proteoforms, including post-translational modifications and variants originating from genomic aberrations. Recent advances in MS-based proteomics have enabled direct, in depth, and quantitative analysis of the expression levels of various cancer-related proteins, as well as their cancer-specific proteoforms, and proteins that fluctuate with cancer initiation and progression in cell lines and tissue samples. Additionally, the integration of proteomic data with genomic, epigenomic, and transcriptomic data has formed the growing field of proteogenomics, which is already yielding new biological and diagnostic knowledge. Deep proteomic profiling provides clinically useful information in various aspects, including understanding the mechanisms of cancer development and progression and discovering targets for diagnosis and drug development. Furthermore, it is expected to make a significant contribution to the promotion of personalized medicine. In this review, recent advances and impacts in MS-based clinical proteomics are highlighted with a focus on oncology.

Identification of CD14 and lipopolysaccharide-binding protein as novel biomarkers for sarcoidosis using proteomics of serum extracellular vesicles.

Sarcoidosis is a complex, polygenic, inflammatory granulomatous multi-organ disease of unknown cause. The granulomatous inflammation in sarcoidosis is driven by the interplay between T cells and macrophages. Extracellular vesicles (EVs) play important roles in intercellular communication. We subjected serum EVs, isolated by size exclusion chromatography, from seven patients with sarcoidosis and five control subjects to non-targeted proteomics analysis. Non-targeted, label-free proteomics analysis detected 2292 proteins in serum EVs; 42 proteins were up-regulated in patients with sarcoidosis relative to control subjects; and 324 proteins were down-regulated. The protein signature of EVs from patients with sarcoidosis reflected disease characteristics such as antigen presentation and immunological disease. Candidate biomarkers were further verified by targeted proteomics analysis (selected reaction monitoring) in 46 patients and 10 control subjects. Notably, CD14 and lipopolysaccharide-binding protein (LBP) were validated by targeted proteomics analysis. Up-regulation of these proteins was further confirmed by immunoblotting, and their expression was strongly increased in macrophages of lung granulomatous lesions. Consistent with these findings, CD14 levels were increased in lipopolysaccharide-stimulated macrophages during multinucleation, concomitant with increased levels of CD14 and LBP in EVs. The area under the curve values of CD14 and LBP were 0.81 and 0.84, respectively, and further increased to 0.98 in combination with angiotensin-converting enzyme and soluble interleukin-2 receptor. These findings suggest that CD14 and LBP in serum EVs, which are associated with granulomatous pathogenesis, can improve the diagnostic accuracy in patients with sarcoidosis.

Escherichia coli-derived outer-membrane vesicles induce immune activation and progression of cirrhosis in mice and humans.

BACKGROUND AND AIMS: Decompensated cirrhosis with fibrosis progression causes portal hypertension followed by an oedematous intestinal tract. These conditions weaken the barrier function against bacteria in the intestinal tract, a condition called leaky gut, resulting in invasion by bacteria and bacterial components. Here, we investigated the role of outer-membrane vesicles (OMVs) of Escherichia coli, which is the representative pathogenic gut-derived bacteria in patients with cirrhosis in the pathogenesis of cirrhosis. METHODS: We investigated the involvement of OMVs in humans using human serum and ascites samples and also investigated the involvement of OMVs from E. coli in mice using mouse liver-derived cells and a mouse cirrhosis model. RESULTS: In vitro, OMVs induced inflammatory responses to macrophages and neutrophils, including the upregulation of C-type lectin domain family 4 member E (Clec4e), and induced the suppression of albumin production in hepatocytes but had a relatively little direct effect on hepatic stellate cells. In a mouse cirrhosis model, administration of OMVs led to increased liver inflammation, especially affecting the activation of macrophages, worsening fibrosis and decreasing albumin production. Albumin administration weakened these inflammatory changes. In addition, multiple antibodies against bacterial components were increased with a progressing Child-Pugh grade, and OMVs were detected in ascites of patients with decompensated cirrhosis. CONCLUSIONS: In conclusion, OMVs induce inflammation, fibrosis and suppression of albumin production, affecting the pathogenesis of cirrhosis. We believe that our study paves the way for the future prevention and treatment of cirrhosis.

Protein profiling of extracellular vesicles from iPSC-derived astrocytes of patients with ALS/PDC in Kii peninsula.

BACKGROUND: Amyotrophic lateral sclerosis/Parkinsonism-dementia complex in Kii peninsula, Japan (Kii ALS/PDC), is an endemic neurodegenerative disease whose causes and pathogenesis remain unknown. However, astrocytes in autopsied cases of Kii ALS/PDC show characteristic lesions. In addition, relationships between extracellular vesicles (EVs) and neurodegenerative diseases are increasingly apparent. Therefore, we focused on proteins in EVs derived from Kii ALS/PDC astrocytes in the present study. METHODS: Induced pluripotent stem cells (iPSCs) derived from three healthy controls (HCs) and three patients with Kii ALS/PDC were differentiated into astrocytes. EVs in the culture medium of astrocytes were collected and subjected to quantitative proteome analysis. RESULTS: Our proteome analysis reveals that EV-containing proteins derived from astrocytes of patients with Kii ALS/PDC show distinctive patterns compared with those of HCs. Moreover, EVs derived from Kii ALS/PDC astrocytes display increased proteins related to proteostasis and decreased proteins related to anti-inflammation. DISCUSSION: Proteins contained in EVs from astrocytes unveil protective support to neurons and may reflect the molecular pathomechanism of Kii ALS/PDC; accordingly, they may be potential biomarker candidates of Kii ALS/PDC.

Changes in the preoperative ocular surface flora with an increase in patient age: A surveillance analysis of bacterial diversity and resistance to fluoroquinolone.

PURPOSE: This study analyzed the relationship between patient age and the prevalence and fluoroquinolone susceptibility of gram-positive cocci from the ocular surface flora before ophthalmic surgery. METHODS: This surveillance study included scraped samples from the conjunctival sac of 8923 eyes of 5490 patients (70.0 ± 13.7 years) without ocular infection before ophthalmologic surgery between August 2018 and December 2020. A review of microbiological records regarding patient age was used to determine the number of isolates and gram-positive species obtained, as well as their fluoroquinolone susceptibility. Fluoroquinolone susceptibility was determined using the Clinical and Laboratory Standards Institute protocols of broth microdilution. Statistical analysis was performed using a generalized additive model and a log-linear model. RESULTS: In total, 9,894 bacterial isolates obtained from scraped samples from the patients were analyzed. The detected species were Staphylococcus epidermidis (31.0%), Staphylococcus aureus (6.1%), Staphylococcus lugdunensis (3.9%), Enterococcus faecalis (5.8%), Corynebacterium species (31.7%), and Cutibacterium acnes (7.5%) and others. The number of species isolated from the ocular surface was increased at the rate of 1.018 per 10 years of age (p < 0.0001). S. epidermidis, S. lugdunensis, E. faecalis, and Corynebacterium species were isolated more often with an increase in patient age. The levofloxacin resistance ratio of methicillin-sensitive S. epidermidis and Corynebacterium species increased at the rate of 1.204 and 1.087 respectively with a 10-year increase in age (both p < 0.0001). CONCLUSION: Gram-positive bacteria in the ocular surface flora (OSF) exhibited gradual changes in diversity and fluoroquinolone resistance with an increase in patient age. It is important to monitor the OSF of the patients before ophthalmologic surgery to prevent refractory ocular postoperative infection.

The T-CEA score: a useful prognostic indicator based on postoperative CEA and pathological T4 levels for patients with stage II-III colorectal cancer.

PURPOSE: To investigate a prognostic score for stage II-III colorectal cancer (CRC) based on post-CEA and pT4 levels. METHODS: Two cohorts of stage II-III CRC patients who underwent curative surgery between 2011 and 2017 were included. The prognostic score (T-CEA score) was calculated as follows: T-CEA-0, post-CEA ≤ 5 ng/mL and pT1-3; T-CEA-1, post-CEA > 5 ng/mL or pT4; T-CEA-2, post-CEA > 5 ng/mL and pT4. RESULTS: The T-CEA scores of the 587 patients were as follows: T-CEA-0 (n = 436; 74%), T-CEA-1 (n = 129; 22%), and T-CEA-2 (n = 10; 2%). The 5-year recurrence-free survival (RFS) rates of the T-CEA-0, 1, and 2 groups were 80.3%, 54.8%, and 0%, respectively (P < 0.01), and the 5-year overall survival (OS) rates were 90.9%, 74.2%, and 0%, respectively (T-CEA-0 vs T-CEA-1: P < 0.01, T-CEA-1 vs T-CEA-2: P = 0.04). Multivariate analysis revealed that an elevated T-CEA score of 1 or 2 was a significant risk factor for poor RFS (HR: 2.89, P < 0.01) and OS (HR: 2.85, P < 0.01). CONCLUSION: The T-CEA score is a reliable and convenient prognostic score for stage II-III CRC.

Development of a dynamic motion platform with two independent drive systems for radiotherapy.

BACKGROUND: There are some motion platforms for radiotherapy quality assurance. However, no platform with two drive systems that can move along three axes is available. PURPOSE: The purpose of this study is to develop a dynamic motion platform with two drive systems capable of three-axis motion and to evaluate its motion performance. METHODS: The developed moving platform had two drive systems that use the same equipment. Each axis of the platform used can support a maximum load of 10 kg. The motors for moving the platform in each direction are capable of a drive stroke up to 40 mm. The drive speed is 30 mm/s at maximum load fluctuation. To evaluate the static positional accuracy of this system with an arbitrary input movement, the XYZ position of each axis was measured using a coordinate measuring machine operating from 0 to 40 mm at 10 mm intervals. In addition, the accuracy of dynamic motion was verified with Sine waveform inputs of different patterns to the three axes for approximately 60 s, and they were compared with the resulting detected signals by SyncTrax. RESULTS: The two drive systems were successfully operated on three axes by using independent control systems. For static position, the accuracies were within 0.2 mm, 0.05 mm, and 0.14 mm for lateral, longitudinal, and vertical directions, respectively. For dynamic motion, the mean absolute errors in the X, Y, and Z axes between the platform inputs and SyncTrax detected signals were 0.14 ± 0.10 mm, 0.16 ± 0.12 mm, and 0.16 ± 0.11 mm, respectively. CONCLUSIONS: A new dynamic platform for radiation therapy with two drive systems capable of three-axis motion was developed, and the positional accuracy of the drive axes was confirmed to be less than 0.2 mm.

Evaluation of computed tomography metal artifact and CyberKnife fiducial recognition for novel size fiducial markers.

PURPOSE: This study aimed to compare fiducial markers used in CyberKnife treatment in terms of metal artifact intensity observed in CT images and fiducial recognition in the CyberKnife system affected by patient body thickness and type of marker. METHODS: Five markers, ACCULOC 0.9 mm × 3 mm, Ball type Gold Anchor (GA) 0.28 mm × 10 mm, 0.28 mm × 20 mm, and novel size GA 0.4 mm × 10 mm, 0.4 mm × 20 mm were evaluated. To evaluate metal artifacts of CT images, two types of CT images of water-equivalent gels with each marker were acquired using Aquilion LB CT scanner, one applied SEMAR (SEMAR-on) and the other did not apply this technique (SEMAR-off). The evaluation metric of artifact intensity (MSD ) which represents a variation of CT values were compared for each marker. Next, 5, 15, and 20 cm thickness of Tough Water (TW) was placed on the gel under the condition of overlapping the vertebral phantom in the Target Locating System, and the live image of each marker was acquired to compare fiducial recognition. RESULTS: The mean MSD of SEMAR-off was 78.80, 74.50, 97.25, 83.29, and 149.64 HU for ACCULOC, GA0.28 mm × 10 mm, 20 mm, and 0.40 mm × 10 mm, 20 mm, respectively. In the same manner, that of SEMAR-on was 23.52, 20.26, 26.76, 24.89, and 33.96 HU, respectively. Fiducial recognition decreased in the order of 5, 15, and 20 cm thickness, and GA 0.4 × 20 mm showed the best recognition at thickness of 20 cm TW. CONCLUSIONS: We demonstrated the potential to reduce metal artifacts in the CT image to the same level for all the markers we evaluated by applying SEMAR. Additionally, the fiducial recognition of each marker may vary depending on the thickness of the patient's body. Particularly, we showed that GA 0.40 × 20 mm may have more optimal recognition for CyberKnife treatment in cases of high bodily thickness in comparison to the other markers.

YBX1 Regulates Satellite II RNA Loading into Small Extracellular Vesicles and Promotes the Senescent Phenotype.

Senescent cells secrete inflammatory proteins and small extracellular vesicles (sEVs), collectively termed senescence-associated secretory phenotype (SASP), and promote age-related diseases. Epigenetic alteration in senescent cells induces the expression of satellite II (SATII) RNA, non-coding RNA transcribed from pericentromeric repetitive sequences in the genome, leading to the expression of inflammatory SASP genes. SATII RNA is contained in sEVs and functions as an SASP factor in recipient cells. However, the molecular mechanism of SATII RNA loading into sEVs is unclear. In this study, we identified Y-box binding protein 1 (YBX1) as a carrier of SATII RNA via mass spectrometry analysis after RNA pull-down. sEVs containing SATII RNA induced cellular senescence and promoted the expression of inflammatory SASP genes in recipient cells. YBX1 knockdown significantly reduced SATII RNA levels in sEVs and inhibited the propagation of SASP in recipient cells. The analysis of the clinical dataset revealed that YBX1 expression is higher in cancer stroma than in normal stroma of breast and ovarian cancer tissues. Furthermore, high YBX1 expression was correlated with poor prognosis in breast and ovarian cancers. This study demonstrated that SATII RNA loading into sEVs is regulated via YBX1 and that YBX1 is a promising target in novel cancer therapy.

Identification of Novel Senescent Markers in Small Extracellular Vesicles.

Senescent cells exhibit several typical features, including the senescence-associated secretory phenotype (SASP), promoting the secretion of various inflammatory proteins and small extracellular vesicles (EVs). SASP factors cause chronic inflammation, leading to age-related diseases. Recently, therapeutic strategies targeting senescent cells, known as senolytics, have gained attention; however, noninvasive methods to detect senescent cells in living organisms have not been established. Therefore, the goal of this study was to identify novel senescent markers using small EVs (sEVs). sEVs were isolated from young and senescent fibroblasts using three different methods, including size-exclusion chromatography, affinity column for phosphatidylserine, and immunoprecipitation using antibodies against tetraspanin proteins, followed by mass spectrometry. Principal component analysis revealed that the protein composition of sEVs released from senescent cells was significantly different from that of young cells. Importantly, we identified ATP6V0D1 and RTN4 as novel markers that are frequently upregulated in sEVs from senescent and progeria cells derived from patients with Werner syndrome. Furthermore, these two proteins were significantly enriched in sEVs from the serum of aged mice. This study supports the potential use of senescent markers from sEVs to detect the presence of senescent cells in vivo.

Plasma exosomal DOK3 reflects immunological states in lung tumor and predicts prognosis of gefitinib treatment.

To identify liquid biomarkers that predict clinical outcomes of epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI), we enrolled patients with EGFR gene mutation-positive non-small-cell lung cancer who were intended to receive gefitinib treatment. Using plasma samples obtained prior to gefitinib treatment from 12 enrolled patients, we performed comprehensive proteomic analysis of plasma exosomes to explore proteins correlating with tumor reduction rate (TRR), progression-free survival (PFS), or overall survival (OS). Of the detected 1769 proteins, 119, 130, or 119 proteins demonstrated a strong correlation (|r| > 0.5) with TRR, PFS, or OS, respectively. Interestingly, 34 (29%), 41 (32%), or 27 (23%) of them, respectively, were functionally involved in the regulation of the immune response. CD8α chain was consistently listed as a molecule positively correlated with PFS and OS, suggesting that the long-lasting effects of gefitinib may be due to the antitumor effects of CD8+ T cells, as well as the induction of immunogenic apoptosis of tumor cells by blocking the EGFR signaling pathway. Notably, Doking Protein 3 (DOK3), a molecule involved in B-cell receptor signaling, and some immunoglobulin and complement molecules exhibited a clear correlation with PFS longevity of gefitinib treatment. Indeed, the strong expression of DOK3 in B cells was confirmed within tertiary lymphoid structures of lung cancer tissues derived from patients with long PFS. These findings suggest that the patients with active B-cell and T-cell immunity as a host immunological feature are more likely to benefit from gefitinib therapy. Circulating exosomal DOK3 has the potential as a predictive marker of response to gefitinib indicating this immunological feature.

Fast and Ultrasensitive Glycoform Analysis by Supercritical Fluid Chromatography-Tandem Mass Spectrometry.

The glycoform of a therapeutic monoclonal antibody (mAb) has a significant impact on its effector function as well as its safety and pharmacokinetics. Glycoform heterogeneity is influenced by various factors, including the producing cells and cell culture processes. Therefore, accurate glycoform characterization is essential for drug design, process optimization, manufacturing, and quality control of therapeutic mAbs. In this study, we developed a fast, quantitative, and highly sensitive analytical platform for glycan profiling by supercritical fluid chromatography-tandem mass spectrometry (SFC-MS/MS) and applied the technique to the glycan structural analysis of mAbs. To achieve both the highest sensitivity and the most comprehensive glycan profiling, we integrated our energy-resolved oxonium ion monitoring (Erexim) method with SFC-MS to construct a new SFC-Erexim technology. An 8 min analysis of bevacizumab, nivolumab, ramucirumab, rituximab, and trastuzumab by SFC-Erexim detected a total of 102 glycoforms, with a detection limit of 5 attomoles. The dynamic range of glycan abundance was over 6 orders of magnitude for bevacizumab analysis by SFC-Erexim compared to 3 orders of magnitude for conventional fluorescence HPLC analysis. This method revealed the glycan profile characteristics and lot-to-lot heterogeneity of various therapeutic mAbs. We were also able to detect a series of structural variations in pharmacologically important glycan structures. The SFC-MS-based glycoform profiling method will provide an ideal platform for the in-depth analysis of precise glycan structure and abundance.

G-quadruplex-forming nucleic acids interact with splicing factor 3B subunit 2 and suppress innate immune gene expression.

G-quadruplex (G4), a non-canonical higher-order structure formed by guanine-rich nucleic acid sequences, affects various genetic events in cis, including replication, transcription and translation. Whereas up-regulation of innate immune/interferon-stimulated genes (ISGs) is implicated in cancer progression, G4-forming oligonucleotides that mimic telomeric repeat-containing RNA suppress ISG induction in three-dimensional (3D) culture of cancer cells. However, it is unclear how G4 suppresses ISG expression in trans. In this study, we found that G4 binding to splicing factor 3B subunit 2 (SF3B2) down-regulated STAT1 phosphorylation and ISG expression in 3D-cultured cancer cells. Liquid chromatography-tandem mass spectrometry analysis identified SF3B2 as a G4-binding protein. Either G4-forming oligonucleotides or SF3B2 knockdown suppressed ISG induction, whereas Phen-DC3, a G4-stabilizing compound, reversed the inhibitory effect of G4-forming oligonucleotides on ISG induction. Phen-DC3 inhibited SF3B2 binding to G4 in vitro. SF3B2-mediated ISG induction appeared to occur independently of RNA splicing because SF3B2 knockdown did not affect pre-mRNA splicing under the experimental conditions, and pharmacological inhibition of splicing by pladienolide B did not repress ISG induction. These observations suggest that G4 disrupts the ability of SF3B2 to induce ISGs in cancer. We propose a new mode for gene regulation, which employs G4 as an inhibitory trans-element.