Macrophage-induced carboxypeptidase A4 promotes the progression of anaplastic thyroid cancer.

BACKGROUND: The density of tumor-associated macrophages (TAMs) in the tumor microenvironment of anaplastic thyroid cancer (ATC) is associated with poor prognosis. However, the crosstalk between macrophages and ATC cells is poorly understood. This study aimed to examine the impact of macrophages on cancer cell phenotypes. We found a new mediator between M2 macrophages and ATC cells through proteomics analysis. METHODS: The role of macrophages in proliferation, migration, and invasion of ATC cells was evaluated using co-culture assay and conditioned medium (CM). Secretory factors in the CM from single or co-culture were identified using LC-MS/MS proteomics analysis. We evaluated the role of the secretory factor in proliferation, migration, and invasion of cancer cells. In vivo xenograft model was used to evaluate the effect of the factor. RESULTS: M2 macrophages significantly increased the proliferation, migration, and invasion of ATC cells, whereas M1 macrophages decreased the proliferation, migration, and invasion of ATC cells. Based on proteomic analysis of CM, we identify carboxypeptidase A4 (CPA4) as a mediator of the crosstalk between macrophages and ATC cells. CPA4 was only detected in the co-culture media of M2 macrophage/8505C, and its expression in cancer cells increased by M2 macrophage. The expression of CPA4 protein was significantly higher in human thyroid cancers, particularly in ATCs, than normal and benign tissues. A bioinformatics analysis of public data revealed that CPA4 expression was associated with poor prognosis and dedifferentiation of thyroid cancer. Knockdown of CPA4 suppressed proliferation, colony formation, migration, and invasion of ATC cells, consistent with the decrease of STAT3, ERK, and AKT/mTOR phosphorylation and EMT marker expression. In addition, the increased expression of CPA4 in cancer cells by M2 macrophage stimulation induced the polarization of macrophages to the M2 phenotype, which formed a positive feedback loop. Xenograft tumors did not develop after CPA4 knockdown. CONCLUSIONS: Our data suggest that CPA4 stimulates the progression of thyroid cancer by mediating between M2 macrophages and ATC cells. CPA4 can be a new therapeutic target for the treatment of patients with ATC.

Dualistic Effects of PRKAR1A as a Potential Anticancer Target in Cancer Cells and Cancer-Derived Stem Cells.

The integration of innovative medical technologies and interdisciplinary collaboration could improve the treatment of cancer, a globally prevalent and often deadly disease. Despite recent advancements, current cancer therapies fail to specifically address recurrence and target cancer stem cells (CSCs), which contribute to relapse. In this study, we utilized three types of cancer cells, from which three types of CSCs were further derived, to conduct a proteomic analysis. Additionally, shared cell surface biomarkers were identified as potential targets for a comprehensive treatment strategy. The selected biomarkers were evaluated through short hairpin RNA treatment, which revealed contrasting functions in cancer cells and CSCs. Knockdown of the identified proteins revealed that they regulate the epithelial-mesenchymal transition (EMT) and stemness via the ERK signaling pathway. Resistance to anticancer agents was consequently reduced, ultimately enhancing the overall anticancer effects of the treatment. Additionally, the significance of these biomarkers in clinical patient outcomes was confirmed using bioinformatics. Our study suggests a novel cancer treatment strategy that addresses the limitations of current anticancer therapies.

Glycocalyx-Mimicking Nanoparticles with Differential Organ Selectivity for Drug Delivery and Therapy.

Organ-selective drug delivery is expected to maximize the efficacy of various therapeutic modalities while minimizing their systemic toxicity. Lipid nanoparticles and polymersomes can direct the organ-selective delivery of mRNAs or gene editing machineries, but their delivery is limited to mostly liver, spleen, and lung. A platform that enables delivery to these and other target organs is urgently needed. Here, a library of glycocalyx-mimicking nanoparticles (GlyNPs) comprising five randomly combined sugar moieties is generated, and direct in vivo library screening is used to identify GlyNPs with preferential biodistribution in liver, spleen, lung, kidneys, heart, and brain. Each organ-targeting GlyNP hit show cellular tropism within the organ. Liver, kidney, and spleen-targeting GlyNP hits equipped with therapeutics effectively can alleviate the symptoms of acetaminophen-induced liver injury, cisplatin-induced kidney injury, and immune thrombocytopenia in mice, respectively. Furthermore, the differential organ targeting of GlyNP hits is influenced not by the protein corona but by the sugar moieties displayed on their surface. It is envisioned that the GlyNP-based platform may enable the organ- and cell-targeted delivery of therapeutic cargoes.

Characterization of lymphocyte-rich hepatocellular carcinoma and the prognostic role of tertiary lymphoid structures.

BACKGROUND & AIMS: Lymphocyte-rich hepatocellular carcinoma (LR-HCC) is largely unknown and a rare subtype of HCC with immune-rich stroma. Tertiary lymphoid structures (TLS), frequently observed in LR-HCC, are known to be prognostically significant in various malignancies; however, their significance in HCC remains unevaluated. METHODS: Clinicopathologic data of 191 cases of surgically resected conventional HCC (C-HCC, n = 160) and LR-HCC (n = 31) were retrieved. Immunohistochemistry, multiplex immunofluorescence staining, RNA sequencing and proteomic analysis were conducted. Differences between the subtypes were statistically evaluated. RESULTS: LR-HCC was significantly correlated to larger tumour size, higher Edmondson-Steiner grade, presence of TLS and higher CD3-, CD8- and FOXP3-positive T cell, high PD-1 and PD-L1 expression (p < .001 for all) compared to C-HCC. Patients with LR-HCC exhibited significantly better overall survival (OS) (p = .044) and recurrence-free survival (RFS) (p = .025) than C-HCC. LR-HCC demonstrated TLS signatures with significantly higher proteomic-based immune scores in 14 of 17 types of tumour-infiltrating immune cells. Furthermore, C-HCC with secondary follicles, the most mature form of TLS, exhibited significantly better OS (p = .031) and RFS (p = .033) than those without. Across the global proteome, LR-HCC was well-differentiated from C-HCC and a map of protein-protein interactions between tumour-infiltrating lymphocytes and HCC in tumour microenvironment was completed. CONCLUSION: LR-HCC is clinicopathologically and molecularly distinct and shows better prognosis compared to C-HCC. Also, the presence of secondary follicle can be an important prognostic marker for better prognosis in both LR-HCC and C-HCC.

Proteomic analysis of breast cancer based on immune subtypes.

BACKGROUND: Immunotherapy is applied to breast cancer to resolve the limitations of survival gain in existing treatment modalities. With immunotherapy, a tumor can be classified into immune-inflamed, excluded and desert based on the distribution of immune cells. We assessed the clinicopathological features, each subtype's prognostic value and differentially expressed proteins between immune subtypes. METHODS: Immune subtyping and proteomic analysis were performed on 56 breast cancer cases with neoadjuvant chemotherapy. The immune subtyping was based on the level of tumor-infiltrating lymphocytes (TILs) and Klintrup criteria. If the level of TILs was ≥ 10%, it was classified as immune-inflamed type without consideration of the Klintrup criteria. In cases of 1-9% TIL, Klintrup criteria 1-3 were classified as the immune-excluded subtype and Klintrup criteria not available (NA) was classified as NA. Cases of 1% TILs and Klintrup 0 were classified as the immune-desert subtype. Mass spectrometry was used to identify differentially expressed proteins in formalin-fixed paraffin-embedded biopsy tissues. RESULTS: Of the 56 cases, 31 (55%) were immune-inflamed, 21 (38%) were immune-excluded, 2 (4%) were immune-desert and 2 (4%) were NA. Welch's t-test revealed two differentially expressed proteins between immune-inflamed and immune-excluded/desert subtypes. Coronin-1A was upregulated in immune-inflamed tumors (adjusted p = 0.008) and α-1-antitrypsin was upregulated in immune-excluded/desert tumors (adjusted p = 0.008). Titin was upregulated in pathologic complete response (pCR) than non-pCR among immune-inflamed tumors (adjusted p = 0.036). CONCLUSIONS: Coronin-1A and α-1-antitrypsin were upregulated in immune-inflamed and immune-excluded/desert subtypes, respectively. Titin's elevated expression in pCR within the immune-inflamed subtype may indicate a favorable prognosis. Further studies involving large representative cohorts are necessary to validate these findings.

PIBF1 regulates trophoblast syncytialization and promotes cardiovascular development.

Proper placental development in early pregnancy ensures a positive outcome later on. The developmental relationship between the placenta and embryonic organs, such as the heart, is crucial for a normal pregnancy. However, the mechanism through which the placenta influences the development of embryonic organs remains unclear. Trophoblasts fuse to form multinucleated syncytiotrophoblasts (SynT), which primarily make up the placental materno-fetal interface. We discovered that endogenous progesterone immunomodulatory binding factor 1 (PIBF1) is vital for trophoblast differentiation and fusion into SynT in humans and mice. PIBF1 facilitates communication between SynT and adjacent vascular cells, promoting vascular network development in the primary placenta. This process affected the early development of the embryonic cardiovascular system in mice. Moreover, in vitro experiments showed that PIBF1 promotes the development of cardiovascular characteristics in heart organoids. Our findings show how SynTs organize the barrier and imply their possible roles in supporting embryogenesis, including cardiovascular development. SynT-derived factors and SynT within the placenta may play critical roles in ensuring proper organogenesis of other organs in the embryo.

Migrasomal autophagosomes relieve endoplasmic reticulum stress in glioblastoma cells.

BACKGROUND: Glioblastoma (GBM) is more difficult to treat than other intractable adult tumors. The main reason that GBM is so difficult to treat is that it is highly infiltrative. Migrasomes are newly discovered membrane structures observed in migrating cells. Thus, they can be generated from GBM cells that have the ability to migrate along the brain parenchyma. However, the function of migrasomes has not yet been elucidated in GBM cells. RESULTS: Here, we describe the composition and function of migrasomes generated along with GBM cell migration. Proteomic analysis revealed that LC3B-positive autophagosomes were abundant in the migrasomes of GBM cells. An increased number of migrasomes was observed following treatment with chloroquine (CQ) or inhibition of the expression of STX17 and SNAP29, which are involved in autophagosome/lysosome fusion. Furthermore, depletion of ITGA5 or TSPAN4 did not relieve endoplasmic reticulum (ER) stress in cells, resulting in cell death. CONCLUSIONS: Taken together, our study suggests that increasing the number of autophagosomes, through inhibition of autophagosome/lysosome fusion, generates migrasomes that have the capacity to alleviate cellular stress.

Understanding the molecular mechanism of pathogenic variants of BIR2 domain in XIAP-deficient inflammatory bowel disease.

X-linked inhibitor of apoptosis protein (XIAP) deficiency causes refractory inflammatory bowel disease. The XIAP protein plays a pivotal role in the pro-inflammatory response through the nucleotide-binding oligomerization domain-containing signaling pathway that is important in mucosal homeostasis. We analyzed the molecular mechanism of non-synonymous pathogenic variants (PVs) of XIAP BIR2 domain. We generated N-terminally green fluorescent protein-tagged XIAP constructs of representative non-synonymous PVs. Co-immunoprecipitation and fluorescence cross-correlation spectroscopy showed that wild-type XIAP and RIP2 preferentially interacted in live cells, whereas all non-synonymous PV XIAPs failed to interact properly with RIP2. Structural analysis showed that various structural changes by mutations, such as hydrophobic core collapse, Zn-finger loss, and spatial rearrangement, destabilized the two loop structures (174-182 and 205-215) that critically interact with RIP2. Subsequently, it caused a failure of RIP2 ubiquitination and loss of protein deficiency by the auto-ubiquitination of all XIAP mutants. These findings could enhance our understanding of the role of XIAP mutations in XIAP-deficient inflammatory bowel disease and may benefit future therapeutic strategies.

18F-ASEM PET/MRI targeting alpha7-nicotinic acetylcholine receptor can reveal skeletal muscle denervation.

BACKGROUND: The increased expression of the nicotinic acetylcholine receptor (nAChR) in muscle denervation is thought to be associated with electrophysiological acetylcholine supersensitivity after nerve injury. Hence, we investigated the utility of the 18F-ASEM alpha7-nAChR targeting radiotracer as a new diagnostic method by visualizing skeletal muscle denervation in mouse models of sciatic nerve injury. METHODS: Ten-week-old C57BL/6 male mice were utilized. The mice were anesthetized, and the left sciatic nerve was resected after splitting the gluteal muscle. One week (n = 11) and three weeks (n = 6) after the denervation, 18F-ASEM positron emission tomography/magnetic resonance imaging (PET/MRI) was acquired. Maximum standardized uptake values (SUVmax) of the tibialis anterior muscle were measured for the denervated side and the control side. Autoradiographic evaluation was performed to measure the mean counts of the denervated and control tibialis anterior muscles at one week. In addition, immunohistochemistry was used to identify alpha7-nAChR-positive areas in denervated and control tibialis anterior muscles at one week (n = 6). Furthermore, a blocking study was conducted with methyllycaconitine (MLA, n = 5). RESULTS: 18F-ASEM PET/MRI showed significantly increased 18F-ASEM uptake in the denervated tibialis anterior muscle relative to the control side one week and three weeks post-denervation. SUVmax of the denervated muscles at one week and three weeks showed significantly higher uptake than the control (P = 0.0033 and 0.0277, respectively). The relative uptake by autoradiography for the denervated muscle was significantly higher than in the control, and immunohistochemistry revealed significantly greater alpha7-nAChR expression in the denervated muscle (P = 0.0277). In addition, the blocking study showed no significant 18F-ASEM uptake in the denervated side when compared to the control (P = 0.0796). CONCLUSIONS: Our results suggest that nAChR imaging with 18F-ASEM has potential as a noninvasive diagnostic method for peripheral nervous system disorders.

Circulating lumican as a potential biomarker for osteosarcopenia in older adults.

In vitro and animal experiments demonstrated that lumican exerts anabolic effects on bone and muscle by stimulating osteoblastogenesis, suppressing osteoclastogenesis and increasing myogenesis. However, the relationship between circulating lumican and musculoskeletal phenotypes in humans remains unclear. We aimed to analyze the relationship between serum lumican levels and osteosarcopenia in older adults. Blood samples were collected from 134 participants (age: 65 years and older) who underwent comprehensive assessment of bone and muscle phenotypes. Osteoporosis and sarcopenia were diagnosed based on World Health Organization and Asian consensus guidelines, respectively. Osteosarcopenia was defined as the simultaneous presence of osteoporosis and sarcopenia. After adjusting for sex, age, and body mass index, older adults with osteosarcopenia had 20.2 % lower serum lumican levels than those without (P = 0.010). The odds ratio (OR) for osteosarcopenia per standard deviation decrease in serum lumican level was 4.17 (P = 0.003). Consistently, higher serum lumican levels were correlated with higher bone mass at all measured sites (P = 0.004 to 0.045) and higher grip strength (P = 0.023). Furthermore, participants in the lowest tertile (T1) had 7.56-fold higher OR for osteosarcopenia (P = 0.024) than those in the highest lumican tertile (T3). In conclusion, these findings clinically validate previous experimental data showing the musculoskeletal protective effects of lumican and suggest that blood lumican levels could be used as a potential biomarker to assess the risk of not only osteosarcopenia but also osteoporosis or sarcopenia in older adults.

Higher Plasma Stromal Cell-Derived Factor 1 Is Associated with Lower Risk for Sarcopenia in Older Asian Adults.

BACKGRUOUND: Despite the protective effects of stromal cell-derived factor 1 (SDF-1) in stimulating muscle regeneration shown in experimental research, there is a lack of clinical studies linking circulating SDF-1 concentrations with muscle phenotypes. In order to elucidate the role of SDF-1 as a potential biomarker reflecting human muscle health, we investigated the association of plasma SDF-1 levels with sarcopenia in older adults. METHODS: This cross-sectional study included 97 community-dwelling participants who underwent a comprehensive geriatric assessment at a tertiary hospital in South Korea. Sarcopenia was defined by specific cutoff values applicable to the Asian population, whereas plasma SDF-1 levels were determined using an enzyme immunoassay. RESULTS: After accounting for sex, age, and body mass index, participants with sarcopenia and low muscle mass exhibited plasma SDF-1 levels that were 21.8% and 18.3% lower than those without these conditions, respectively (P=0.008 and P=0.009, respectively). Consistently, higher plasma SDF-1 levels exhibited a significant correlation with higher skeletal muscle mass index (SMI) and gait speed (both P=0.043), and the risk of sarcopenia and low muscle mass decreased by 58% and 55% per standard deviation increase in plasma SDF-1 levels, respectively (P=0.045 and P=0.030, respectively). Furthermore, participants in the highest SDF-1 tertile exhibited significantly higher SMI compared to those in the lowest tertile (P=0.012). CONCLUSION: These findings clinically corroborate earlier experimental discoveries highlighting the muscle anabolic effects of SDF- 1 and support the potential role of circulating SDF-1 as a biomarker reflecting human muscle health in older adults.

Systemic antibiotics cause deterioration of emphysema associated with exaggerated inflammation and autophagy.

The interaction between the microbial environment and the host is important for immune homeostasis. Recent research suggests that microbiota dysbiosis can be involved in respiratory diseases. Emphysema is a chronic inflammatory disease, but it is unclear whether dysbiosis caused by antibiotics can affect disease progression. Here, we tried to elucidate the effect of systemic antibiotics on smoking-exposed emphysema models. In this study, the antibiotic mixture caused more alveolar destruction and airspace expansion in the smoking group than in the smoking only or control groups. This emphysema aggravation as a result of antibiotic exposure was associated with increased levels of inflammatory cells, IL-6, IFNγ and protein concentrations in bronchoalveolar lavage fluid. Proteomics analysis indicated that autophagy could be involved in antibiotic-associated emphysema aggravation, and increased protein levels of LC3B, atg3, and atg7 were identified by Western blotting. In microbiome and metabolome analyses, the composition of the gut microbiota was different with smoking and antibiotic exposure, and the levels of short-chain fatty acids (SCFAs), including acetate and propionate, were reduced by antibiotic exposure. SCFA administration restored emphysema development with reduced inflammatory cells, IL-6, and IFNγ and decreased LC3B, atg3, and atg7 levels. In conclusion, antibiotics can aggravate emphysema, and inflammation and autophagy may be associated with this aggravation. This study provides important insight into the systemic impact of microbial dysbiosis and the therapeutic potential of utilizing the gut microbiota in emphysema.

Urine SERPINC1/ORM1 as biomarkers for early detection of lupus nephritis in MRL-lpr mice.

Background: To evaluate the usefulness of urine SERPINC1 and ORM1 as biomarkers for early detection of lupus nephritis (LN). Methods: Using proteomics, we screened for potential urine biomarkers that differentiate LN from systemic lupus erythematosus (SLE) patients without nephritis. In addition, urine levels of target biomarkers were measured by ELISA in 13- and 23-week-old MRL-lpr (murine model for LN) and MRL/MpJ mice. Histological analysis was also performed on the kidneys of 23-week-old mice. Results: Urine SERPINC1 and ORM1 were elevated in SLE patients with newly diagnosed LN compared with SLE patients without LN (SERPINC1, AUC=.892, P<.001; ORM1, AUC=.886, P<.001). Levels of urine SERPINC1 and ORM1 were also significantly higher in MRL-lpr mice than in MRL/MpJ mice at 13 and 23 weeks (SERPINC1: p<.01 and p<.001 at 13 and 23 weeks, respectively; ORM1: p<.01 at 13 and 23 weeks). In contrast, a significant difference in urine albumin between the two groups was only observed at 23 weeks (p<.001) not at 13 weeks (p=.83). Regarding the kidney pathology of MPL-lpr mice, urine ORM1 and urine albumin, but not urine SERPINC1, were positively correlated with the activity index (ORM1, rho =.879, p<.001; albumin, rho =.807, p=.003) and chronicity index (ORM1, rho =.947, p<.001; albumin, rho =.869, p<.001). Conclusion: We propose that urine SERPINC1 and ORM1 are novel biomarkers for early LN.

Neuronal MHC-I complex is destabilized by amyloid-ß and its implications in Alzheimer's disease.

BACKGROUNDS: The expression of major histocompatibility complex I (MHC-I) in neurons has recently been shown to regulate neurite outgrowth and synaptic plasticity. However, its contribution to neurodegenerative diseases such as Alzheimer's disease (AD) remains largely unknown. METHODS: In this study, we investigated the relationship between impaired MHC-I-ß2M complex and AD in vitro and human AD samples. Interaction between protein was identified by liquid chromatography-tandem mass spectrometry and confirmed by immunoprecipitation. Single-chain trimer of MHC-I-ß2M was generated to study the effect of stabilization of MHC-I-ß2M complex on NCAM1 signaling. RESULTS: MHC-I is destabilized in the brains of AD patients and neuronal cells treated with oligomeric ß-amyloid (Aß). Specifically, Aß oligomers disassemble the MHC-I-ß2-microglobulin (ß2M) complex, leading to reduced interactions with neural cell adhesion molecule 1 (NCAM1), a novel interactor of neuronal MHC-I, and decreased signaling. Inhibition of MHC-I-ß2M complex destabilization by non-dissociable MHC-I-ß2M-peptide complex restored MHC-I-NCAM1 signaling in neuronal cells. CONCLUSIONS: The current study demonstrated that disruption of MHC-1-NCAM1 signaling by Aß induced disassembly of MHC-I-ß2M complex is involved in the pathophysiology of AD. Moreover, our findings suggest modulation of MHC-I stability may be a potential therapeutic target for restoring synaptic function in AD.

The deubiquitinase UCHL3 mediates p300-dependent chemokine signaling in alveolar type II cells to promote pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic, fatal, fibrotic, interstitial lung disease of unknown cause. Despite extensive studies, the underlying mechanisms of IPF development remain unknown. Here, we found that p300 was upregulated in multiple epithelial cells in lung samples from patients with IPF and mouse models of lung fibrosis. Lung fibrosis was significantly diminished by the alveolar type II (ATII) cell-specific deletion of the p300 gene. Moreover, we found that ubiquitin C-terminal hydrolase L3 (UCHL3)-mediated deubiquitination of p300 led to the transcriptional activation of the chemokines Ccl2, Ccl7, and Ccl12 through the cooperative action of p300 and C/EBPß, which consequently promoted M2 macrophage polarization. Selective blockade of p300 activity in ATII cells resulted in the reprogramming of M2 macrophages into antifibrotic macrophages. These findings demonstrate a pivotal role for p300 in the development of lung fibrosis and suggest that p300 could serve as a promising target for IPF treatment.

Lack of association between serum myonectin levels and sarcopenia in older Asian adults.

Myonectin is a muscle-secreted factor that helps maintain homeostasis in the body by regulating several functions, including lipid metabolism. Previous studies suggested that myonectin may play a role in muscle health in an autocrine manner, but its impact on human skeletal muscle is still unclear. We aimed to investigate the relationship of serum myonectin levels with sarcopenia and related muscle parameters. We conducted a cross-sectional study of 142 older adults whose muscle mass, grip strength, gait speed, chair stands, and short physical performance battery (SPPB) were evaluated in the geriatric clinic of a tertiary medical center. Sarcopenia was defined based on Asian-specific cutoff values, and circulating myonectin levels were measured using an enzyme immunoassay. Before and after adjusting for age, sex, and body mass index, the serum myonectin level was not significantly different when the patients were stratified by status of sarcopenia, muscle mass, muscle strength, and physical performance. Furthermore, whether given as a continuous variable or divided into quartile groups, the serum myonectin level had no association with the skeletal muscle mass, grip strength, gait speed, chair stand test, or SPPB score. Our findings did not confirm the potential role of myonectin in muscle metabolism observed in experimental research. Thus, serum myonectin levels cannot predict the risk of sarcopenia in older Asian adults.

Generating Detailed Spectral Libraries for Canine Proteomes Obtained from Serum and Urine.

Domestic dogs (Canis lupus familiaris) are popular companion animals. Increase in medical expenses associated with them and demand for extending their lifespan in a healthy manner has created the need to develop new diagnostic technology. Companion dogs also serve as important animal models for non-clinical research as they can provide various biological phenotypes. Proteomics have been increasingly used on dogs and humans to identify novel biomarkers of various diseases. Despite the growing applications of proteomics in liquid biopsy in veterinary medicine, no publicly available spectral assay libraries have been created for the proteome of canine serum and urine. In this study, we generated spectral assay libraries for the two-representative liquid-biopsy samples using mid-pH fractionation that allows in-depth understanding of proteome coverage. The resultant canine serum and urine spectral assay libraries include 1,132 and 4,749 protein groups and 5,483 and 25,228 peptides, respectively. We built these complimentary accessible resources for proteomic biomarker discovery studies through ProteomeXchange with the identifier PXD034770.

Insulin-Like Growth Factor-1 Promotes Synaptogenesis Signaling, a Major Dysregulated Pathway in Malformation of Cortical Development, in a Rat Model.

Malformation of cortical development (MCD) is one of the main causes of intractable epilepsy in childhood. We explored a treatment based on molecular changes using an infant rat model of methylazoxymethanol (MAM)-induced MCD established by injecting MAM at gestational day 15. The offspring were sacrificed on postnatal day (P) 15 for proteomic analysis, which revealed significant downregulation in the synaptogenesis signaling pathway in the cortex of MCD rats. Recombinant human insulin-growth factor-1 (rhIGF-1) was injected from P12 to P14 twice daily and the effect of IGF1 on N-methyl-D-aspartate (NMDA)-induced spasms (15 mg/kg of NMDA, i.p.) was tested; the onset of P15 single spasm was significantly delayed (p = 0.002) and the number of spasms decreased (p < 0.001) in rhIGF1-pretreated rats (n = 17) compared to those in VEH-treated rats (n = 18). Electroencephalographic monitoring during spasms showed significantly reduced spectral entropy and event-related spectral dynamics of fast oscillation in rhIGF-1 treated rats. Magnetic resonance spectroscopy of the retrosplenial cortex showed decreased glutathione (GSH) (p = 0.039) and significant developmental changes in GSH, phosphocreatine (PCr), and total creatine (tCr) (p = 0.023, 0.042, 0.015, respectively) after rhIGF1 pretreatment. rhIGF1 pretreatment significantly upregulated expression of cortical synaptic proteins such as PSD95, AMPAR1, AMPAR4, NMDAR1, and NMDAR2A (p < 0.05). Thus, early rhIGF-1 treatment could promote synaptic protein expression, which was significantly downregulated by prenatal MAM exposure, and effectively suppress NMDA-induced spasms. Early IGF1 treatment should be further investigated as a therapeutic strategy in infants with MCD-related epilepsy.

Magnetic transferrin nanoparticles (MTNs) assay as a novel isolation approach for exosomal biomarkers in neurological diseases.

BACKGROUND: Brain-derived exosomes released into the blood are considered a liquid biopsy to investigate the pathophysiological state, reflecting the aberrant heterogeneous pathways of pathological progression of the brain in neurological diseases. Brain-derived blood exosomes provide promising prospects for the diagnosis of neurological diseases, with exciting possibilities for the early and sensitive diagnosis of such diseases. However, the capability of traditional exosome isolation assays to specifically isolate blood exosomes and to characterize the brain-derived blood exosomal proteins by high-throughput proteomics for clinical specimens from patients with neurological diseases cannot be assured. We report a magnetic transferrin nanoparticles (MTNs) assay, which combined transferrin and magnetic nanoparticles to isolate brain-derived blood exosomes from clinical samples. METHODS: The principle of the MTNs assay is a ligand-receptor interaction through transferrin on MTNs and transferrin receptor on exosomes, and electrostatic interaction via positively charged MTNs and negatively charged exosomes to isolate brain-derived blood exosomes. In addition, the MTNs assay is simple and rapid (< 35 min) and does not require any large instrument. We confirmed that the MTNs assay accurately and efficiently isolated exosomes from serum samples of humans with neurodegenerative diseases, such as dementia, Parkinson's disease (PD), and multiple sclerosis (MS). Moreover, we isolated exosomes from serum samples of 30 patients with three distinct neurodegenerative diseases and performed unbiased proteomic analysis to explore the pilot value of brain-derived blood protein profiles as biomarkers. RESULTS: Using comparative statistical analysis, we found 21 candidate protein biomarkers that were significantly different among three groups of neurodegenerative diseases. CONCLUSION: The MTNs assay is a convenient approach for the specific and affordable isolation of extracellular vesicles from body fluids for minimally-invasive diagnosis of neurological diseases.

Proteomic analysis predicts anti-angiogenic resistance in recurred glioblastoma.

BACKGROUND: Recurrence is common in glioblastoma multiforme (GBM) because of the infiltrative, residual cells in the tumor margin. Standard therapy for GBM consists of surgical resection followed by chemotherapy and radiotherapy, but the median survival of GBM patients remains poor (~ 1.5 years). For recurrent GBM, anti-angiogenic treatment is one of the common treatment approaches. However, current anti-angiogenic treatment modalities are not satisfactory because of the resistance to anti-angiogenic agents in some patients. Therefore, we sought to identify novel prognostic biomarkers that can predict the therapeutic response to anti-angiogenic agents in patients with recurrent glioblastoma. METHODS: We selected patients with recurrent GBM who were treated with anti-angiogenic agents and classified them into responders and non-responders to anti-angiogenic therapy. Then, we performed proteomic analysis using liquid-chromatography mass spectrometry (LC-MS) with formalin-fixed paraffin-embedded (FFPE) tissues obtained from surgical specimens. We conducted a gene-ontology (GO) analysis based on protein abundance in the responder and non-responder groups. Based on the LC-MS and GO analysis results, we identified potential predictive biomarkers for anti-angiogenic therapy and validated them in recurrent glioblastoma patients. RESULTS: In the mass spectrometry-based approach, 4957 unique proteins were quantified with high confidence across clinical parameters. Unsupervised clustering analysis highlighted distinct proteomic patterns (n = 269 proteins) between responders and non-responders. The GO term enrichment analysis revealed a cluster of genes related to immune cell-related pathways (e.g., TMEM173, FADD, CD99) in the responder group, whereas the non-responder group had a high expression of genes related to nuclear replisome (POLD) and damaged DNA binding (ERCC2). Immunohistochemistry of these biomarkers showed that the expression levels of TMEM173 and FADD were significantly associated with the overall survival and progression-free survival of patients with recurrent GBM. CONCLUSIONS: The candidate biomarkers identified in our protein analysis may be useful for predicting the clinical response to anti-angiogenic agents in patients with recurred GBM.