A Gelatin/Alginate Double Network Hydrogel Nerve Guidance Conduit Fabricated by a Chemical-Free Gamma Radiation for Peripheral Nerve Regeneration.

Nerve guidance conduits (NGCs) are widely developed using various materials for the functional repair of injured or diseased peripheral nerves. Especially, hydrogels are considered highly suitable for the fabrication of NGCs due to their beneficial tissue-mimicking characteristics (e.g., high water content, softness, and porosity). However, the practical applications of hydrogel-based NGCs are hindered due to their poor mechanical properties and complicated fabrication processes. To bridge this gap, a novel double-network (DN) hydrogel using alginate and gelatin by a two-step crosslinking process involving chemical-free gamma irradiation and ionic crosslinking, is developed. DN hydrogels (1% alginate and 15% gelatin), crosslinked with 30 kGy gamma irradiation and barium ions, exhibit substantially improved mechanical properties, including tensile strength, elastic modulus, and fracture stain, compared to single network (SN) gelatin hydrogels. Additionally, the DN hydrogel NGC exhibits excellent kink resistance, mechanical stability to successive compression, suture retention, and enzymatic degradability. In vivo studies with a sciatic defect rat model indicate substantially improved nerve function recovery with the DN hydrogel NGC compared to SN gelatin and commercial silicone NGCs, as confirm footprint analysis, electromyography, and muscle weight measurement. Histological examination reveals that, in the DN NGC group, the expression of Schwann cell and neuronal markers, myelin sheath, and exon diameter are superior to the other controls. Furthermore, the DN NGC group demonstrates increased muscle fiber formation and reduced fibrotic scarring. These findings suggest that the mechanically robust, degradable, and biocompatible DN hydrogel NGC can serve as a novel platform for peripheral nerve regeneration and other biomedical applications, such as implantable tissue constructs.

A New Understanding of Long Non-Coding RNA in Hepatocellular Carcinoma-From m6A Modification to Blood Biomarkers.

With recent advancements in biological research, long non-coding RNAs (lncRNAs) with lengths exceeding 200 nucleotides have emerged as pivotal regulators of gene expression and cellular phenotypic modulation. Despite initial skepticism due to their low sequence conservation and expression levels, their significance in various biological processes has become increasingly apparent. We provided an overview of lncRNAs and discussed their defining features and modes of operation. We then explored their crucial function in the hepatocarcinogenesis process, elucidating their complex involvement in hepatocellular carcinoma (HCC). The influential role of lncRNAs within the HCC tumor microenvironment is emphasized, illustrating their potential as key modulators of disease dynamics. We also investigated the significant influence of N6-methyladenosine (m6A) modification on lncRNA function in HCC, enhancing our understanding of both their roles and their upstream regulators. Additionally, the potential of lncRNAs as promising biomarkers was discussed in liver cancer diagnosis, suggesting a novel avenue for future research and clinical application. Finally, our work underscored the dual potential of lncRNAs as both contributors to HCC pathogenesis and innovative tools for its diagnosis. Existing challenges and prospective trajectories in lncRNA research are also discussed, emphasizing their potential in advancing liver cancer research.

Tumor Endothelial Cells-Associated Integrin Alpha-6 as a Promising Biomarker for Early Detection and Prognosis of Hepatocellular Carcinoma.

HCC remains a lethal cancer type, with early detection being critical for improved patient outcomes. This study introduces a comprehensive methodological approach to identify the ITGA6 gene as a potential blood marker for early HCC (eHCC) detection. We initially analyzed the GSE114564 dataset encompassing various stages of liver disease, identifying 972 differentially expressed genes in HCC. A refined analysis yielded 59 genes specifically differentially expressed in early HCC, including ITGA6. Subsequent validation in multiple datasets confirmed the consistent upregulation of ITGA6 in HCC. In addition, when analyzing progression-free survival (PFS) within the entire patient cohort and overall survival (OS) specifically among patients classified as tumor grade G1, the group of patients characterized by high expression levels of ITGA6 displayed an elevated risk ratio in relation to prognosis. Further analyses demonstrated the predominant expression of ITGA6 in TECs and its enrichment in angiogenesis-related pathways. Additionally, positive correlations were found between ITGA6 expression and pro-tumorigenic immune cells, but not with anti-tumorigenic immune cells. Our study elucidates the potential of ITGA6 as a blood-based marker for HCC early detection and diagnosis and its complex interplay with the tumor microenvironment. Further research may lead to novel strategies for HCC management and patient care.

GNAQ-Regulated ZO-1 and ZO-2 Act as Tumor Suppressors by Modulating EMT Potential and Tumor-Repressive Microenvironment in Lung Cancer.

Epithelial-to-mesenchymal transition (EMT) plays a critical role in the development and progression of lung cancer by promoting its invasiveness and metastasis. Using integrative analyses of the public lung cancer database, we found that the expression levels of the tight junction proteins, zonula occluden (ZO)-1 and ZO-2, were lower in lung cancer tissues, including both lung adenocarcinoma and lung squamous cell carcinoma than in normal lung tissues analyzed using The Cancer Genome Atlas (TCGA). Although the ectopic expression or knockdown of ZO-1 and ZO-2 did not affect the growth of lung cancer cells, they significantly regulated cell migration and invasion. When M0 macrophages were co-cultured with ZO-1 or ZO-2 knockdown Calu-1 cells, M2-like polarization was efficiently induced. Conversely, co-culture of M0 THP-1 cells with A549 cells stably expressing ZO-1 or ZO-2 significantly reduced M2 differentiation. We also identified G protein subunit alpha q (GNAQ) as a potential ZO-1- and ZO-2-specific activator through analysis of correlated genes with the TCGA lung cancer database. Our results suggest that the GNAQ-ZO-1/2 axis may play a tumor-suppressive role in lung cancer development and progression and highlight ZO-1 and ZO-2 as key EMT- and tumor microenvironment-suppressive proteins. These findings provide new insights for the development of targeted therapies for lung cancer.

Aberrantly Expressed MicroRNAs in Cancer-Associated Fibroblasts and Their Target Oncogenic Signatures in Hepatocellular Carcinoma.

Cancer-associated fibroblasts (CAFs) contribute to tumor progression, and microRNAs (miRs) play an important role in regulating the tumor-promoting properties of CAFs. The objectives of this study were to clarify the specific miR expression profile in CAFs of hepatocellular carcinoma (HCC) and identify its target gene signatures. Small-RNA-sequencing data were generated from nine pairs of CAFs and para-cancer fibroblasts isolated from human HCC and para-tumor tissues, respectively. Bioinformatic analyses were performed to identify the HCC-CAF-specific miR expression profile and the target gene signatures of the deregulated miRs in CAFs. Clinical and immunological implications of the target gene signatures were evaluated in The Cancer Genome Atlas Liver Hepatocellular Carcinoma (TCGA_LIHC) database using Cox regression and TIMER analysis. The expressions of hsa-miR-101-3p and hsa-miR-490-3p were significantly downregulated in HCC-CAFs. Their expression in HCC tissue gradually decreased as HCC stage progressed in the clinical staging analysis. Bioinformatic network analysis using miRWalks, miRDB, and miRTarBase databases pointed to TGFBR1 as a common target gene of hsa-miR-101-3p and hsa-miR-490-3p. TGFBR1 expression was negatively correlated with miR-101-3p and miR-490-3p expression in HCC tissues and was also decreased by ectopic miR-101-3p and miR-490-3p expression. HCC patients with TGFBR1 overexpression and downregulated hsa-miR-101-3p and hsa-miR-490-3p demonstrated a significantly poorer prognosis in TCGA_LIHC. TGFBR1 expression was positively correlated with the infiltration of myeloid-derived suppressor cells, regulatory T cells, and M2 macrophages in a TIMER analysis. In conclusion, hsa-miR-101-3p and hsa-miR-490-3p were substantially downregulated miRs in CAFs of HCC, and their common target gene was TGFBR1. The downregulation of hsa-miR-101-3p and hsa-miR-490-3p, as well as high TGFBR1 expression, was associated with poor clinical outcome in HCC patients. In addition, TGFBR1 expression was correlated with the infiltration of immunosuppressive immune cells.

An agonistic anti-Tie2 antibody suppresses the normal-to-tumor vascular transition in the glioblastoma invasion zone.

Tumor progression is intimately associated with the vasculature, as tumor proliferation induces angiogenesis and tumor cells metastasize to distant organs via blood vessels. However, whether tumor invasion is associated with blood vessels remains unknown. As glioblastoma (GBM) is featured by aggressive invasion and vascular abnormalities, we characterized the onset of vascular remodeling in the diffuse tumor infiltrating zone by establishing new spontaneous GBM models with robust invasion capacity. Normal brain vessels underwent a gradual transition to severely impaired tumor vessels at the GBM periphery over several days. Increasing vasodilation from the tumor periphery to the tumor core was also found in human GBM. The levels of vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2) showed a spatial correlation with the extent of vascular abnormalities spanning the tumor-invading zone. Blockade of VEGFR2 suppressed vascular remodeling at the tumor periphery, confirming the role of VEGF-VEGFR2 signaling in the invasion-associated vascular transition. As angiopoietin-2 (ANGPT2) was expressed in only a portion of the central tumor vessels, we developed a ligand-independent tunica interna endothelial cell kinase 2 (Tie2)-activating antibody that can result in Tie2 phosphorylation in vivo. This agonistic anti-Tie2 antibody effectively normalized the vasculature in both the tumor periphery and tumor center, similar to the effects of VEGFR2 blockade. Mechanistically, this antibody-based Tie2 activation induced VE-PTP-mediated VEGFR2 dephosphorylation in vivo. Thus, our study reveals that the normal-to-tumor vascular transition is spatiotemporally associated with GBM invasion and may be controlled by Tie2 activation via a novel mechanism of action.

ADAR1-dependent miR-3144-3p editing simultaneously induces MSI2 expression and suppresses SLC38A4 expression in liver cancer.

Aberrant adenosine-to-inosine (A-to-I) RNA editing, catalyzed by adenosine deaminase acting on double-stranded RNA (ADAR), has been implicated in various cancers, but the mechanisms by which microRNA (miRNA) editing contributes to cancer development are largely unknown. Our multistage hepatocellular carcinogenesis transcriptome data analyses, together with publicly available data, indicated that ADAR1 was the most profoundly dysregulated gene among RNA-editing enzyme family members in liver cancer. Targeted inactivation of ADAR1 inhibited the in vitro tumorigenesis of liver cancer cells. An integrative computational analyses of RNA-edited hotspots and the known editing frequency of miRNAs suggested that the miRNA miR-3144-3p was edited by ADAR1 during liver cancer progression. Specifically, ADAR1 promoted A-to-I editing of canonical miR-3144-3p to replace the adenosine at Position 3 in the seed region with a guanine (ED_miR-3144-3p(3_A < G)) in liver cancer cells. We then demonstrated that Musashi RNA-binding protein 2 (MSI2) was a specific target of miR-3144-3p and that MSI2 overexpression was due to excessive ADAR1-dependent over-editing of canonical miR-3144-3p in liver cancer. In addition, target prediction analyses and validation experiments identified solute carrier family 38 member 4 (SLC38A4) as a specific gene target of ED_miR-3144-3p(3_A < G). The ectopic expression of both ADAR1 and the ED_miR-3144-3p(3_A < G) mimic enhanced mitotic activities, and ADAR1 suppressed SLC38A4 expression in liver cancer cells. Treatments with mouse-specific ADAR1-, MSI2-siRNA-, or SLC38A4-expressing plasmids suppressed tumorigenesis and tumor growth in a mouse model of spontaneous liver cancer. Our findings suggest that the aberrant regulation of ADAR1 augments oncogenic MSI2 effects by excessively editing canonical miR-3144-3p and that the resultant ED_miR-3144-3p(3_A < G) simultaneously suppresses tumor suppressor SLC38A4 expression, contributing to hepatocellular carcinogenesis.

Regulation of MDM2 E3 ligase-dependent vascular calcification by MSX1/2.

Vascular calcification increases morbidity and mortality in patients with cardiovascular and renal diseases. Previously, we reported that histone deacetylase 1 prevents vascular calcification, whereas its E3 ligase, mouse double minute 2 homolog (MDM2), induces vascular calcification. In the present study, we identified the upstream regulator of MDM2. By utilizing cellular models and transgenic mice, we confirmed that E3 ligase activity is required for vascular calcification. By promoter analysis, we found that both msh homeobox 1 (Msx1) and msh homeobox 2 (Msx2) bound to the MDM2 promoter region, which resulted in transcriptional activation of MDM2. The expression levels of both Msx1 and Msx2 were increased in mouse models of vascular calcification and in calcified human coronary arteries. Msx1 and Msx2 potentiated vascular calcification in cellular and mouse models in an MDM2-dependent manner. Our results establish a novel role for MSX1/MSX2 in the transcriptional activation of MDM2 and the resultant increase in MDM2 E3 ligase activity during vascular calcification.

Oat Extract Avenanthramide-C Reverses Hippocampal Long-Term Potentiation Decline in Tg2576 Mice.

Memory deterioration in Alzheimer's disease (AD) is thought to be underpinned by aberrant amyloid ß (Aß) accumulation, which contributes to synaptic plasticity impairment. Avenanthramide-C (Avn-C), a polyphenol compound found predominantly in oats, has a range of biological properties. Herein, we performed methanolic extraction of the Avns-rich fraction (Fr. 2) from germinated oats using column chromatography, and examined the effects of Avn-C on synaptic correlates of memory in a mouse model of AD. Avn-C was identified in Fr. 2 based on 1H-NMR analysis. Electrophysiological recordings were performed to examine the effects of Avn-C on the hippocampal long-term potentiation (LTP) in a Tg2576 mouse model of AD. Avn-C from germinated oats restored impaired LTP in Tg2576 mouse hippocampal slices. Furthermore, Avn-C-facilitated LTP was associated with changes in the protein levels of phospho-glycogen synthase kinase-3ß (p-GSK3ß-S9) and cleaved caspase 3, which are involved in Aß-induced synaptic impairment. Our findings suggest that the Avn-C extract from germinated oats may be beneficial for AD-related synaptic plasticity impairment and memory decline.

Avenanthramide C as a novel candidate to alleviate osteoarthritic pathogenesis.

Osteoarthritis (OA) is a degenerative disorder that can result in the loss of articular cartilage. No effective treatment against OA is currently available. Thus, interest in natural health products to relieve OA symptoms is increasing. However, their qualities such as efficacy, toxicity, and mechanism are poorly understood. In this study, we determined the efficacy of avenanthramide (Avn)-C extracted from oats as a promising candidate to prevent OA progression and its mechanism of action to prevent the expression of matrix-metalloproteinases (MMPs) in OA pathogenesis. Interleukin-1 beta (IL-1ß), a proinflammatory cytokine as a main causing factor of cartilage destruction, was used to induce OAlike condition of chondrocytes in vitro. Avn-C restrained IL-1ß- mediated expression and activity of MMPs, such as MMP-3, -12, and -13 in mouse articular chondrocytes. Moreover, Avn-C alleviated cartilage destruction in experimental OA mouse model induced by destabilization of the medial meniscus (DMM) surgery. However, Avn-C did not affect the expression of inflammatory mediators (Ptgs2 and Nos) or anabolic factors (Col2a1, Aggrecan, and Sox9), although expression levels of these genes were upregulated or downregulated by IL-1ß, respectively. The inhibition of MMP expression by Avn-C in articular chondrocytes was mediated by p38 kinase and c-Jun N-terminal kinase (JNK) signaling, but not by ERK or NF-κB. Interestingly, Avn-C added with SB203580 and SP600125 as specific inhibitors of p38 kinase and JNK, respectively, enhanced its inhibitory effect on the expression of MMPs in IL-1ß treated chondrocytes. Taken together, these results suggest that Avn-C is an effective candidate to prevent OA progression and a natural health product to relieve OA pathogenesis. [BMB Reports 2021; 54(10): 528-533].

SMARCA4 oncogenic potential via IRAK1 enhancer to activate Gankyrin and AKR1B10 in liver cancer.

SWItch/Sucrose Non-Fermentable (SWI/SNF) is a multiprotein complex essential for the regulation of eukaryotic gene expression. SWI/SNF complex genes are genetically altered in over 20% of human malignancies, but the aberrant regulation of the SWI/SNF subunit genes and subsequent dysfunction caused by abnormal expression of subunit gene in cancer, remain poorly understood. Among the SWI/SNF subunit genes, SMARCA4, SMARCC1, and SMARCA2 were identified to be overexpressed in human hepatocellular carcinoma (HCC). Modulation of SMARCA4, SMARCC1, and SMARCA2 inhibited in vitro tumorigenesis of HCC cells. However, SMARCA4-targeting elicited remarkable inhibition in an in vivo Ras-transgenic mouse HCC model (Ras-Tg), and high expression levels of SMARCA4 significantly associated with poor prognosis in HCC patients. Furthermore, most HCC patients (72-86%) showed SMARCA4 overexpression compared to healthy controls. To identify SMARCA4-specific active enhancers, mapping, and analysis of chromatin state in liver cancer cells were performed. Integrative analysis of SMARCA4-regulated genes and active chromatin enhancers suggested 37 genes that are strongly activated by SMARCA4 in HCC. Through chromatin immunoprecipitation-qPCR and luciferase assays, we demonstrated that SMARCA4 activates Interleukin-1 receptor-associated kinase 1 (IRAK1) expression through IRAK1 active enhancer in HCC. We then showed that transcriptional activation of IRAK1 induces oncoprotein Gankyrin and aldo-keto reductase family 1 member B10 (AKR1B10) in HCC. The regulatory mechanism of the SMARCA4-IRAK1-Gankyrin, AKR1B10 axis was further demonstrated in HCC cells and in vivo Ras-Tg mice. Our results suggest that aberrant overexpression of SMARCA4 causes SWI/SNF to promote IRAK1 enhancer to activate oncoprotein Gankyrin and AKR1B10, thereby contributing to hepatocarcinogenesis.

Orphan nuclear receptor ERR-γ regulates hepatic FGF23 production in acute kidney injury.

Fibroblast growth factor 23 (FGF23), a hormone generally derived from bone, is important in phosphate and vitamin D homeostasis. In acute kidney injury (AKI) patients, high-circulating FGF23 levels are associated with disease progression and mortality. However, the organ and cell type of FGF23 production in AKI and the molecular mechanism of its excessive production are still unidentified. For insight, we investigated folic acid (FA)-induced AKI in mice. Interestingly, simultaneous with FGF23, orphan nuclear receptor ERR-γ expression is increased in the liver of FA-treated mice, and ectopic overexpression of ERR-γ was sufficient to induce hepatic FGF23 production. In patients and in mice, AKI is accompanied by up-regulated systemic IL-6, which was previously identified as an upstream regulator of ERR-γ expression in the liver. Administration of IL-6 neutralizing antibody to FA-treated mice or of recombinant IL-6 to healthy mice confirms IL-6 as an upstream regulator of hepatic ERR-γ-mediated FGF23 production. A significant (P < 0.001) interconnection between high IL-6 and FGF23 levels as a predictor of AKI in patients that underwent cardiac surgery was also found, suggesting the clinical relevance of the finding. Finally, liver-specific depletion of ERR-γ or treatment with an inverse ERR-γ agonist decreased hepatic FGF23 expression and plasma FGF23 levels in mice with FA-induced AKI. Thus, inverse agonist of ERR-γ may represent a therapeutic strategy to reduce adverse plasma FGF23 levels in AKI.

Oral carbohydrate solution cause an inflammatory response when aspirated into the lungs in mice.

PURPOSE: Many studies have been published on the beneficial effects of oral carbohydrate solutions (OCS) administered prior to surgery. However, the risk of pulmonary aspiration cannot be excluded in all patients undergoing anesthesia. But, there are few studies on the safety of OCS at lung aspiration. METHODS: Experiments were conducted with mice (Nine- to ten-week-old male BALB/c mice weighted 23-26 g). Lung aspiration was performed by intratracheal administration of OCS and its major constituents, fructose and maltodextrin. Bronchoalveolar lavage fluid (BALF) was collected 3 and 24 h after lung aspiration. The level of Tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and macrophage inflammatory protein-2 (MIP-2) were measured in BALF. The total white blood cell, neutrophil counts, wet to dry ratio and histological examination were performed in BALF and lung tissue, respectively, at 24 h after aspiration. RESULTS: The OCS increased the level of TNF-α, IL-6 and MIP-2 at 3 h and the neutrophil count at 24 h in BALFs, compared to a phosphate-buffered saline (PBS) group. The increase in IL-6 level induced by OCS was maintained for 24 h. The OCS also increased the number of white blood cells and the percentage of neutrophils in BALFs. Compared to fructose, maltodextrin significantly increased the production of MIP-2 in BALFs. OCS and maltodextrin also increased neutrophil recruitment in lung tissue. CONCLUSION: Aspiration of OCS may cause inflammation of the lungs. The preoperative use of OCS may require caution under specific clinical conditions, such as patients at risk of lung aspiration.

Identification of a novel senomorphic agent, avenanthramide C, via the suppression of the senescence-associated secretory phenotype.

Senescent cells are deeply involved in the induction of tissue damage and aging-related diseases. The identification of factors that eliminate senescent cells or inhibit the senescence-associated secretory phenotype (SASP) in these cells is necessary. Here, we report an avenanthramice C (Avn C) extracted from oat as a new SASP modulator. Treatment with Avn C led to a significant reduction in the levels of markers of senescent cells, with no toxicity observed. The SASP was also inhibited by Avn C treatment, similar to non-senescent cells, and the suppression of cell division by autocrine signals associated with SASP was restored. To investigate the mechanism underlying SASP inhibition by Avn C, we analyzed the effect of Avn C in lipopolysaccharide (LPS)-induced inflammation in non-senescent cells. Avn C inhibited nuclear factor κB (NF-κB) activity and the secretion of inflammatory cytokines before or after LPS treatment. Although the activity of MAP kinases, which are NF-κB upstream signals, was inhibited by Avn C in LPS-induced inflammation, only p38 activity was specifically inhibited in senescent cells. Interestingly, the inhibition of p38 in senescent cells was observed through Avn C-induced 5'-adenosine monophosphate-activated protein kinase (AMPK) activity. Avn C-induced inhibition of the SASP is triggered by senescence-related stress.

Time-Domain Investigation of Switchable Filter Wide-Band Antenna for Microwave Breast Imaging.

This paper investigates the time-domain performance of a switchable filter impulse radio ultra-wideband (IR-UWB) antenna for microwave breast imaging applications. A miniaturized CPW-fed integrated filter antenna with switchable performance in the range of the Worldwide Interoperability for Microwave Access (WiMAX) and Wireless Local Area Network (WLAN) bands could operate well within a 3.0 to 11 GHz frequency range. The time-domain performance of the filter antenna was investigated in comparison to that of the designed reference wideband antenna. By comparing both antennas' time-domain characteristics, it was seen that the switchable filter antenna had good time-domain resolution along with the frequency-domain operation. Additionally, the time-domain investigation revealed that the switchable filter wide-band antenna performed similarly to the reference wide band antenna. This antenna was also utilized for a tumor detection application, and it was seen that the switchable filter wide-band antenna could detect a miniaturized irregularly shaped tumor easily, which is quite promising. Such an antenna with a good time-domain resolution and tumor detection capability will be a good candidate and will find potential applications in microwave breast imaging.

VEGFR2 signaling drives meningeal vascular regeneration upon head injury.

Upon severe head injury (HI), blood vessels of the meninges and brain parenchyma are inevitably damaged. While limited vascular regeneration of the injured brain has been studied extensively, our understanding of meningeal vascular regeneration following head injury is quite limited. Here, we identify key pathways governing meningeal vascular regeneration following HI. Rapid and complete vascular regeneration in the meninges is predominantly driven by VEGFR2 signaling. Substantial increase of VEGFR2 is observed in both human patients and mouse models of HI, and endothelial cell-specific deletion of Vegfr2 in the latter inhibits meningeal vascular regeneration. We further identify the facilitating, stabilizing and arresting roles of Tie2, PDGFRß and Dll4 signaling, respectively, in meningeal vascular regeneration. Prolonged inhibition of this angiogenic process following HI compromises immunological and stromal integrity of the injured meninges. These findings establish a molecular framework for meningeal vascular regeneration after HI, and may guide development of wound healing therapeutics.

Excessive Astrocytic GABA Causes Cortical Hypometabolism and Impedes Functional Recovery after Subcortical Stroke.

Glucose hypometabolism in cortical structures after functional disconnection is frequently reported in patients with white matter diseases such as subcortical stroke. However, the molecular and cellular mechanisms have been poorly elucidated. Here we show, in an animal model of internal capsular infarct, that GABA-synthesizing reactive astrocytes in distant cortical areas cause glucose hypometabolism via tonic inhibition of neighboring neurons. We find that reversal of aberrant astrocytic GABA synthesis, by pharmacological inhibition and astrocyte-specific gene silencing of MAO-B, reverses the reduction in cortical glucose metabolism. Moreover, induction of aberrant astrocytic GABA synthesis by cortical injection of putrescine or adenovirus recapitulates cortical hypometabolism. Furthermore, MAO-B inhibition causes a remarkable recovery from post-stroke motor deficits when combined with a rehabilitation regimen. Collectively, our data indicate that cortical glucose hypometabolism in subcortical stroke is caused by aberrant astrocytic GABA and MAO-B inhibition and that attenuating cortical hypometabolism can be a therapeutic approach in subcortical stroke.

Ultrasensitive detection of malignant melanoma using PET molecular imaging probes.

Malignant melanoma has one of the highest mortality rates of any cancer because of its aggressive nature and high metastatic potential. Clinical staging of the disease at the time of diagnosis is very important for the prognosis and outcome of melanoma treatment. In this study, we designed and synthesized the 18F-labeled pyridine-based benzamide derivatives N-(2-(dimethylamino)ethyl)-5-[18F]fluoropicolinamide ([18F]DMPY2) and N-(2-(dimethylamino)ethyl)-6-[18F]fluoronicotinamide ([18F]DMPY3) to detect primary and metastatic melanoma at an early stage and evaluated their performance in this task. [18F]DMPY2 and [18F]DMPY3 were synthesized by direct radiofluorination of the bromo precursor, and radiochemical yields were ∼15-20%. Cell uptakes of [18F]DMPY2 and [18F]DMPY3 were >103-fold and 18-fold higher, respectively, in B16F10 (mouse melanoma) cells than in negative control cells. Biodistribution studies revealed strong tumor uptake and retention of [18F]DMPY2 (24.8% injected dose per gram of tissue [ID/g] at 60 min) and [18F]DMPY3 (11.7%ID/g at 60 min) in B16F10 xenografts. MicroPET imaging of both agents demonstrated strong tumoral uptake/retention and rapid washout, resulting in excellent tumor-to-background contrast in B16F10 xenografts. In particular, [18F]DMPY2 clearly visualized almost all metastatic lesions in lung and lymph nodes, with excellent image quality. [18F]DMPY2 demonstrated a significantly higher tumor-to-liver ratio than [18F]fluorodeoxyglucose ([18F]FDG) and the previously reported benzamide tracers N-[2-(diethylamino)-ethyl]-5-[18F]fluoropicolinamide ([18F]P3BZA) and N-[2-(diethylamino)-ethyl]-4-[18F]fluorobenzamide ([18F]FBZA) in B16F10-bearing or SK-MEL-3 (human melanoma)-bearing mice. In conclusion, [18F]DMPY2 might have strong potential for the diagnosis of early stage primary and metastatic melanoma using positron emission tomography (PET).

Glial TIM-3 Modulates Immune Responses in the Brain Tumor Microenvironment.

T-cell immunoglobulin and mucin domain-containing molecule 3 (TIM-3), a potential immunotherapeutic target for cancer, has been shown to display diverse characteristics in a context-dependent manner. Thus, it would be useful to delineate the precise functional features of TIM-3 in a given situation. Here, we report that glial TIM-3 shows distinctive properties in the brain tumor microenvironment. TIM-3 was expressed on both growing tumor cells and their surrounding cells including glia and T cells in an orthotopic mouse glioma model. The expression pattern of TIM-3 was distinct from those of other immune checkpoint molecules in tumor-exposed and tumor-infiltrating glia. Comparison of cells from tumor-bearing and contralateral hemispheres of a glioma model showed that TIM-3 expression was lower in tumor-infiltrating CD11b+CD45mid glial cells but higher in tumor-infiltrating CD8+ T cells. In TIM-3 mutant mice with intracellular signaling defects and Cre-inducible TIM-3 mice, TIM-3 affected the expression of several immune-associated molecules including iNOS and PD-L1 in primary glia-exposed conditioned media (CM) from brain tumors. Further, TIM-3 was cross-regulated by TLR2, but not by TLR4, in brain tumor CM- or Pam3CSK4-exposed glia. In addition, following exposure to tumor CM, IFNγ production was lower in T cells cocultured with TIM-3-defective glia than with normal glia. Collectively, these findings suggest that glial TIM-3 actively and distinctively responds to brain tumor, and plays specific intracellular and intercellular immunoregulatory roles that might be different from TIM-3 on T cells in the brain tumor microenvironment. SIGNIFICANCE: TIM-3 is typically thought of as a T-cell checkpoint receptor. This study demonstrates a role for TIM-3 in mediating myeloid cell responses in glioblastoma.