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Glial cell activation precedes neuronal cell death during brain aging and the progression of neurodegenerative diseases. Under neuroinflammatory stress conditions, lipocalin-2 (LCN2), also known as neutrophil gelatinase-associated lipocalin or 24p3, is produced and secreted by activated microglia and reactive astrocytes. Lcn2 expression levels are known to be increased in various cells, including reactive astrocytes, through the activation of the NF-κB signaling pathway. In the central nervous system, as LCN2 exerts neurotoxicity when secreted from reactive astrocytes, many researchers have attempted to identify various strategies to inhibit LCN2 production, secretion, and function to minimize neuroinflammation and neuronal cell death. These strategies include regulation at the transcriptional, posttranscriptional, and posttranslational levels, as well as blocking its functions using neutralizing antibodies or antagonists of its receptor. The suppression of NF-κB signaling is a strategy to inhibit LCN2 production, but it may also affect other cellular activities, raising questions about its effectiveness and feasibility. Recently, LCN2 was found to be a target of the autophagy‒lysosome pathway. Therefore, autophagy activation may be a promising therapeutic strategy to reduce the levels of secreted LCN2 and overcome neurodegenerative diseases. In this review, we focused on research progress on astrocyte-derived LCN2 in the central nervous system., (© 2023. The Author(s).)

Opisthorchis viverrini infection is an emerging disease in Cambodia, especially in central and southeastern areas. However, its status in northern areas bordering Lao PDR has been relatively unknown. The present study was performed to investigate the status of O. viverrini infection among people in Preah Vihear and Stung Treng provinces through fecal examination to detect eggs and recovery of adult flukes from some of the egg-positive cases. Fecal examinations were performed on a total of 1101 people from 10 villages in the 2 provinces using the Kato-Katz thick smear technique. For recovery of adult flukes and other helminth parasites 10 volunteers positive for eggs of Opisthorchis viverrini and/or minute intestinal flukes (Ov/MIF), in Kampong Sangkae village, Preah Vihear province, were administered a single oral dose of 40 mg/kg praziquantel plus 5-10 mg/kg of pyrantel pamoate and purged with 40-50 g magnesium salts. Adult trematodes, together with nematodes and cestodes expelled in diarrheic stools were collected under a stereomicroscope or with the naked eye. The proportion of egg-positive cases for overall liver and intestinal helminths was high but not notably different between the 2 provinces, 65.5% in Preah Vihear versus 64.7% in Stung Treng. The average proportion of Ov/MIF egg-positive cases was 59.8%. A total of 315 adult specimens of O. viverrini were recovered from the 10 volunteers (4-98 specimens per individual; mean, 32). A smaller number of Haplorchis taichui adults, an intestinal fluke, were found mixed-infected in 7 (103 specimens in total; 1-31 per individual; mean, 15) of the 10 volunteers. Adult specimens of hookworms, Enterobius vermicularis, Trichostrongylus sp., and a Taenia tapeworm strobila were recovered in some cases. Based on the results, it has been confirmed that the surveyed areas in Preah Vihear and Stung Treng provinces, Cambodia, are highly endemic areas of O. viverrini infection with a low-grade mixed infection with H. taichui., (© 2023. The Author(s).)

Toxoplasma gondii is an intracellular protozoan parasite that can modulate the microenvironment of infected hosts and is known to be associated with the incidence of brain tumor growth. In this study, we suggested that the exosomal microRNA-21 derived from Toxoplasma infection would contribute to the growth of brain tumors. Exosomes of BV2 microglial cells infected with Toxoplasma were characterized and confirmed internalization to U87 glioma cells. Exosomal miRNA expression profiles were analyzed using microRNA array and miR-21A-5p associated with Toxoplasma and tumor sorted. We also examined the mRNA level of tumor-associated genes in U87 glioma cells by changing the level of miR-21 within exosomes and the effects of exosomes on the proliferation of human U87 glioma cells. Expression of miRNA-21 was increased and anti-tumorigenic genes (FoxO1, PTEN, and PDCD4) were decreased in exosomes within T. gondii-infected U87 glioma cells. Toxoplasma-infected BV2-derived exosomes induced proliferation of U87 glioma cells. The exosomes induced the growth of U87 cells in a mouse tumor model. We suggest that the increased exosomal miR-21 from Toxoplasma-infected BV2 microglial cells may play an important role as a cell growth promotor of U87 glioma cells through a down-regulation of anti-tumorigenic genes., (© 2022. The Author(s).)

Background: While immune checkpoint inhibitors are becoming a standard of care for multiple types of cancer, the majority of patients do not respond to this form of immunotherapy. New approaches are required to overcome resistance to immunotherapies., Methods: We investigated the effects of adenoviral p53 (Ad-p53) gene therapy in combination with immune checkpoint inhibitors and selective IL2 or IL15 CD122/132 agonists in the aggressive B16F10 tumor model resistant to immunotherapies. To assess potential mechanisms of action, pre- and post- Ad-p53 treatment biopsies were evaluated for changes in gene-expression profiles by Nanostring IO 360 assays., Results: The substantial synergy of "triplet" Ad-p53 + CD122/132 + anti-PD-1 therapy resulted in potential curative effects associated with the complete tumor remissions of both the primary and contralateral tumors. Interestingly, contralateral tumors, which were not injected with Ad-p53 showed robust abscopal effects resulting in statistically significant decreases in tumor size and increased survival (p < 0.001). None of the monotherapies or doublet treatments induced the complete tumor regressions. Ad-p53 treatment increased interferon, CD8 + T cell, immuno-proteosome antigen presentation, and tumor inflammation gene signatures. Ad-p53 treatment also decreased immune-suppressive TGF-beta, beta-catenin, macrophage, and endothelium gene signatures, which may contribute to enhanced immune checkpoint inhibitor (CPI) efficacy. Unexpectedly, a number of previously unidentified, strongly p53 downregulated genes associated with stromal pathways and IL10 expression identified novel anticancer therapeutic applications., Conclusions: These results imply the ability of Ad-p53 to induce efficacious local and systemic antitumor immune responses with the potential to reverse resistance to immune checkpoint inhibitor therapy when combined with CD122/132 agonists and immune checkpoint blockade. Our findings further imply that Ad-p53 has multiple complementary immune mechanisms of action, which support future clinical evaluation of triplet Ad-p53, CD122/132 agonist, and immune checkpoint inhibitor combination treatment., (© 2021. The Author(s).)

Testis development, including early embryonic gonad formation and late postnatal spermatogenesis, is essential for the reproduction of higher metazoans to generate fertile gametes, called sperm. We have previously reported that the polyubiquitin gene Ubb is required for fertility in both male and female mice. In particular, the Ubb-null male mice showed an azoospermia phenotype due to arrest of spermatogenesis at the pachytene stage. Here, we analyzed the whole testis proteome at postnatal day 20 to define the molecular mediators of the male-infertility phenotype caused by Ubb knockout. From the identified proteome, 564 proteins were significantly and differentially expressed in Ubb-knockout testes and, among these, 36 downregulated proteins were involved at different stages of spermatogenesis. We also found that levels of piRNA metabolic process-related proteins, including Piwil2 and Tdrd1, were downregulated in Ubb-null testes through functional gene ontology analysis. Further, protein-protein interaction mapping revealed that 24 testis development-related proteins, including Hsp90aa1, Eef1a1, and Pabpc1, were directly influenced by the depletion of ubiquitin. In addition, the reduced mRNA levels of these proteins were observed in Ubb-knockout testes, which closely resembled the global downregulation of piRNA-metabolic gene expression at the transcriptional and post-transcriptional levels. Together with proteomic and transcriptional analyses, our data suggest that Ubb expression is essential for the maintenance of testicular RNA-binding regulators and piRNA-metabolic proteins to complete spermatogenesis in mice., (© 2021. The Author(s).)

Reduced levels of cellular ubiquitin (Ub) pools due to disruption of the polyubiquitin gene Ubb lead to dysregulation of neural stem cell (NSC) differentiation and impaired neuronal maturation in cells isolated from Ubb -/- mouse embryonic brains. However, it is currently unknown whether Ub is required for the specific stage of neuronal development or whether it plays a pleiotropic role throughout the process. To answer this question, we aimed to downregulate Ubb expression temporally during neuronal development, which could not be achieved in Ubb -/- cells. Therefore, we exploited lentivirus-mediated knockdown (KD) of Ubb at different stages of neuronal development, and investigated their phenotypes. Here, we report the outcome of Ubb KD on two independent culture days in vitro (DIV): DIV1 and DIV7. We observed that NSCs did not differentiate properly via Ubb KD on DIV1, but the maturation of already differentiated neurons was intact via Ubb KD on DIV7. Intriguingly, Ubb KD activated Notch signaling when it had been suppressed, but exerted no effect when it had already been activated. Therefore, our study suggests that Ub plays a pivotal role in NSC differentiation to suppress Notch signaling, but not in the subsequent maturation stages of neurons that had already been differentiated.

Current treatments of hepatocellular carcinoma (HCC) are ineffective and unsatisfactory in many aspects. Cancer-targeting gene virotherapy using oncolytic adenoviruses (OAds) armed with anticancer genes has shown efficacy and safety in clinical trials. Nowadays, both inhibitor of growth 4 (ING4), as a multimodal tumor suppressor gene, and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), as a potent apoptosis-inducing gene, are experiencing a renaissance in cancer gene therapy. Herein we investigated the antitumor activity and safety of mono- and combined therapy with OAds armed with ING4 (Ad-ΔB/ING4) and TRAIL (Ad-ΔB/TRAIL) gene, respectively, on preclinical models of human HCC. OAd-mediated expression of ING4 or TRAIL transgene was confirmed. Ad-ΔB/TRAIL and/or Ad-ΔB/ING4 exhibited potent killing effect on human HCC cells (HuH7 and Hep3B) but not on normal liver cells. Most importantly, systemic therapy with Ad-ΔB/ING4 plus Ad-ΔB/TRAIL elicited more eradicative effect on an orthotopic mouse model of human HCC than their monotherapy, without causing obvious overlapping toxicity. Mechanistically, Ad-ΔB/ING4 and Ad-ΔB/TRAIL were remarkably cooperated to induce antitumor apoptosis and immune response, and to repress tumor angiogenesis. This is the first study showing that concomitant therapy with Ad-ΔB/ING4 and Ad-ΔB/TRAIL may provide a potential strategy for HCC therapy and merits further investigations to realize its possible clinical translation.

Supercoiling DNA (folding DNA into a more compact molecule) from open circular forms requires significant bending energy. The double helix is coiled into a higher order helix form; thus it occupies a smaller footprint. Compact packing of DNA is essential to improve the efficiency of gene delivery, which has broad implications in biology and pharmaceutical research. Here we show that low-intensity pulsed ultrasound can pack open circular DNA into supercoil form. Plasmid DNA subjected to 5.4 mW/cm(2) intensity ultrasound showed significant (p-values <0.001) supercoiling compared to DNA without exposure to ultrasound. Radiation force induced from ultrasound and dragging force from the fluid are believed to be the main factors that cause supercoiling. This study provides the first evidence to show that low-intensity ultrasound can directly alter DNA topology. We anticipate our results to be a starting point for improved non-viral gene delivery.

Gene therapy using RNA interference can be directed against tumors through various strategies, but has been hindered owing to the inefficiency of non-viral delivery. To evaluate the antitumor effects of adenine nucleotide translocase-2 (ANT2) short hairpin RNA (shRNA) by intraperitoneal injection using the polyethylenimine (PEI) and an ultrasound gene delivery method, human breast carcinoma MDA-MB-231 cells were injected subcutaneously into NOG (NOD/Shi-scid/IL-2Rγ(null)) mice. The results showed greater tumor regression (*P<0.05) as well as an increased survival rate in the group receiving ANT2 shRNA+two types of enhancer relative to the groups receiving ANT2 shRNA without enhancer. These findings demonstrate that the introduction of PEI and ultrasound with SonoVue exerted enhanced antitumor effects in vivo. Although the combination of jet-PEI and ultrasound provided the best results with respect to tumor regression, the antitumor effects from the individual enhancers were approximately equivalent. In addition, we confirmed that there was no toxicity on aspartate aminotransferase and alanine aminotransferase levels in the liver and albumin, blood urea nitrogen or creatine kinase levels in the kidney following the various gene delivery methods.