Case report: Ghost cell odontogenic carcinoma in a dog: diagnostics and surgical outcome.

A 6 year-old spayed female Poodle presented with a mandibular mass. Radiographic examination revealed osteolysis from the right mandibular canine to the fourth premolar, along with horizontal bone loss and dorsal displacement of the right mandibular first and second premolars. Skull cone beam computed tomography revealed osteolysis at the level of the right mandibular canine and fourth premolar. A destructive bone lesion was observed in the apical area of the right mandibular canine, with mass invasion of the interradicular bone of the right mandibular first molar near the mandibular canal. Consequently, unilateral total mandibulectomy and skin flap surgery were performed. Histopathological examination revealed poorly demarcated and infiltrative neoplastic epithelial cells that formed small islands and trabeculae. Neoplastic cells exhibited the malignant features of cytological atypia and high mitotic activity. Furthermore, the neoplastic epithelial cells frequently showed ghost cell changes and were diagnosed as ghost cell odontogenic carcinoma (GCOC). The dog was followed up for 1 year, during which no severe complications or local recurrence was observed, except for slight mandibular drift, tongue protrusion, and drooling. This case report describes the clinical features, diagnostic imaging, and histologic features of an unreported GCOC in a dog and the favorable outcome following surgical resection.

Preventive effect of teriparatide on medication-related osteonecrosis of the jaw in rats.

This study aimed to investigate the preventive effect of teriparatide (TPD) administration on medication-related osteonecrosis of the jaw (MRONJ) before tooth extraction due to periodontal lesions in bilaterally ovariectomized female rats treated with zoledronic acid. Thirty skeletally mature Sprague-Dawley rats were randomly divided into three groups: control (CONT, n = 10), zoledronic acid (ZA, n = 10), and zoledronic acid and teriparatide (ZA-TPD, n = 10). The rats were sacrificed 8 weeks after tooth extraction. Micro-computed tomography analysis of the tibia showed that bone mineral density was highest in the CONT, followed by that in the ZA and ZA-TPD groups (CONT/ZA, p = 0.009; CONT/ZA-TPD, p < 0.001; ZA/ZA-TPD, p < 0.001). In the trabecular bone analysis of the extraction site, significant differences in specific bone surface (CONT/ZA, p = 0.010; CONT/ZA-TPD, p = 0.007; ZA/ZA-TPD, p = 0.002) and trabecular thickness (CONT/ZA-TPD, p = 0.002; ZA/ZA-TPD, p = 0.002) were observed. Histological analyses of the extraction sites revealed characteristic MRONJ lesions in the ZA group. Osteonecrosis, inflammatory cells, and sequestrum were less frequently observed in the ZA-TPD group than in the ZA group. In conclusion, TPD administration before tooth extraction helped reduce the occurrence of MRONJ in rats treated with zoledronic acid, confirming its preventative effects.

Preparation and characterization of lysozyme loaded liposomal dry powder inhalation using non-ionic surfactants.

Delivering protein drugs through dry powder inhalation (DPI) remains a significant challenge. Liposomes offer a promising solution, providing protection for proteins from external environment and controlled release capabilities. Furthermore, the use of non-ionic surfactants plays a crucial role in protecting the activity of proteins because of how the surfactants positioning themselves at the liquid-gas interface during the spray-drying process. In this study, lysozyme-loaded liposomal DPI formulations were prepared using various non-ionic surfactants, including polysorbate 80, poloxamer 188, poloxamer 407, and sucrose stearate. Lysozyme solution and 1,2-distearoyl-sn-glycero-3-phosphatidylcholine liposomes were subjected through high-pressure homogenization to form lysozyme-loaded liposomes. Formulations of homogenized lysozyme liposomes were spray-dried and further characterized. The particle size of reconstituted liposomal lysozyme DPI was from 129.5 to 816.9 nm. The formulations showed encapsulation efficiency up to 32.5% with zeta potential value of around - 30 mV, and spherical structures were observed. The aerosol dispersion performance of the dry powder inhalers was evaluated with emitted doses reaching up to 103% and fine particle fractions up to 28.4%. Significantly higher lysozyme activity was confirmed in formulation with drug to PS 80 ratio of 1: 0.5 w/w (92.1%) compared to that of formulation containing no surfactant (59.8%). The formulation stood out as the only formulation that maintained protein activity while demonstrating good aerosol performance.

Promoting angiogenesis and diabetic wound healing through delivery of protein transduction domain-BMP2 formulated nanoparticles with hydrogel.

Decreased angiogenesis contributes to delayed wound healing in diabetic patients. Recombinant human bone morphogenetic protein-2 (rhBMP2) has also been demonstrated to promote angiogenesis. However, the short half-lives of soluble growth factors, including rhBMP2, limit their use in wound-healing applications. To address this limitation, we propose a novel delivery model using a protein transduction domain (PTD) formulated in a lipid nanoparticle (LNP). We aimed to determine whether a gelatin hydrogel dressing loaded with LNP-formulated PTD-BMP2 (LNP-PTD-BMP2) could enhance the angiogenic function of BMP2 and improve diabetic wound healing. In vitro, compared to the control and rhBMP2, LNP-PTD-BMP2 induced greater tube formation in human umbilical vein endothelial cells and increased the cell recruitment capacity of HaCaT cells. We inflicted large, full-thickness back skin wounds on streptozotocin-induced diabetic mice and applied gelatin hydrogel (GH) cross-linked by microbial transglutaminase containing rhBMP2, LNP-PTD-BMP2, or a control to these wounds. Wounds treated with LNP-PTD-BMP2-loaded GH exhibited enhanced wound closure, increased re-epithelialization rates, and higher collagen deposition than those with other treatments. Moreover, LNP-PTD-BMP2-loaded GH treatment resulted in more CD31- and α-SMA-positive cells, indicating greater neovascularization capacity than rhBMP2-loaded GH or GH treatments alone. Furthermore, in vivo near-infrared fluorescence revealed that LNP-PTD-BMP2 has a longer half-life than rhBMP2 and that BMP2 localizes around wounds. In conclusion, LNP-PTD-BMP2-loaded GH is a viable treatment option for diabetic wounds.

Micellized protein transduction domain-bone morphogenetic protein-2 accelerates bone healing in a rat tibial distraction osteogenesis model.

The clinical application of growth factors such as recombinant human bone morphogenetic protein-2 (rh-BMP-2), for functional bone regeneration remains challenging due to limited in vivo efficacy and adverse effects of previous modalities. To overcome the instability and short half-life of rh-BMP-2 in vivo, we developed a novel osteogenic supplement by fusing a protein transduction domain (PTD) with BMP-2, effectively creating a prodrug of BMP-2. In this study, we first created an improved PTD-BMP-2 formulation using lipid nanoparticle (LNP) micellization, resulting in downsizing from micrometer to nanometer scale and achieving a more even distribution. The micellized PTD-BMP-2 (mPTD-BMP-2) demonstrated improved distribution and aggregation profiles. As a prodrug of BMP-2, mPTD-BMP-2 successfully activated Smad1/5/8 and induced mineralization with osteogenic gene induction in vitro. In vivo pharmacokinetic analysis revealed that mPTD-BMP-2 had a much more stable pharmacokinetic profile than rh-BMP-2, with a 7.5-fold longer half-life. The in vivo BMP-responsive element (BRE) reporter system was also successfully activated by mPTD-BMP-2. In the in vivo rat tibia distraction osteogenesis (DO) model, micro-computed tomography (micro-CT) scan findings indicated that mPTD-BMP-2 significantly increased bone volume, bone surface, axis moment of inertia (MOI), and polar MOI. Furthermore, it increased the expression of osteogenesis-related genes, and induced bone maturation histologically. Based on these findings, mPTD-BMP-2 could be a promising candidate for the next-generation osteogenesis drug to promote new bone formation in DO surgery. STATEMENT OF SIGNIFICANCE: This study introduces micellized bone morphogenetic protein-2 (mPTD-BMP-2), a next-generation osteogenic supplement that combines protein transduction domain (PTD) and nano-sized micelle formulation technique to improve transduction efficiency and stability. The use of PTD represents a novel approach, and our results demonstrate the superiority of mPTD-BMP-2 over rh-BMP-2 in terms of in vivo pharmacokinetic profile and osteogenic potential, particularly in a rat tibial model of distraction osteogenesis. These findings have significant scientific impact and potential clinical applications in the treatment of bone defects that require distraction osteogenesis. By advancing the field of osteogenic supplements, our study has the potential to contribute to the development of more effective treatments for musculoskeletal disorders.

Bone morphogenetic protein-7 attenuates pancreatic damage under diabetic conditions and prevents progression to diabetic nephropathy via inhibition of ferroptosis.

Background: Approximately 30% of diabetic patients develop diabetic nephropathy, a representative microvascular complication. Although the etiological mechanism has not yet been fully elucidated, renal tubular damage by hyperglycemia-induced expression of transforming growth factor-ß (TGF-ß) is known to be involved. Recently, a new type of cell death by iron metabolism called ferroptosis was reported to be involved in kidney damage in animal models of diabetic nephropathy, which could be induced by TGF-ß. Bone morphogenetic protein-7 (BMP7) is a well-known antagonist of TGF-ß inhibiting TGF-ß-induced fibrosis in many organs. Further, BMP7 has been reported to play a role in the regeneration of pancreatic beta cells in diabetic animal models. Methods: We used protein transduction domain (PTD)-fused BMP7 in micelles (mPTD-BMP7) for long-lasting in vivo effects and effective in vitro transduction and secretion. Results: mPTD-BMP7 successfully accelerated the regeneration of diabetic pancreas and impeded progression to diabetic nephropathy. With the administration of mPTD-BMP7, clinical parameters and representative markers of pancreatic damage were alleviated in a mouse model of streptozotocin-induced diabetes. It not only inhibited the downstream genes of TGF-ß but also attenuated ferroptosis in the kidney of the diabetic mouse and TGF-ß-stimulated rat kidney tubular cells. Conclusion: BMP7 impedes the progression of diabetic nephropathy by inhibiting the canonical TGF-ß pathway, attenuating ferroptosis, and helping regenerate diabetic pancreas.

Glycogen Synthase Kinase-3 Interaction Domain Enhances Phosphorylation of SARS-CoV-2 Nucleocapsid Protein.

A structural protein of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), nucleocapsid (N) protein is phosphorylated by glycogen synthase kinase (GSK)-3 on the serine/arginine (SR) rich motif located in disordered regions. Although phosphorylation by GSK-3ß constitutes a critical event for viral replication, the molecular mechanism underlying N phosphorylation is not well understood. In this study, we found the putative alpha-helix L/FxxxL/AxxRL motif known as the GSK-3 interacting domain (GID), found in many endogenous GSK-3ß binding proteins, such as Axins, FRATs, WWOX, and GSKIP. Indeed, N interacts with GSK-3ß similarly to Axin, and Leu to Glu substitution of the GID abolished the interaction, with loss of N phosphorylation. The N phosphorylation is also required for its structural loading in a virus-like particle (VLP). Compared to other coronaviruses, N of Sarbecovirus lineage including bat RaTG13 harbors a CDK1-primed phosphorylation site and Gly-rich linker for enhanced phosphorylation by GSK-3ß. Furthermore, we found that the S202R mutant found in Delta and R203K/G204R mutant found in the Omicron variant allow increased abundance and hyper-phosphorylation of N. Our observations suggest that GID and mutations for increased phosphorylation in N may have contributed to the evolution of variants.

Dual inhibition of CPT1A and G6PD suppresses glioblastoma tumorspheres.

PURPOSE: Limited treatment options are currently available for glioblastoma (GBM), an extremely lethal type of brain cancer. For a variety of tumor types, bioenergetic deprivation through inhibition of cancer-specific metabolic pathways has proven to be an effective therapeutic strategy. Here, we evaluated the therapeutic effects and underlying mechanisms of dual inhibition of carnitine palmitoyltransferase 1A (CPT1A) and glucose-6-phosphate dehydrogenase (G6PD) critical for fatty acid oxidation (FAO) and the pentose phosphate pathway (PPP), respectively, against GBM tumorspheres (TSs). METHODS: Therapeutic efficacy against GBM TSs was determined by assessing cell viability, neurosphere formation, and 3D invasion. Liquid chromatography-mass spectrometry (LC-MS) and RNA sequencing were employed for metabolite and gene expression profiling, respectively. Anticancer efficacy in vivo was examined using an orthotopic xenograft model. RESULTS: CPT1A and G6PD were highly expressed in GBM tumor tissues. Notably, siRNA-mediated knockdown of both genes led to reduced viability, ATP levels, and expression of genes associated with stemness and invasiveness. Similar results were obtained upon combined treatment with etomoxir and dehydroepiandrosterone (DHEA). Transcriptome analyses further confirmed these results. Data from LC-MS analysis showed that this treatment regimen induced a considerable reduction in the levels of metabolites associated with the TCA cycle and PPP. Additionally, the combination of etomoxir and DHEA inhibited tumor growth and extended survival in orthotopic xenograft model mice. CONCLUSION: Our collective findings support the utility of dual suppression of CPT1A and G6PD with selective inhibitors, etomoxir and DHEA, as an efficacious therapeutic approach for GBM.

Acceleration of bone formation by octacalcium phosphate composite in a rat tibia critical-sized defect.

Background: The osteogenic capabilities and biodegradability of octacalcium phosphate (OCP) composites make them unique. Despite the excellent characteristics of OCP, their use is limited due to handling difficulties. In this study, we aimed to evaluate and compare three types of OCPs (cemented OCP (C-OCP), C-OCP with collagen (OCP/Col), and synthetic OCP (S-OCP) with alginate (OCP/Alg)) versus commercially available ß-tricalcium phosphate (ß-TCP) regarding their potential to accelerate bone formation in defective rat tibias. Methods: The specimens with OCP composite were manufactured into 5 â€‹mm cubes and inserted into the segmental defects of rat tibias fixed with an external fixator. In addition, 3 â€‹mm-hole defects in rat tibias were evaluated to compare the graft material properties in different clinical situations. Serial X-ray studies were evaluated weekly and the tibias were harvested at postoperative 6 weeks or 8 weeks for radiologic evaluation. Histological and histomorphometric analyses were performed to evaluate the acceleration of bone formation. Results: In the critical-defect model, OCP/Alg showed bone bridges between segmentally resected bone ends that were comparable to those of ß-TCP. However, differences were observed in the residual graft materials. Most ß-TCP was maintained until 8 weeks postoperatively; however, OCP/Alg was more biodegradable. In addition calcification in the ß-TCP occurred at the directly contacted area between graft particles and bony ingrowth was observed in the region adjacent resected surface of tibia. In contrast, no direct bony ingrowth was observed in OCP-based materials, but osteogenesis induced from resected surface of tibia was more active. In the hole-defect model, OCP/Col accelerated bone formation. ß-TCP and OCP/Alg showed similar patterns with relatively higher biodegradability. In histology, among the OCP-based materials, directly contacted new bone was formed only in OCP/Alg group. The new bone formation in the periphery area of graft materials was much more active in the OCP-based materials, and the newly formed bone showed a thicker trabecular and more mature appearance than the ß-TCP group. Conclusions: In this study, OCP/Alg was equivalent to ß-TCP in the acceleration of bone formation with better biodegradability appropriate for clinical situations in different circumstances. Our OCP/Col composite showed fast degradation, which makes it unsuitable for use in mechanical stress conditions in clinical orthopedic settings. The Translational Potential of this Article: In our research, we compared our various manufactured OCP composites to commercially available ß-TCP in critical-defect rat tibia model. OCP/Col showed acceleration in hole-defect model as previous studies in dental field but in our critical-sized defect model it resorbed fast without acceleration of bony union. OCP/Alg showed matched results compared to ß-TCP and relatively fast resorption so we showed market value in special clinical indication depending on treatment strategy. This is the first OCP composite study in orthopaedics with animal critical-sized tibia bone study and further study should be considered for clinical application based on this study.

Etomoxir, a carnitine palmitoyltransferase 1 inhibitor, combined with temozolomide reduces stemness and invasiveness in patient-derived glioblastoma tumorspheres.

INTRODUCTION: The importance of fatty acid oxidation (FAO) in the bioenergetics of glioblastoma (GBM) is being realized. Etomoxir (ETO), a carnitine palmitoyltransferase 1 (CPT1) inhibitor exerts cytotoxic effects in GBM, which involve interrupting the FAO pathway. We hypothesized that FAO inhibition could affect the outcomes of current standard temozolomide (TMZ) chemotherapy against GBM. METHODS: The FAO-related gene expression was compared between GBM and the tumor-free cortex. Using four different GBM tumorspheres (TSs), the effects of ETO and/or TMZ was analyzed on cell viability, tricarboxylate (TCA) cycle intermediates and adenosine triphosphate (ATP) production to assess metabolic changes. Alterations in tumor stemness, invasiveness, and associated transcriptional changes were also measured. Mouse orthotopic xenograft model was used to elucidate the combinatory effect of TMZ and ETO. RESULTS: GBM tissues exhibited overexpression of FAO-related genes, especially CPT1A, compared to the tumor-free cortex. The combined use of ETO and TMZ further inhibited TCA cycle and ATP production than single uses. This combination treatment showed superior suppression effects compared to treatment with individual agents on the viability, stemness, and invasiveness of GBM TSs, as well as better downregulation of FAO-related gene expression. The results of in vivo study showed prolonged survival outcomes in the combination treatment group. CONCLUSION: ETO, an FAO inhibitor, causes a lethal energy reduction in the GBM TSs. When used in combination with TMZ, ETO effectively reduces GBM cell stemness and invasiveness and further improves survival. These results suggest a potential novel treatment option for GBM.

Risk Factors of Microscopically Tumor-Free Surgical Margins for Recurrence and Survival of Oral Squamous Cell Carcinoma Patients.

Objectives: The concept of adequate surgical margins remains controversial in oral squamous cell carcinoma (OSCC) surgery. This study aimed to identify surgical margin-related indicators that might impact recurrence and survival of OSCC patients. Materials and Methods: Histopathological examination was performed using hematoxylin-eosin-stained surgical margin tissue sections in 235 OSCC patients. Axin2 and Snail expression at the surgical margin was detected by immunohistochemistry. The impact of the Axin2-Snail cascade on tumorigenesis of the immortalized human oral keratinocyte (IHOK) line was investigated in vivo. Results: The width and dysplasia of surgical margins were not significantly associated with the outcome of OSCC patients. In a multivariate analysis using variable clinicopathologic factors and with Axin2 and Snail expression as cofactors, higher age (hazard ratio [HR]:1.050; P=0.047), Axin2 (HR:6.883; P=0.014), and Snail abundance (HR:5.663; P=0.009) had independent impacts on worsened overall survival. Similarly, lesion site in retromolar trigone (HR:4.077; P=0.010), upper (HR:4.332; P=0.005) and lower gingiva (HR:3.545; P=0.012), presence of extranodal extension (HR:9.967; P<0.001), perineural invasion (HR:3.627; P=0.024), and Snail abundance (HR:3.587; P<0.001) had independent impacts on worsened recurrence-free survival. Furthermore, Axin2 knockdown induced decreased Snail expression and attenuated tumorigenesis in the IHOK line. Conclusion: Histopathological examination of surgical margins may not be reliable to predict OSCC patient outcome. Molecular analysis may provide a more accurate risk assessment of surgical margins in OSCC. In particular, Axin2 and Snail are potential predictive biomarkers for the risk assessment of surgical margins in OSCC.

Potential Therapeutic Role of Bone Morphogenic Protein 7 (BMP7) in the Pathogenesis of Graves' Orbitopathy.

Purpose: We investigated a role of bone morphogenic protein 7 (BMP7), a member of the TGF-ß superfamily on pathogenic mechanism of Graves' orbitopathy (GO). The therapeutic effects of BMP7 on inflammation and fibrosis were evaluated in cultured Graves' orbital fibroblasts. Methods: Expression of BMP7 was compared in cultured orbital tissue explants from GO (n = 12) and normal control (n = 12) subjects using real-time PCR. Orbital fibroblasts were cultured from orbital connective tissues obtained from GO (n = 3) and normal control patients (n = 3). Cells were pretreated with recombinant human BMP7 (rhBMP7) before stimulation with TGF-ß, IL-1ß, and TNF-α. Fibrosis-related proteins and inflammatory cytokines were analyzed by Western blotting. The activation of signaling molecules in inflammation and fibrosis was also analyzed. Results: The expressions of BMP7 mRNA were lower in GO orbital tissues than control. Fibrosis-related proteins, fibronectin, collagen 1α, and α-SMA induced by TGF-ß were suppressed by treating rhBMP7, and rhBMP7 upregulated TGF-ß induced SMAD1/5/8 protein expression, whereas downregulated SMAD2/3. Increased pro-inflammatory molecules, IL-6, IL-8, and intercellular adhesion molecule-1 (ICAM-1) by IL-1ß or TNF-α were blocked by rhBMP7 treatment, and the expression of phosphorylated NFκB and Akt was suppressed by rhBMP7 treatment. Conclusions: BMP7 transcript levels were downregulated in Graves' orbital tissues. Exogenous BMP7 treatment showed inhibitory effects on the production of profibrotic proteins and proinflammatory cytokines in orbital fibroblasts. Our results provide a molecular basis of BMP7 as a new potential therapeutic agent through the opposing mechanism of profibrotic TGF-ß/SMAD signaling and proinflammatory cytokine production.

A micellized bone morphogenetic protein-7 prodrug ameliorates liver fibrosis by suppressing transforming growth factor-ß signaling.

Bone morphogenetic protein-7 (BMP-7) antagonizes transforming growth factor-ß (TGF-ß), which is critically involved in liver fibrogenesis. Here, we designed a micelle formulation consisting of a protein transduction domain (PTD) fused BMP-7 polypeptide (mPTD-BMP-7) to enhance endocytic delivery, and investigated its ability to ameliorate liver fibrosis. The mPTD-BMP-7 formulation was efficiently delivered into cells via endocytosis, where it inhibited TGF-ß mediated epithelial-mesenchymal transition. After successfully demonstrating delivery of fluorescently labeled mPTD-BMP-7 into the murine liver in vivo, we tested the mPTD-BMP-7 formulation in a murine liver fibrosis model, developed by repeated intraperitoneal injection of hepatotoxic carbon tetrachloride, twice weekly from 4 to 16 weeks. mPTD-BMP-7 effects were tested by injecting the mPTD-BMP-7 formulation (or vehicle control) into the lateral tail at a dose of 50 (n=8) or 500 µg/kg (n=10), also twice per week from 4 to 16 weeks. Vehicle-treated control mice developed fibrous septa surrounding the liver parenchyma and marked portal-to-portal bridging with occasional nodules, whereas mice treated with mPTD-BMP-7 showed only fibrous expansion of some portal areas, with or without short fibrous septa. Using the Ishak scoring system, we found that the fibrotic burden was significantly lower in mPTD-BMP-7 treated mice than in control mice (all P<0.001). Treatment with mPTD-BMP-7 protected tight junctions between hepatocytes and reduced extracellular matrix protein levels. It also significantly decreased mRNA levels of collagen 1A, smooth muscle α-actin, and connective tissue growth factor compared with that in control mice (all P<0.001). Collectively, out results indicate that mPTD-BMP-7, a prodrug formulation of BMP-7, ameliorates liver fibrosis by suppressing the TGF-ß signaling pathway in a murine liver fibrosis model.

Niclosamide and Pyrvinium Are Both Potential Therapeutics for Osteosarcoma, Inhibiting Wnt-Axin2-Snail Cascade.

Osteosarcoma, the most common primary bone malignancy, is typically related to growth spurts during adolescence. Prognosis is very poor for patients with metastatic or recurrent osteosarcoma, with survival rates of only 20-30%. Epithelial-mesenchymal transition (EMT) is a cellular mechanism that contributes to the invasion and metastasis of cancer cells, and Wnt signaling activates the EMT program by stabilizing Snail and ß-catenin in tandem. Although the Wnt/Snail axis is known to play significant roles in the progression of osteosarcoma, and the anthelmintic agents, niclosamide and pyrvinium, have been studied as inhibitors of the Wnt pathway, their therapeutic effects and regulatory mechanisms in osteosarcoma remain unidentified. In this study, we show that both niclosamide and pyrvinium target Axin2, resulting in the suppression of EMT by the inhibition of the Wnt/Snail axis in osteosarcoma cells. Axin2 and Snail are abundant in patient samples and cell lines of osteosarcoma. The treatment of niclosamide and pyrvinium inhibits the migration of osteosarcoma cells at nanomolar concentrations. These results suggest that Axin2 and Snail are candidate therapeutic targets in osteosarcoma, and that anthelminthic agents, niclosamide and pyrvinium, may be effective for osteosarcoma patients.

Competing Endogenous RNA of Snail and Zeb1 UTR in Therapeutic Resistance of Colorectal Cancer.

The epithelial-mesenchymal transition (EMT) comprises an important biological mechanism not only for cancer progression but also in the therapeutic resistance of cancer cells. While the importance of the protein abundance of EMT-inducers, such as Snail (SNAI1) and Zeb1 (ZEB1), during EMT progression is clear, the reciprocal interactions between the untranslated regions (UTRs) of EMT-inducers via a competing endogenous RNA (ceRNA) network have received little attention. In this study, we found a synchronized transcript abundance of Snail and Zeb1 mediated by a non-coding RNA network in colorectal cancer (CRC). Importantly, the trans-regulatory ceRNA network in the UTRs of EMT inducers is mediated by competition between tumor suppressive miRNA-34 (miR-34) and miRNA-200 (miR-200). Furthermore, the ceRNA network consisting of the UTRs of EMT inducers and tumor suppressive miRs is functional in the EMT phenotype and therapeutic resistance of colon cancer. In The Cancer Genome Atlas (TCGA) samples, we also found genome-wide ceRNA gene sets regulated by miR-34a and miR-200 in colorectal cancer. These results indicate that the ceRNA networks regulated by the reciprocal interaction between EMT gene UTRs and tumor suppressive miRs are functional in CRC progression and therapeutic resistance.

Prediction of African Swine Fever Virus Inhibitors by Molecular Docking-Driven Machine Learning Models.

African swine fever virus (ASFV) causes a highly contagious and severe hemorrhagic viral disease with high mortality in domestic pigs of all ages. Although the virus is harmless to humans, the ongoing ASFV epidemic could have severe economic consequences for global food security. Recent studies have found a few antiviral agents that can inhibit ASFV infections. However, currently, there are no vaccines or antiviral drugs. Hence, there is an urgent need to identify new drugs to treat ASFV. Based on the structural information data on the targets of ASFV, we used molecular docking and machine learning models to identify novel antiviral agents. We confirmed that compounds with high affinity present in the region of interest belonged to subsets in the chemical space using principal component analysis and k-means clustering in molecular docking studies of FDA-approved drugs. These methods predicted pentagastrin as a potential antiviral drug against ASFVs. Finally, it was also observed that the compound had an inhibitory effect on AsfvPolX activity. Results from the present study suggest that molecular docking and machine learning models can play an important role in identifying potential antiviral drugs against ASFVs.

Metformin and Niclosamide Synergistically Suppress Wnt and YAP in APC-Mutated Colorectal Cancer.

The Wnt and Hippo pathways are tightly coordinated and understanding their reciprocal regulation may provide a novel therapeutic strategy for cancer. Anti-helminthic niclosamide is an effective inhibitor of Wnt and is now in a phase II trial for advanced colorectal cancer (CRC) patients. We found that Axin2, an authentic target gene of canonical Wnt, acts as aYAP phosphorylation activator in APC-mutated CRC. While niclosamide effectively suppresses Wnt, it also inhibits Hippo, limiting its therapeutic potential for CRC. To overcome this limitation, we utilized metformin, a clinically available AMPK activator. This combinatory approach not only suppresses canonical Wnt activity, but also inhibits YAP activity in CRC cancer cells and in patient-derived cancer organoid through the suppression of cancer stemness. Further, combinatory oral administration suppressed in vivo tumorigenesis and the cancer progression of APC-MIN mice models. Our observations provide not only a reciprocal link between Wnt and Hippo, but also clinically available novel therapeutics that are able to target Wnt and YAP in APC-mutated CRC.

Exploring the chemical space of protein-protein interaction inhibitors through machine learning.

Although protein-protein interactions (PPIs) have emerged as the basis of potential new therapeutic approaches, targeting intracellular PPIs with small molecule inhibitors is conventionally considered highly challenging. Driven by increasing research efforts, success rates have increased significantly in recent years. In this study, we analyze the physicochemical properties of 9351 non-redundant inhibitors present in the iPPI-DB and TIMBAL databases to define a computational model for active compounds acting against PPI targets. Principle component analysis (PCA) and k-means clustering were used to identify plausible PPI targets in regions of interest in the active group in the chemical space between active and inactive iPPI compounds. Notably, the uniquely defined active group exhibited distinct differences in activity compared with other active compounds. These results demonstrate that active compounds with regions of interest in the chemical space may be expected to provide insights into potential PPI inhibitors for particular protein targets.

Exosome-based delivery of super-repressor IκBα ameliorates kidney ischemia-reperfusion injury.

Ischemia-reperfusion injury is a major cause of acute kidney injury. Recent studies on the pathophysiology of ischemia-reperfusion-induced acute kidney injury showed that immunologic responses significantly affect kidney ischemia-reperfusion injury and repair. Nuclear factor (NF)-ĸB signaling, which controls cytokine production and cell survival, is significantly involved in ischemia-reperfusion-induced acute kidney injury, and its inhibition can ameliorate ischemic acute kidney injury. Using EXPLOR, a novel, optogenetically engineered exosome technology, we successfully delivered the exosomal super-repressor inhibitor of NF-ĸB (Exo-srIĸB) into B6 wild type mice before/after kidney ischemia-reperfusion surgery, and compared outcomes with those of a control exosome (Exo-Naïve)-injected group. Exo-srIĸB treatment resulted in lower levels of serum blood urea nitrogen, creatinine, and neutrophil gelatinase-associated lipocalin in post-ischemic mice than in the Exo-Naïve treatment group. Systemic delivery of Exo-srIĸB decreased NF-ĸB activity in post-ischemic kidneys and reduced apoptosis. Post-ischemic kidneys showed decreased gene expression of pro-inflammatory cytokines and adhesion molecules with Exo-srIĸB treatment as compared with the control. Intravital imaging confirmed the uptake of exosomes in neutrophils and macrophages. Exo-srIĸB treatment also significantly affected post-ischemic kidney immune cell populations, lowering neutrophil, monocyte/macrophage, and T cell frequencies than those in the control. Thus, modulation of NF-ĸB signaling through exosomal delivery can be used as a novel therapeutic method for ischemia-reperfusion-induced acute kidney injury.

Tissue-specific biomarkers in gingival crevicular fluid are correlated with external root resorption caused by constant mechanical load: an in vivo study.

OBJECTIVES: This study investigated the association of changes in cementum protein-1 (CEMP-1), dentine phosphoprotein (DPP), and c-terminal cross-linked telopeptide of type I collagen (CTX-I) levels in human gingival crevicular fluid (GCF) under constant load with external root resorption volume and amount of tooth movement. MATERIALS AND METHODS: In total, 11 healthy adult patients (mean age, 23.5 years [range, 18.3-37.7]; four men and seven women) were enrolled. GCF samples were obtained from premolars at T0, T1 (1 day), T2 (1 week), T3 (2 weeks), T4 (4 weeks), and T5 (8 weeks) under constant 100-gm buccal tipping force. Opposite premolars were used as controls. Teeth were extracted at T5, followed by quantification of external root resorption volume and histological analysis. RESULTS: In the test group, T5/T0 ratios of CEMP-1 and DPP levels, differential CEMP-1 levels between T5 and T0, and differential DPP levels between T2 and T0 correlated positively with root resorption volume (r = 0.734, 0.730, 0.627, and 0.612, respectively, all p < 0.05). CEMP-1 levels at T0 and T3 correlated negatively with root resorption volume (r = -0.603 and -0.706; all p < 0.05). CTX-I levels at T5 correlated positively with the amount of tooth movement (r = 0.848, p < 0.01). CONCLUSIONS: Alterations in CEMP-1 and DPP levels in human GCF at specific timepoints during orthodontic treatment may be associated with different degrees of external root resorption. CLINICAL RELEVANCE: This study demonstrates that changes in the levels of tissue-specific biomarkers in GCF may facilitate early detection of external root resorption during orthodontic tooth movement.