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Background: Bone grafts are extensively used for repairing bone defects and voids in orthopedics and dentistry. Moldable bone grafts offer a promising solution for treating irregular bone defects, which are often difficult to fill with traditional rigid grafts. However, practical applications have been limited by insufficient mechanical strength and rapid degradation. Methods: This study developed a ceramic composite bone graft composed of calcium sulfate (CS), β-tricalcium phosphate (β-TCP) with/without graphene oxide (GO) nano-particles. The biomechanical properties, degradation rate, and in-vitro cellular responses were investigated. In addition, the graft was implanted in-vivo in a critical-sized calvarial defect model. Results: The results showed that the compressive strength significantly improved by 135% and the degradation rate slowed by 25.5% in comparison to the control model. The addition of GO nanoparticles also improved cell compatibility and promoted osteogenic differentiation in the in-vitro cell culture study and was found to be effective at promoting bone repair in the in-vivo animal model. Conclusions: The mixed ceramic composites presented in this study can be considered as a promising alternative for bone graft applications. [ABSTRACT FROM AUTHOR]

Simple Summary: Pancreatic cancer is surrounded by a dense fibrotic environment due to the activity of pancreatic stellate cells (PSCs). This hinders the effectiveness of treatment by limiting drug penetration and oxygen delivery. To better understand this, PSCs and cancer cells were studied in laboratory models using radiotracer imaging techniques. The results indicated that while glucose uptake and hypoxia were similar between cancer cells alone and in combination with PSCs, a specific marker of fibrosis (FAPα) was significantly higher in PSC-rich environments. In mice, the marker in question decreased in tumors with only PSCs, indicating that the cells had died. Conversely, the biomarker increased over time in tumors containing both cancer cells and PSCs, suggesting that mouse cells had invaded. This study sheds light on how the tumor environment influences metabolism and thus provides insights for more targeted theranostic applications. In turn, this approach supports the development of more reliable models. Background/Objective Pancreatic stellate cells (PSCs) in pancreatic adenocarcinoma (PDAC) are producing extracellular matrix, which promotes the formation of a dense fibrotic microenvironment. This makes PDAC a highly heterogeneous tumor-stroma-driven entity, associated with reduced perfusion, limited oxygen supply, high interstitial fluid pressure, and limited bioavailability of therapeutic agents. Methods In this study, spheroid and tumor xenograft models of human PSCs and PanC-1 cells were characterized radiopharmacologically using a combined positron emission tomography (PET) radiotracer approach. [18F]FDG, [18F]FMISO, and [18F]FAPI-74 were employed to monitor metabolic activity, hypoxic metabolic state, and functional expression of fibroblast activation protein alpha (FAPα), a marker of activated PSCs. Results In vitro, PanC-1 and multi-cellular tumor spheroids demonstrated comparable glucose uptake and hypoxia, whereas FAPα expression was significantly higher in PSC spheroids. In vivo, glucose uptake as well as the transition to hypoxia were comparable in PanC-1 and multi-cellular xenograft models. In mice injected with PSCs, FAPα expression decreased over a period of four weeks post-injection, which was attributed to the successive death of PSCs. In contrast, FAPα expression increased in both PanC-1 and multi-cellular xenograft models over time due to invasion of mouse fibroblasts. Conclusion The presented models are suitable for subsequently characterizing stromal cell-induced metabolic changes in tumors using noninvasive molecular imaging techniques. [ABSTRACT FROM AUTHOR]

Simple Summary: DNA-dependent protein kinase (DNA-PK) is a key player in repairing DNA damage. Ku proteins detect DNA damage and activate DNA-PK. Blocking DNA-PK can make cancer treatments such as radiation more effective. We have developed Ku–DNA binding inhibitors (Ku-DBis) that stop DNA-PK activation, making cancer cells more sensitive to radiation. We recently discovered new Ku-DBis that enter cells better than predecessor compounds, allowing cellular and in vivo analyses. Some non-small cell lung cancer (NSCLC) cells, especially those lacking ATM, were very sensitive to these inhibitors and radiation. However, cells lacking BRCA1 were resistant, which reduced the effectiveness of radiation. In animal studies of NSCLC, Ku-DBi treatment inhibited DNA-PK and enhanced a radiation-dependent decrease in tumor cell proliferation. This is the first time a Ku-targeted inhibitor has been shown to work in living organisms, suggesting their potential application in cancer treatment. Background: DNA-dependent protein kinase (DNA-PK) is a validated cancer therapeutic target involved in DNA damage response (DDR) and non-homologous end-joining (NHEJ) repair of DNA double-strand breaks (DSBs). Ku serves as a sensor of DSBs by binding to DNA ends and activating DNA-PK. Inhibition of DNA-PK is a common strategy to block DSB repair and improve efficacy of ionizing radiation (IR) therapy and radiomimetic drug therapies. We have previously developed Ku–DNA binding inhibitors (Ku-DBis) that block in vitro and cellular NHEJ activity, abrogate DNA-PK autophosphorylation, and potentiate cellular sensitivity to IR. Results and Conclusions: Here we report the discovery of oxindole Ku-DBis with improved cellular uptake and retained potent Ku-inhibitory activity. Variable monotherapy activity was observed in a panel of non-small cell lung cancer (NSCLC) cell lines, with ATM-null cells being the most sensitive and showing synergy with IR. BRCA1-deficient cells were resistant to single-agent treatment and antagonistic when combined with DSB-generating therapies. In vivo studies in an NSCLC xenograft model demonstrated that the Ku-DBi treatment blocked IR-dependent DNA-PKcs autophosphorylation, modulated DDR, and reduced tumor cell proliferation. This represents the first in vivo demonstration of a Ku-targeted DNA-binding inhibitor impacting IR response and highlights the potential therapeutic utility of Ku-DBis for cancer treatment. [ABSTRACT FROM AUTHOR]

Mitochondria are dynamic bioenergetic hubs that become compromised with age. In neurons, declining mitochondrial axonal transport has been associated with reduced cellular health. However, it is still unclear to what extent the decline of mitochondrial transport and function observed during ageing are coupled, and if somal and axonal mitochondria display compartment‐specific features that make them more susceptible to the ageing process. It is also not known whether the biophysical state of the cytoplasm, thought to affect many cellular functions, changes with age to impact mitochondrial trafficking and homeostasis. Focusing on the mouse peripheral nervous system, we show that age‐dependent decline in mitochondrial trafficking is accompanied by reduction of mitochondrial membrane potential and intramitochondrial viscosity, but not calcium buffering, in both somal and axonal mitochondria. Intriguingly, we observe a specific increase in cytoplasmic viscosity in the neuronal cell body, where mitochondria are most polarised, which correlates with decreased cytoplasmic diffusiveness. Increasing cytoplasmic crowding in the somatic compartment of DRG neurons grown in microfluidic chambers reduces mitochondrial axonal trafficking, suggesting a mechanistic link between the regulation of cytoplasmic viscosity and mitochondrial dynamics. Our work provides a reference for studying the relationship between neuronal mitochondrial homeostasis and the viscoelasticity of the cytoplasm in a compartment‐dependent manner during ageing. [ABSTRACT FROM AUTHOR]

Objectives: The pathogenesis of temporomandibular joint osteoarthritis (TMJOA) remains not fully understood. Our previous studies demonstrated that miR‐21‐5p may participate in the TMJOA development and the interaction between circRNA‐ACAP2 (CircACAP2) and miR‐21‐5p. Our present study aimed to explore the biological functions and regulatory mechanisms of CircACAP2 in TMJOA. Materials and Methods: The differential expression pattern of CircACAP2 in OA and normal tissues or cells was detected. CircACAP2 biological functions experiments were performed in chondrocytes by overexpression and interference techniques. The interaction of CircACAP2 with miR‐21‐5p and downstream target mRNA, polymorphic adenoma gene 1 (PLAG1), was predicted by bioinformatic databases and then demonstrated by dual‐luciferase reporter assay. The biological role of CircACAP2 in TMJOA was investigated and validated in a mouse model. Results: The expression level of CircACAP2 was markedly reduced in OA cartilage and directly related to chondrocyte proliferation and apoptosis as well as ECM metabolism in the cartilage. CircACAP2 functioned in chondrocytes via targeting miR‐21‐5p and PLAG1. Overexpressing of CircACAP2 alleviated TMJOA in mouse models. Conclusions: The present study unveiled that CircACAP2/miR‐21‐5p/PLAG1 axis may play an important regulatory role in TMJOA progression, which may highlight a potentially effective intervention and therapeutic strategy for the treatment of TMJOA. [ABSTRACT FROM AUTHOR]

This study investigated the antiobesity effects of black ginger extract (BGE) in high-fat diet (HFD)-induced obese mice. Mice were divided into six groups: normal diet control (NC, AIN-93G normal diet), 60% HFD control (HFD), HFD containing metformin at 250 mg/kg b.w. (Met, positive control), and HFD containing BGE at 5, 10, or 20 mg/kg b.w. for 15 weeks. BGE administration significantly prevented HFD-induced increases in weight gain, organ weight, and adipose tissue mass. Furthermore, it resulted in decreased adipogenesis and lipogenesis-related factors, including phosphorylated mitogen-activated protein kinase, peroxisome proliferator-activated receptor gamma, CCAAT/enhancer-binding proteins, sterol regulatory element-binding protein 1, phosphorylated cAMP response element-binding protein, glucose-6-phosphate dehydrogenase, fatty acid synthase, dephosphorylated ATP-citrate lyase, dephosphorylated acetyl-CoA carboxylase, and lipoprotein lipase, in white adipose tissues. Moreover, BGE administration enhanced lipolysis in white adipose tissue, as evidenced by elevated levels of adipose triglyceride lipase, phosphorylated hormone-sensitive lipase, and protein kinase A, along with reduced levels of perilipin and phosphodiesterase 3B. BGE induced thermogenesis in brown adipose tissues, as reflected by the increased expression of AMP-activated protein kinase, uncoupling protein 1, and carnitine palmitoyltransferase 1 and decreased levels of fatty acid-binding protein 4. In conclusion, this study provides comprehensive evidence supporting the antiobesity effects of BGE, elucidating the underlying molecular mechanisms involved in preventing weight gain, suppressing adipogenesis, promoting lipolysis, and stimulating thermogenesis. These findings suggest the potential therapeutic utility of BGE in combating obesity and associated metabolic disorders (KHGASP-2023-034). [ABSTRACT FROM AUTHOR]

Decidual macrophages residing at the maternal-fetal interface have been recognized as pivotal factors for maintaining normal pregnancy; however, they are also key target cells of Toxoplasma gondii (T. gondii) in the pathology of T. gondii-induced adverse pregnancy. Trem2, as a functional receptor on macrophage surface, recognizes and binds various kinds of pathogens. The role and underlying mechanism of Trem2 in T. gondii infection remain elusive. In the present study, we found that T. gondii infection downregulated Trem2 expression and that Trem2-/- mice exhibited more severe adverse pregnancy outcomes than wildtype mice. We also demonstrated that T. gondii infection resulted in increased decidual macrophages, which were significantly reduced in the Trem2-/- pregnant mouse model as compared to wildtype control animals. We further described the inhibited proliferation, migration, and invasion functions of trophoblast cell by T. gondii antigens through macrophages as an "intermediate bridge", while this inhibition can be rescued by Trem2 agonist HSP60. Concurrently, Trem2 deficiency in bone marrow-derived macrophages (BMDMs) heightened the inhibitory effect of TgAg on the migration and invasion of trophoblast cells, accompanied by higher pro-inflammatory factors (IL-1β, IL-6 and TNF-α) but a lower chemokine (CXCL1) in T. gondii antigens-treated BMDMs. Furthermore, compelling evidence from animal models and in vitro cell experiments suggests that T. gondii inhibits the Trem2-Syk-PI3K signaling pathway, leading to impaired function of decidual macrophages. Therefore, our findings highlight Trem2 signaling as an essential pathway by which decidual macrophages respond to T. gondii infection, suggesting Trem2 as a crucial sensor of decidual macrophages and potential therapeutic target in the pathology of T. gondii-induced adverse pregnancy. Author summary: T. gondii is a critically important intracellular protozoan that is a major cause of adverse pregnancy outcomes in humans and livestock. The pathology of adverse pregnancy induced by T. gondii involves the dysfunction of decidual macrophages. Trem2, a functional immune receptor on the surface of decidual macrophages, governs its survival and phagocytosis. Here, we sought to elucidate whether Trem2 signaling is involved in the pathology of T. gondii-induced adverse pregnancy. We found that T. gondii infection led to the downregulation of Trem2 expression in macrophages, both in vivo and in vitro. Trem2-/- mice exhibit more severe adverse pregnancy outcomes after T. gondii infection. We further described the inhibited proliferation, migration, and invasion functions of trophoblast cell by T. gondii antigens through macrophages as an "intermediate bridge", while this inhibition can be rescued by Trem2 agonist HSP60. In parallel, Trem2 deficiency in BMDMs heightened the inhibitory effect of TgAg on the migration and invasion of trophoblast cells. Our work uncovers an important factor involved in the pathological mechanism of T. gondii-induced adverse pregnancy, and research on Trem2 may provide new preventive and therapeutic targets for toxoplasmosis. [ABSTRACT FROM AUTHOR]

The role of tweety homolog 3 (TTYH3) has been studied in several cancers, including hepatocellular carcinoma, cholangiocarcinoma, and gastric cancer. The results showed that TTYH3 is highly expression in cervical cancer tissues and cells and high TTYH3 expression correlates with poor prognosis in patients with cervical cancer. TTYH3 markedly reduced the apoptosis rate and promoted proliferation, migration, and invasion. Silencing of TTYH3 has been shown to have an inhibitory effect on cervical cancer progression. Moreover, TTYH3 enhanced EMT and activated Wnt/β-catenin signaling. Furthermore, TTYH3 knockdown inhibited the tumor growth in vivo. In conclusion, TTYH3 promoted cervical cancer progression by activating the Wnt/β-catenin signaling. HIGHLIGHTS: TTYH3 is upregulated in cervical cancer tissue and cells. TTYH3 promotes cervical cancer cell proliferation. TTYH3 inhibits cervical cancer cell apoptosis. TTYH3 induces cervical cancer cell migration and invasion. TTYH3 activates the Wnt signaling in cervical cancer cell. [ABSTRACT FROM AUTHOR]

Background: An important signaling pathway connecting illness and natural immunity is the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway, but aberrant activation of this pathway is associated with the development of autoimmune and inflammatory diseases. Hence, targeted inhibition of the activation of the cGAS-STING pathway is potentially valuable in the treatment of disease. The primary active component of Salvia miltiorrhiza is total tanshinone (TTN). Research has indicated that TTN possesses noteworthy anti-inflammatory properties. However, the protective mechanism of TTN against acute liver injury (ALI) and autoimmune diseases is unknown. Methods: A model of aberrant activation of the cGAS-STING pathway was established in various cells and treated with TTN, and the expression of cGAS-STING pathway-related proteins, type I interferon, interferon stimulated genes and inflammatory factors was assessed by western blotting, real-time qPCR. Immunofluorescence analysis of the effect of TTN on the entry of associated proteins into the nucleus following aberrant activation of the cGAS-STING pathway. The effect of TTN on STING oligomerisation was investigated using 2'-3'-cyclic GMP-AMP (2',3'-cGAMP) to induce STING oligomerisation. Western blotting was used to examine the impact of TTN on the interactions of STING, tank-binding kinase 1 (TBK1), and interferon regulatory factor 3 (IRF3) after HA or Flag-labelled plasmids were transfected into HEK-293 T cells. A dimethylxanthenone-4-acetic acid (DMXAA) -induced activation model of the cGAS-STING pathway in mice was established to study the effect of TTN on aberrant activation of the cGAS-STING pathway in vivo. On the other hand, an animal model of lipopolysaccharide/D-galactosamine (LPS/D-GaIN)-induced ALI and an autoimmune disease model induced by trex1 knockout were established to study the effects of TTN on inflammatory and autoimmune diseases mediated by the cGAS-STING pathway in vivo. Results: In several models of aberrant activation of the cGAS-STING pathway, TTN significantly inhibited the phosphorylation of STING and IRF3, thereby suppressing the expression of type I interferon, interferon-stimulated genes and inflammatory factors. Additionally, TTN prevented P65 and IRF3 from entering the nucleus after the cGAS-STING signalling pathway was abnormally activated. Subsequent research indicated that TTN was not involved in the oligomerization of STING or the integration of STING-TBK1 and TBK1-IRF3. However, TTN was found to have a substantial effect on the binding process between STING and IRF3. On the other hand, DMXAA-induced STING activation and activation of downstream signalling in vivo are inhibited by TTN. Furthermore, TTN exhibits positive treatment effects on autoimmune diseases caused by deficiency of trex1 and LPS/D-GaIN-induced ALI. Conclusion: Our research indicates that TTN effectively treats ALI and autoimmune illnesses mediated by the cGAS-STING pathway by inhibiting the abnormal activation of this pathway. [ABSTRACT FROM AUTHOR]

53BP1 plays a crucial role in regulating DNA damage repair pathway choice and checkpoint signaling in somatic cells; however, its role in meiosis has remained enigmatic. In this study, we demonstrate that the Caenorhabditis elegans ortholog of 53BP1, HSR-9 , associates with chromatin in both proliferating and meiotic germ cells. Notably, HSR-9 is enriched on the X chromosome pair in pachytene oogenic germ cells. HSR-9 is also present at kinetochores during both mitotic and meiotic divisions but does not appear to be essential for monitoring microtubule–kinetochore attachments or tension. Using cytological markers of different steps in recombinational repair, we found that HSR-9 influences the processing of a subset of meiotic double-stranded breaks into COSA-1 -marked crossovers. Additionally, HSR-9 plays a role in meiotic X chromosome segregation under conditions where X chromosomes fail to pair, synapse, and recombine. Together, these results highlight that chromatin-associated HSR-9 has both conserved and unique functions in the regulation of meiotic chromosome behavior. [ABSTRACT FROM AUTHOR]

Simple Summary: Aquaporins (AQPs) have emerged as potential predictors of response to cancer therapy and as targets for increasing sensitivity to treatment. However, systemic therapeutic inhibition using pan-AQP or AQP-specific inhibitors has not been characterized for efficacy in treating breast cancer or as a component of combination treatment. We evaluated AQP inhibition using established AQP inhibitors in cytotoxicity and pharmacologic assays. This study identified AQPs as a targetable vulnerability in breast cancer. AQP inhibitors can increase therapeutic efficacy, both as single agents and in a combination therapy strategy, in treating breast cancer progression and metastasis. These results provide significant insights that support the future development of improved AQP inhibitors. AQPs contribute to breast cancer progression and metastasis. We previously found that genetic inhibition of Aqp7 reduces primary tumor burden and metastasis in breast cancer. In this study, we utilized two AQP inhibitors, Auphen and Z433927330, to evaluate the efficacy of therapeutic inhibition of AQPs in breast cancer treatment. The inhibitors were evaluated in breast cancer for both cytotoxicity and metabolic stability assays across both murine and human breast cancer cell lines. Both AQP inhibitors also affected the expression of other AQP transcripts and proteins, which demonstrates compensatory regulation between AQP family members. As a single agent, Auphen treatment in vivo extended overall survival but did not impact primary or metastatic tumor burden. However, Auphen treatment made cells more responsive to chemotherapy (doxorubicin) or endocrine treatment (tamoxifen, fulvestrant). In fact, treatment with Tamoxifen reduced overall AQP7 protein expression. RNA-seq of breast cancer cells treated with Auphen identified mitochondrial metabolism genes as impacted by Auphen and may contribute to reducing mammary tumor progression, lung metastasis, and increased therapeutic efficacy of endocrine therapy in breast cancer. Interestingly, we found that Auphen and tamoxifen cooperate to reduce breast cancer cell viability, which suggests that Auphen treatment makes the cells more susceptible to Tamoxifen. Together, this study highlights AQPs as therapeutic vulnerabilities of breast cancer metastasis that are promising and should be exploited. However, the pharmacologic results suggest additional chemical refinements and optimization of AQP inhibition are needed to make these AQP inhibitors appropriate to use for therapeutic benefit in overcoming endocrine therapy resistance. [ABSTRACT FROM AUTHOR]

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Background: Mitochondrial (mt) heteroplasmy can cause adverse biological consequences when deleterious mtDNA mutations accumulate disrupting "normal" mt-driven processes and cellular functions. To investigate the heteroplasmy of such mtDNA changes, we developed a moderate throughput mt isolation procedure to quantify the mt single-nucleotide variant (SNV) landscape in individual mouse neurons and astrocytes. In this study, we amplified mt-genomes from 1645 single mitochondria isolated from mouse single astrocytes and neurons to (1) determine the distribution and proportion of mt-SNVs as well as mutation pattern in specific target regions across the mt-genome, (2) assess differences in mtDNA SNVs between neurons and astrocytes, and (3) study co-segregation of variants in the mouse mtDNA. Results: (1) The data show that specific sites of the mt-genome are permissive to SNV presentation while others appear to be under stringent purifying selection. Nested hierarchical analysis at the levels of mitochondrion, cell, and mouse reveals distinct patterns of inter- and intra-cellular variation for mt-SNVs at different sites. (2) Further, differences in the SNV incidence were observed between mouse neurons and astrocytes for two mt-SNV 9027:G > A and 9419:C > T showing variation in the mutational propensity between these cell types. Purifying selection was observed in neurons as shown by the Ka/Ks statistic, suggesting that neurons are under stronger evolutionary constraint as compared to astrocytes. (3) Intriguingly, these data show strong linkage between the SNV sites at nucleotide positions 9027 and 9461. Conclusions: This study suggests that segregation as well as clonal expansion of mt-SNVs is specific to individual genomic loci, which is important foundational data in understanding of heteroplasmy and disease thresholds for mutation of pathogenic variants. [ABSTRACT FROM AUTHOR]

• Natural killer (NK) cell activity is low and NK cell maturation is suppressed in diabetic mice. • Interferon-γ production of splenocyte is low in diabetic mice. • Transforming growth factor-β expression in spleen is high in diabetic mice. • Enhanced NK cell activity may be associated with changes in proportion of mature NK cells. • Vitamin D supplementation enhanced interleukin-12 production. • Vitamin D supplementation enhanced Bcl2 and Tbx21 expression in NK cells. Type 2 diabetes mellitus negatively affects the immune system, resulting in reduced natural killer (NK) cell activity. Vitamin D has been shown to regulate innate and adaptive immune cells. However, the effects of vitamin D on NK cells remain inconclusive, especially in the context of diabetes. We hypothesized that dietary vitamin D 3 supplementation can enhance NK cell activity in diabetic mice. Therefore, we investigated the effects of dietary vitamin D 3 on NK cell activity in control and diabetic mice and explored the mechanisms of NK cell activity modulation by vitamin D 3. Control (CON) and diabetic mice (db/db) were randomly divided into 2 groups, then fed either a control diet (948 IU vitamin D 3 /kg diet, vDC) or a diet supplemented with vitamin D 3 (9,477 IU vitamin D 3 /kg diet, vDS) for 8 weeks. Diabetic mice exhibited lower NK cell activity than control mice. The vDS group had significantly higher NK cell activity than the vDC group in both control and diabetic mice. The vDS group had a higher percentage of CD11b single-positive NK cells than the vDC group (CON-vDS 34%; db/db-vDS 30%; CON-vDC 27%; db/db-vDC 22%). The intracellular expression of splenic TGF-β was significantly higher in the db/db group than in the CON group. Overall, vDS group had higher Bcl2 and Tbx21 mRNA expressions than the vDC group. In conclusion, the present study shows that NK cell activity is impaired under diabetic conditions, possibly due to the reduced percentage of mature NK cells. Moreover, NK activity is enhanced by dietary supplementation in both control and diabetic mice that may be associated with changes in the proportion of mature NK cells. The purpose of the study is to investigate the effects of vitamin D on NK cell activity in diabetic conditions. The results showed that diabetic mice showed impaired NK cell activity with lower percentage of mature NK cells and NKG2D expression. However, dietary vitamin D 3 -supplementated group showed enhanced NK cell activity in diabetic mice, along with increased production of NK-stimulating cytokines and increased levels of mRNAs associated with NK cell maturation.Abbreviations: Bcl-2, B-cell lymphoma/leukemia-2; CD, cluster of differentiation; IFN-γ, Interferon gamma; IL, interleukin; NK cells, Natural killer cells; NKG2D, Natural killer group 2D; Tbx21, T-box transcription factor 21; TGF-β, Transforming growth factor beta. [Display omitted] [ABSTRACT FROM AUTHOR]

• High-fat diet during early life reshapes the gut microbiome later in life. • Early-life gut dysbiosis associates with disrupted mammary microenvironment later. • Shift of gut microbiome during early life may influence breast health later on. The influence of gut microbiota on gut health is well-documented, but it remains obscure for extraintestinal diseases such as breast cancer. Moreover, it is entirely unknown how gut dysbiosis during early life contributes to breast tumorigenesis later in life. In this study, we hypothesized that a high-fat diet during early life leads to alterations in the gut microbiome and is associated with disruptions in the mammary microenvironment. Female C57BL/6 mice were fed a low-fat diet (10% kcal fat) or a high-fat diet (HF, 60% kcal fat) for 8 weeks from the age of 4 to 12 weeks, which is equivalent to human childhood and adolescence. Twelve mice were sacrificed immediately after the 8-week feeding, the remainder were euthanized after switching to a normal lifecycle-supporting diet for an additional 12 weeks; the gut microbiome was then sequenced. The 8-week HF diet feeding altered the beta-diversity (Bray & Jaccard P <.01), and the difference remained significant after switching the diet (Bray & Jaccard P <.05). Immediately after HF feeding, a greater number of microbial taxa (>50) were altered, and about half of the taxa (25) remained significantly changed after switching the diet. The abundance of Alistipes, Bilophila , and Rikenellaceae stood out as significantly associated with multiple metabolic and inflammatory biomarkers in mammary tissue, including aromatase, Ccl2 , and Cox2. In conclusion, an 8-week early-life HF feeding reshaped the gut microbiome, which connected with disrupted mammary microenvironments. A high-fat diet during early life (4–12 weeks of age in mice), corresponding to childhood and adolescence in humans, significantly influences the gut microbiome and consequently affects the mammary microenvironment, which may ultimately lead to increase the risk of young-onset breast cancer later in life (24 weeks of age in mice), equivalent to the young to middle ages of adulthood in humans. [Display omitted] [ABSTRACT FROM AUTHOR]

Background: The aim of this study is to investigate the co-culture effects of human endometrial mesenchymal stem cells (EnMSCs) with mouse oocytes to enhance their maturation and development by using the hanging drop and sodium alginate hydrogel methods. Materials and Methods: In this experimental study, we prepared human EnMSCs (2.5x105 cells/mL) and co-cultured them with partially denuded mouse oocytes by the hanging drop (n=120) and sodium alginate hydrogel (n=120) methods. Control oocytes (n=230, total) were cultured in both systems in the absence of human EnMSCs for 18 hours. Both survival and maturation rates of the oocytes were analysed morphologically. After insemination with capacitated sperm, the fertiliza tion and development of the embryos up to the blastocyst stage were assessed and compared statistically for all of the study groups via one-way ANOVA and the t tests. Results: Oocytes cultured in the hanging drop method had a significantly higher survival rate than their control group (92.60 ± 4.36% vs. 84.20 ± 3.12%, P=0.018). There were no significant differences between the two experimental groups in terms of survival. The mean percent of oocytes that reached the metaphase II (MII) stage was 64.35 ± 3.19% and fertilised was 62.25 ± 4.43% in the hanging drop method; these rates were 63.43 ± 1.92% and 58.14 ± 4.14 in sodium alginate hydrogel method, respectively. These rates were higher than their controls (P<0.050), but there were no statistical differences between the two experimental groups (P>0.050). Among the studied groups, the highest significant blastocyst rate (32.55 ± 2.18%) was observed in the hanging drop experimental group (P=0.0017). Conclusion: The results of this study show that human EnMSCs improve the survival, maturation, and development rates of oocytes and they could have future clinical applications. [ABSTRACT FROM AUTHOR]

Simple Summary: Targeted therapies can induce strong tumor regression, but they typically fail to eradicate metastatic cancers. The elucidation of the causes that enable cancers to survive within a residual disease is essential for finding eradication strategies. The ability of cancers to survive eradication reflects not only cell-intrinsic sensitivities, but also microenvironmental effects. Paracrine signals produced by fibroblast, non-neoplastic cells that make tumor stroma, can provide strong but spatially limited therapy protection. Even though this phenomenon is well-established, its contribution to the ability of tumors to escape eradication remains poorly understood. To address this gap of knowledge, we developed an in silico model that recapitulates the effect of stroma on therapy responses in tumor tissues. This model enabled us to evaluate the contribution of spatial aspects of stroma-mediated resistance. Our analyses reveal that stroma dispersal might be the most important yet overlooked aspect of stromal resistance that determines the overall tumor responses to therapy. The response of tumors to anti-cancer therapies is defined not only by cell-intrinsic therapy sensitivities but also by local interactions with the tumor microenvironment. Fibroblasts that make tumor stroma have been shown to produce paracrine factors that can strongly reduce the sensitivity of tumor cells to many types of targeted therapies. Moreover, a high stroma/tumor ratio is generally associated with poor survival and reduced therapy responses. However, in contrast to advanced knowledge of the molecular mechanisms responsible for stroma-mediated resistance, its effect on the ability of tumors to escape therapeutic eradication remains poorly understood. To a large extent, this gap of knowledge reflects the challenge of accounting for the spatial aspects of microenvironmental resistance, especially over longer time frames. To address this problem, we integrated spatial inferences of proliferation-death dynamics from an experimental animal model of targeted therapy responses with spatial mathematical modeling. With this approach, we dissected the impact of tumor/stroma distribution, magnitude and distance of stromal effects. While all of the tested parameters affected the ability of tumor cells to resist elimination, spatial patterns of stroma distribution within tumor tissue had a particularly strong impact. [ABSTRACT FROM AUTHOR]

Objective: To explore the effect of Dickkopf-1 (DKK1) expression on CD4+ T cell polarization in malignant tumors and its potential value as a target for cancer immunotherapy. Methods: Bioinformatics algorithm was used to analyze the expression of DKK1 in multiple types of tumor tissues and adjacent tissues, and the correlation between the expression of DKK1 and the prognosis of cancer patients and immune infiltration of tumor microenvironment. The effect of DKK1 protein on the phenotype of CD4+ T cells was analyzed by flow cytometry in vitro. A melanoma B16F10 cell mouse subcutaneous transplantation tumor model was established to observe the effects of blocking DKK1 on the growth of mouse transplantation tumor and immune cell infiltration and phenotype in transplantation tumor tissues. Results: The mRNA expression levels of DKK1 in many kinds of tumor tissues were significantly higher than those in adjacent tissues. High expression of DKK1 was related to the poor prognosis of most tumor patients. In most types of tumors DKK1 played important negative regulatory role in CD4+ T cell anti-tumor immune response (P<0.05 or P<0.01). The results of flow cytometry in vitro showed that DKK1 protein stimulation significantly decreased the expression levels of T-bet, IFN-γ and CD107a in CD4+ T cells (all P<0.01). In the mouse subcutaneous melanoma model, it was found that blocking DKK1 could significantly inhibit the growth of transplanted tumors (P<0.01) and effectively improve the anti-tumor immune responses including the proportion of Th1 cells (T-bet+CD4+) increasing significantly (P<0.001), the proportion of effector CD8+ T cells (CD44+CD62L-) increasing significantly (P<0.01), and the proportion of Th2 cells (GATA3+CD4+) and Treg cells decreasing significantly (both P<0.01). Conclusion: Blocking DKK1 can effectively promote the phenotypic polarization of CD4+ T cells to Th1. DKK1 has potential value as a target for tumor immunotherapy. [ABSTRACT FROM AUTHOR]

Background: Triple Negative Breast Cancer (TNBC) is a particular type of breast cancer with the highest mortality rate. Essential oils are concerned more and more as potential anti-cancer drugs. Methods: TNBC cells were treated with different concentrations of navel orange peel essential oil (NOPEO), and then a variety of experiments were performed to investigate the changes in the growth and progression of TNBC cells. MTT assay was performed to detect the proliferation of TNBC cells. The changes of cell cycle and apoptosis were analyzed by FACS. In order to explored the migration of TNBC cells, scratch wound assay was carried out. Western blotting and qPCR were used to examine the expression of proteins and mRNA of related genes. Furthermore, RNA-seq was used to analyze the altered genes and explored the possible signal pathway. Results: NOPEO demonstrated dose- and time-dependent suppression of TNBC cell growth. TNBC cells showed an increased percentage of G2/M-phase cells and the protein levels of CyclinB1 and CyclinD1 were decreased after NOPEO treatment. The apoptotic cells were increased in the NOPEO treated TNBC cells. The migration mobility was significantly inhibited by NOPEO. In total, 1376 genes were found to be up-regulated and 1335 genes were down-regulated after NOPEO treatment. According to KEGG and GO pathways, the differentially expressed genes were related to MAPK, Jak/stat and FoxQ signaling pathways. Conclusion: This investigation explored the bio-activity and molecular mechanisms of NOPEO against TNBC cells. These results indicated that NOPEO could suppress TNBC growth and migration perhaps via the MAPK and Jak/stat signaling pathways, which may provide theoretical reference for anticancer drug development. NOPEO may be a potential natural product for the chemotherapeutic of TNBC. [ABSTRACT FROM AUTHOR]

Ploidy is relevant to numerous biological phenomena, including development, metabolism, and tissue regeneration. Single-cell RNA-seq and other omics studies are revolutionizing our understanding of biology, yet they have largely overlooked ploidy. This is likely due to the additional assay step required for ploidy measurement. Here, we developed a statistical method to infer ploidy from single-cell ATAC-seq data, addressing this gap. When applied to data from human and mouse cell atlases, our method enabled systematic detection of polyploidy across diverse cell types. This method allows for the integration of ploidy analysis into single-cell studies. Additionally, this method can be adapted to detect the proliferating stage in the cell cycle and copy number variations in cancer cells. The software is implemented as the scPloidy package of the R software and is freely available from CRAN. [ABSTRACT FROM AUTHOR]

Simple Summary: Patients with glioblastoma have very few treatment options; even with best treatment, including surgery, radiotherapy and chemotherapy, overall survival time is poor. Cancer cells need very high levels of energy to maintain their rapid growth, and a key component of this is a chemical called NAD. We can block the production of NAD in cancer cells by using inhibitors of specific substances in cells, limiting the growth of the tumour. This study highlights how cancer cells might exhibit resistance to inhibitors of NAD production, and how we can select a subset of patients that are uniquely responsive to these types of inhibitors. Furthermore, this study demonstrates that these inhibitors improve the effectiveness of the type of chemotherapy most often used to treat patients with aggressive brain tumours, called temozolomide. Patients may therefore benefit from the use of inhibitors of NAD production in combination with temozolomide. Glioblastoma (GBM) cells require high levels of nicotinamide adenine dinucleotide (NAD) to fuel metabolic reactions, regulate their cell cycle and support DNA repair in response to chemotherapy and radiation. Inhibition of a key enzyme in NAD biosynthesis, NAMPT, has demonstrated significant anti-neoplastic activity. Here, we sought to characterise NAD biosynthetic pathways in GBM to determine resistance mechanisms to NAD inhibitors. GBM cells were treated with the NAMPT inhibitor FK866 with and without NAD precursors, and were analysed by qPCR, Western blot and proliferation assays (monolayer and spheroid). We also measured changes in the cell cycle, apoptosis, NAD/NADH levels and energy production. We performed orthoptic xenograft experiments in athymic nude mice to test the efficacy of FK866 in combination with temozolomide (TMZ). We show that the expression of key genes involved in NAD biosynthesis is highly variable across GBM tumours. FK866 inhibits proliferation, reduces NAD levels and limits oxidative metabolism, leading to G2/M cell cycle arrest; however, this can be reversed by supplementation with specific NAD precursors. Furthermore, FK866 potentiates the effects of radiation and TMZ in vitro and in vivo. NAMPT inhibitors should be considered for the treatment of GBM, with patients stratified based on their expression of key enzymes in other NAD biosynthetic pathways. [ABSTRACT FROM AUTHOR]

Computational simulations with data-driven physiological detail can foster a deeper understanding of the neural mechanisms involved in cognition. Here, we utilize the wealth of cellular properties from Hippocampome.org to study neural mechanisms of spatial coding with a spiking continuous attractor network model of medial entorhinal cortex circuit activity. The primary goal is to investigate if adding such realistic constraints could produce firing patterns similar to those measured in real neurons. Biological characteristics included in the work are excitability, connectivity, and synaptic signaling of neuron types defined primarily by their axonal and dendritic morphologies. We investigate the spiking dynamics in specific neuron types and the synaptic activities between groups of neurons. Modeling the rodent hippocampal formation keeps the simulations to a computationally reasonable scale while also anchoring the parameters and results to experimental measurements. Our model generates grid cell activity that well matches the spacing, size, and firing rates of grid fields recorded in live behaving animals from both published datasets and new experiments performed for this study. Our simulations also recreate different scales of those properties, e.g., small and large, as found along the dorsoventral axis of the medial entorhinal cortex. Computational exploration of neuronal and synaptic model parameters reveals that a broad range of neural properties produce grid fields in the simulation. These results demonstrate that the continuous attractor network model of grid cells is compatible with a spiking neural network implementation sourcing data-driven biophysical and anatomical parameters from Hippocampome.org. The software (version 1.0) is released as open source to enable broad community reuse and encourage novel applications. [ABSTRACT FROM AUTHOR]

Recently, dose delivery technology has rapidly evolved with flattening filter-free beams (FFF), and the biological effects of high dose rates are a matter of interest. We hypothesized that FFF beams at different dose rates obtained with modern linear accelerators have different effects on the TME. The B16–F10 melanoma syngeneic tumor model was established, and mice were randomized to 2 different doses (2 Gy and 10 Gy) and 3 different dose rates (1 Gy/min, 6 Gy/min, and 14 Gy/min) along with the control group. Euthanasia was performed on the seventh day after RT, and intracardiac blood was collected for a comet assay. Tumors were harvested and examined histomorphologically and immunohistochemically. Statistical analyses were performed using SPSS software version 23 (SPSS Inc., Chicago, IL, USA). The daily growth rate was uniform, and no difference was observed between tumor volumes across all three dose rates for each dose. Deoxyribonucleic acid (DNA) damage in blood mononuclear cells was not affected by dose or dose rate. In the TME histomorphological examination, the number of mitosis is less in the 10 Gy arm, whereas the pleomorphism score was greater. Nevertheless, varying dose rates had no effect on the number of mitosis or the pleomorphism score. The severity of the inflammation, cell densities in the TME, and expression of immunohistochemical markers were comparable across all doses and dose rates. In our study involving the B16–F10 syngeneic tumor model, varying dose rates obtained with FFF beams had no effect on tumor volume, blood mononuclear cell DNA damage, or TME parameters. However, in order to fully understand the biological impacts of novel techniques, our study should be validated with alternative preclinical setups. • We built a syngeneic tumor model with B16–F10 malignant melanoma cells. • We investigated the impact of varying FFF dose rates (1-6-14 Gy/min) on TME. • There was no effect of dose or dose rate on DNA damage in blood mononuclear cells. • Low, moderate, and high dose rates showed no variation in tumor sizes. • No different effects were observed on TME. [ABSTRACT FROM AUTHOR]

Aims: Evidence is accumulating of the therapeutic benefits of mesenchymal stromal cells (MSCs) in diabetes‐related conditions. We have identified a novel population of stromal cells within islets of Langerhans – islet stellate cells (ISCs) – which have a similar morphology to MSCs. In this study we characterize mouse ISCs and compare their morphology and function to MSCs to determine whether ISCs may also have therapeutic potential in diabetes. Methods: ISCs isolated from mouse islets were compared to mouse bone marrow MSCs by analysis of cell morphology; expression of cell‐surface markers and extracellular matrix (ECM) components; proliferation; apoptosis; paracrine activity; and differentiation into adipocytes, chondrocytes and osteocytes. We also assessed the effects of co‐culture with ISCs or MSCs on the insulin secretory capacity of islet beta cells. Results: Although morphological similar, ISCs were functionally distinct from MSCs. Thus, ISCs were less proliferative and more apoptotic; they had different expression levels of important paracrine factors; and they were less efficient at differentiation down multiple lineages. Co‐culture of mouse islets with ISCs enhanced glucose induced insulin secretion more effectively than co‐culture with MSCs. Conclusions: ISCs are a specific sub‐type of islet‐derived stromal cells that possess biological behaviors distinct from MSCs. The enhanced beneficial effects of ISCs on islet beta cell function suggests that they may offer a therapeutic target for enhancing beta cell functional survival in diabetes. [ABSTRACT FROM AUTHOR]

Objective: The aim of this study was to investigate the association between autoinducer‐2 (AI‐2) of oral microbial flora and the alveolar bone destruction in periodontitis to determine if AI‐2 may have the potential that monitor periodontitis and predict bone loss. Background: Plaque biofilm was the initiating factor of periodontitis and the essential factor of periodontal tissue destruction. The formation of biofilms depended on the complex regulation of the quorum sensing (QS) system, in which bacteria could sense changes in surrounding bacterial density by secreting the autoinducer (AI) to regulate the corresponding physiological function. Most oral bacteria also communicated with each other to form biofilms administrating the QS system, which implied that the QS system of periodontal pathogens was related to periodontitis, but the specific relationship was unknown. Method: We collected the gingival crevicular fluid (GCF) samples and measured the concentration of AI‐2 in samples using the Vibrio harveyi BB180 bioluminescent‐reporter system. To explore the interaction between AI‐2 and bone metabolism, we utilized AI‐2 purified from Fusobacterium nucleatum to investigate the impact of F. nucleatum AI‐2 on osteoclast differentiation. Moreover, we constructed murine periodontitis models and multi‐species biofilm models to study the association between AI‐2 and periodontal disease progression. Results: The AI‐2 concentration in GCF samples increased along with periodontal disease progression (p <.0001). F. nucleatum AI‐2 promoted osteoclast differentiation in a dose‐dependent manner. In the periodontitis mice model, the CEJ‐ABC distance in the F. nucleatum AI‐2 treatment group was higher than that in the simple ligation group (p <.01), and the maxilla of the mice in the group exhibited significantly lower BMD and BV/TV values (p <.05). Conclusions: We demonstrated that the AI‐2 concentration varied with the alveolar bone destruction in periodontitis, and it may have the potential for screening periodontitis. F. nucleatum AI‐2 promoted osteoclast differentiation in a dose‐dependent manner and aggravated bone loss. [ABSTRACT FROM AUTHOR]

Background and objective: Forkhead box‐O 1 (FOXO1) is a transcription factor actively involved in oral wound healing at the epithelial barrier. However, less is known regarding the role of FOXO1 during the tissue repair response in the connective tissue compartment. This study explored the involvement of FOXO1 in the modulation of fibroblast activity related to wound healing. Methods: Primary cultures of human gingival fibroblasts were obtained from four healthy young donors. Myofibroblastic differentiation, collagen gel contraction, cell migration, cell spreading, and integrin activation were evaluated in the presence or absence of a FOXO1 inhibitor (AS1842856). Variations in mRNA and proteins of interest were evaluated through qRT‐PCR and western blot, respectively. Distribution of actin, α‐smooth muscle actin, and β1 integrin was evaluated using immunofluorescence. FOXO1 and TGF‐β1 expression in gingival wound healing was assessed by immunohistochemistry in gingival wounds performed in C57BL/6 mice. Images were analyzed using ImageJ/Fiji. ANOVA or Kruskal‐Wallis test followed by Tukey's or Dunn's post‐hoc test was performed. All data are expressed as mean ± SD. p <.05 was considered statistically significant. Results: FOXO1 inhibition caused a decrease in the expression of the myofibroblastic marker α‐SMA along with a reduction in fibronectin, type I collagen, TGF‐β1, and β1 integrin mRNA level. The FOXO1 inhibitor also caused decreases in cell migration, cell spreading, collagen gel contraction, and β1 integrin activation. FOXO1 and TGF‐β1 were prominently expressed in gingival wounds in fibroblastic cells located at the wound bed. Conclusion: The present study indicates that FOXO1 plays an important role in the modulation of several wound‐healing functions in gingival fibroblast. Moreover, our findings reveal an important regulatory role for FOXO1 on the differentiation of gingival myofibroblasts, the regulation of cell migration, and collagen contraction, all these functions being critical during tissue repair and fibrosis. [ABSTRACT FROM AUTHOR]

Background: Myocarditis refers to an autoimmune inflammatory response of the myocardium with characterization of self-reactive CD4+ T cell activation, which lacks effective treatment and has a poor prognosis. Acacetin is a natural flavonoid product that has been reported to have anti-inflammatory effects. However, acacetin has not been investigated in myocarditis. Methods: Oral acacetin treatment was administered in an experimental autoimmune myocarditis model established with myosin heavy chain-alpha peptide. Echocardiography, pathological staining, and RT-qPCR were used to detect cardiac function, myocardial injury, and inflammation levels. Flow cytometry was utilized to detect the effect of acacetin on CD4+ T cell function. RNA-seq, molecular docking, and microscale thermophoresis (MST) were employed to investigate potential mechanisms. Seahorse analysis, mitoSOX, JC-1, and mitotracker were utilized to detect the effect of acacetin on mitochondrial function. Results: Acacetin attenuated cardiac injury and fibrosis as well as heart dysfunction, and reduced cardiac inflammatory cytokines and ratio of effector CD4+ T and Th17 cells. Acacetin inhibited CD4+ T cell activation, proliferation, and Th17 cell differentiation. Mechanistically, the effects of acacetin were related to reducing mitochondrial complex II activity thereby inhibiting mitochondrial respiration and mitochondrial reactive oxygen species in CD4+ T cells. Conclusion: Acacetin may be a valuable therapeutic drug in treating CD4+ T cell-mediated myocarditis. [ABSTRACT FROM AUTHOR]

The primary inflammatory process in atherosclerosis, a major contributor to cardiovascular disease, begins with monocyte adhering to vascular endothelial cells. Actinidia arguta (kiwiberry) is an edible fruit that contains various bioactive components. While A. arguta extract (AAE) has been recognized for its anti-inflammatory characteristics, its specific inhibitory effect on early atherogenic events has not been clarified. We used tumor necrosis factor-α (TNF-α)–stimulated human umbilical vein endothelial cells (HUVECs) for an in vitro model. AAE effectively hindered the attachment of THP-1 monocytes and reduced the expression of vascular cell adhesion molecule-1 (VCAM-1) in HUVECs. Transcriptome analysis revealed that AAE treatment upregulated phosphatase and tensin homolog (PTEN), subsequently inhibiting phosphorylation of AKT and glycogen synthase kinase 3β (GSK3β) in HUVECs. AAE further hindered phosphorylation of AKT downstream of the nuclear factor kappa B (NF-κB) signaling pathway, leading to suppression of target gene expression. Oral administration of AAE suppressed TNF-α–stimulated VCAM-1 expression, monocyte-derived macrophage infiltration, and proinflammatory cytokine expression in C57BL/6 mouse aortas. Myo-inositol, identified as the major compound in AAE, played a key role in suppressing THP-1 monocyte adhesion in HUVECs. These findings suggest that AAE could serve as a nutraceutical for preventing atherosclerosis by inhibiting its initial pathogenesis. [ABSTRACT FROM AUTHOR]

Background: Ovarian cancer (OVCA) is prevalent in female reproductive organs. Despite recent advances, clinical outcomes remain poor, warranting fresh treatment avenues. Honokiol has an inhibitory effect on proliferation, invasion, and survival of cancer cells in vitro and in vivo. Therefore, this study intended to explore specific molecular mechanism by which honokiol affected OVCA progression. Methods: Bioinformatics analyzed the drug honokiol that bound to OTU deubiquitinase, ubiquitin aldehyde binding 2 (OTUB2). Cellular thermal shift assay (CETSA) verified the binding relationship between honokiol and OTUB2. Cell counting kit 8 (CCK‐8) tested the IC50 value and cell viability of OVCA cells after honokiol treatment. Corresponding assay kits determined malonic dialdehyde (MDA) and Fe2+ levels in OVCA cells. Flow cytometry measured reactive oxygen species levels. Western blot detected OTUB2, SLC7A11, and transcriptional co‐activators Yes‐associated protein (YAP) expression, and quantitative polymerase chain reaction (qPCR) detected OTUB2 expression. Immunohistochemistry (IHC) detected the expression level of Ki67 protein in tumor tissues. Results: Honokiol was capable of inducing ferroptosis in OVCA cells. CETSA confirmed that honokiol could bind to OTUB2. Further cell functional and molecular experiments revealed that honokiol induced ferroptosis in OVCA cells via repression of YAP signaling pathway through binding to OTUB2. In addition, in vivo experiments have confirmed that honokiol could inhibit the growth of OVCA. Conclusion: Honokiol induced ferroptosis in OVCA cells via repression of YAP signaling pathway through binding to OTUB2, implicating that OTUB2 may be an effective target for OVCA treatment, and our study results may provide new directions for development of more effective OVCA treatment strategies. [ABSTRACT FROM AUTHOR]

Simple Summary: Patients with treatable cancers all too often return to the clinic with untreatable tumors, requiring highly toxic secondary treatments or palliative care. In this study, we aim to determine whether activation of the Anaphase Promoting Complex (APC) in multiple-drug-resistant (MDR) breast cancer cells will re-sensitize them to chemotherapeutic agents. The APC is required for the targeted degradation of proteins that inhibit passage through mitosis. We found that APC activity is indeed impaired in MDR cells and that chemical activation of the APC increased the sensitivity of these cells to doxorubicin. Mitotic progression was slowed in MDR cells, compared to matched parental drug sensitive cells, with an accumulation of APC substrate proteins. APC activation in nocodazole-arrested cells resulted in increased passage through mitosis with lower APC substrate levels. Mice growing a patient-derived xenografted (PDX) tumor were treated with increasing doses of a chemical APC activator, resulting in a dose-dependent reduction in tumor size. Taken together, our data show that APC activity is reduced in MDR cells, with APC activation resulting in a species- and cancer-type-independent reversal of the MDR phenotype. The development of multiple-drug-resistant (MDR) cancer all too often signals the need for toxic alternative therapy or palliative care. Our recent in vivo and in vitro studies using canine MDR lymphoma cancer cells demonstrate that the Anaphase Promoting Complex (APC) is impaired in MDR cells compared to normal canine control and drug-sensitive cancer cells. Here, we sought to establish whether this phenomena is a generalizable mechanism independent of species, malignancy type, or chemotherapy regime. To test the association of blunted APC activity with MDR cancer behavior, we used matched parental and MDR MCF7 human breast cancer cells, and a patient-derived xenograft (PDX) model of human triple-negative breast cancer. We show that APC activating mechanisms, such as APC subunit 1 (APC1) phosphorylation and CDC27/CDC20 protein associations, are reduced in MCF7 MDR cells when compared to chemo-sensitive matched cell lines. Consistent with impaired APC function in MDR cells, APC substrate proteins failed to be effectively degraded. Similar to our previous observations in canine MDR lymphoma cells, chemical activation of the APC using Mad2 Inhibitor-1 (M2I-1) in MCF7 MDR cells enhanced APC substrate degradation and resensitized MDR cells in vitro to the cytotoxic effects of the alkylating chemotherapeutic agent, doxorubicin (DOX). Using cell cycle arrest/release experiments, we show that mitosis is delayed in MDR cells with elevated substrate levels. When pretreated with M2I-1, MDR cells progress through mitosis at a faster rate that coincides with reduced levels of APC substrates. In our PDX model, mice growing a clinically MDR human triple-negative breast cancer tumor show significantly reduced tumor growth when treated with M2I-1, with evidence of increased DNA damage and apoptosis. Thus, our results strongly support the hypothesis that APC impairment is a driver of aggressive tumor development and that targeting the APC for activation has the potential for meaningful clinical benefits in treating recurrent cases of MDR malignancy. [ABSTRACT FROM AUTHOR]

Simple Summary: Further investigation into the molecular mechanisms of chemotherapy resistance in pancreatic cancer is crucial for improving the prognosis of pancreatic cancer patients. Extensive research has demonstrated a close association between ferroptosis and gemcitabine resistance in various tumor types, including pancreatic cancer. We first show that high expression of Mitotic checkpoint serine/threonine kinase BUB1 in pancreatic cancer and its association with poor prognosis. Results also suggested that BUB1 suppresses ferroptosis in pancreatic cancer cells, and BUB1 knockdown significantly enhances the sensitivity of drug-resistant pancreatic cancer cells to ferroptosis. Furthermore, BUB1 promotes gemcitabine resistance in pancreatic cancer cells. We believe that our study makes a significant contribution to the literature because it was previously unclear whether BUB1 plays a key role in pancreatic cancer ferroptosis. The development of chemotherapy resistance severely limits the therapeutic efficacy of gemcitabine (GEM) in pancreatic cancer (PC), and the dysregulation of ferroptosis is a crucial factor in the development of chemotherapy resistance. BUB1 Mitotic Checkpoint Serine/Threonine Kinase (BUB1) is highly overexpressed in PC patients and is closely associated with patient prognosis. However, none of the literature reports the connection between BUB1 and ferroptosis. The molecular mechanisms underlying GEM resistance are also not well understood. Therefore, this study first established the high expression levels of BUB1 in PC patients, then explored the role of BUB1 in the process of ferroptosis, and finally investigated the mechanisms by which BUB1 regulates ferroptosis and contributes to GEM resistance in PC cells. In this study, downregulation of BUB1 enhanced the sensitivity of PC cells to Erastin, and inhibited cell proliferation and migration. Mechanistically, BUB1 could inhibit the expression levels of Neurofibromin 2 (NF2) and MOB kinase activator 1 (MOB1), and promote Yes-associated protein (YAP) expression, thereby inhibiting ferroptosis and promoting GEM resistance in PC cells. Furthermore, the combination of BUB1 inhibition with GEM exhibited a synergistic therapeutic effect. These findings reveal the mechanisms underlying the development of GEM chemotherapy resistance based on ferroptosis and suggest that the combined use of BUB1 inhibitors may be an effective approach to enhance GEM efficacy. [ABSTRACT FROM AUTHOR]

Simple Summary: The metabolism of cancer cells and the tumor microenvironment are of increasing interest as part of ongoing efforts to develop potential adjuvant therapies to be used along with conventional chemotherapy and radiation. In this report, the antitumor properties of two compounds that affect glucose metabolism, dichloroacetate and quercetin, are examined. Both DCA and quercetin, a naturally occurring plant flavonoid found in fruits and vegetables, demonstrate inhibitory effects on the growth of head and neck cancer both in cell culture and in a preclinical mouse model of head and neck cancer. These two compounds have synergistic antitumor effects when combined, both in vitro and in vivo. The drug combination inhibited tumor growth and induced cell death with the maintenance of an unfavorable tumor microenvironment. The altered tumor microenvironment appears to enhance immune-mediated clearance of tumors. Thus, this study supports additional preclinical research to further explore the antitumor effects of DCA and quercetin. Elevated glucose uptake and production of lactate are common features of cancer cells. Among many tumor-promoting effects, lactate inhibits immune responses and is positively correlated with radioresistance. Dichloroacetate (DCA) is an inhibitor of pyruvate dehydrogenase kinase that decreases lactate production. Quercetin is a flavonoid compound found in fruits and vegetables that inhibits glucose uptake and lactate export. We investigated the potential role and mechanisms of DCA, quercetin, and their combination, in the treatment of HPV-positive head and neck squamous cell carcinoma, an antigenic cancer subtype in need of efficacious adjuvant therapies. C57Bl/6-derived mouse oropharyngeal epithelial cells, a previously developed mouse model that was retrovirally transduced with HPV type-16 E6/E7 and activated Ras, were used to assess these compounds. Both DCA and quercetin inhibited colony formation and reduced cell viability, which were associated with mTOR inhibition and increased apoptosis through enhanced ROS production. DCA and quercetin reduced tumor growth and enhanced survival in immune-competent mice, correlating with decreased proliferation as well as decreased acidification of the tumor microenvironment and reduction of Foxp (+) Treg lymphocytes. Collectively, these data support the possible clinical application of DCA and quercetin as adjuvant therapies for head and neck cancer patients. [ABSTRACT FROM AUTHOR]

INTRODUCTION This study aimed to investigate the effects of nicotine on the activation of pancreatic stellate cells (PSCs) and pancreatic fibrosis in chronic pancreatitis (CP), along with its underlying molecular mechanisms. METHODS This was an in vivo and in vitro study. In vitro, PSCs were cultured to study the effects of nicotine on their activation and oxidative stress. Transcriptome sequencing was performed to identify potential signaling pathways involved in nicotine action. And the impact of nicotine on mitochondrial Ca2+ levels and Ca2+ transport-related proteins in PSCs was analyzed. The changes in nicotine effects were observed after the knockdown of the mitochondrial calcium uniporter (MCU) in PSCs. In vivo experiments were conducted using a mouse model of CP to assess the effects of nicotine on pancreatic fibrosis and oxidative stress in mice. The alterations in nicotine effects were observed after treatment with the MCU inhibitor Ru360. RESULTS In vitro experiments demonstrated that nicotine promoted PSCs activation, characterized by increased cell proliferation, elevated α-SMA and collagen expression. Nicotine also increased the production of reactive oxygen species (ROS) and cellular malondialdehyde (MDA), exacerbating oxidative stress damage. Transcriptome sequencing revealed that nicotine may exert its effects through the calcium signaling pathway, and it was verified that nicotine elevated mitochondrial Ca2+ levels and upregulated MCU expression. Knockdown of MCU reversed the effects of nicotine on mitochondrial calcium homeostasis, improved mitochondrial oxidative stress damage and structural dysfunction, thereby alleviating the activation of PSCs. In vivo validation experiments showed that nicotine significantly aggravated pancreatic fibrosis in CP mice, promoted PSCs activation, exacerbated pancreatic tissue oxidative stress, and increased MCU expression. However, treatment with Ru360 significantly mitigated these effects. CONCLUSIONS This study confirms that nicotine upregulates the expression of MCU, leading to mitochondrial calcium overload and exacerbating oxidative stress in PSCs, and ultimately promoting PSCs activation and exacerbating pancreatic fibrosis in CP. [ABSTRACT FROM AUTHOR]

Background: Photodynamic therapy (PDT) relies on the light activation of a photosensitizers to generate reactive oxygen species such as singlet oxygen, but its effect on cancer therapy is limited dramatically by hypoxia in the tumor microenvironment. Objectives: To determine the potential of a nano‐photosensitizer loaded salvianolic acid B (SalB) and 5‐aminolevulinic acid (ALA) for enhancing the efficacy of PDT in oral squamous cell carcinoma Cal27 cells and leukoplakia Leuk1 cells. Results: Singlet oxygen sensor green (SOSG) assay showed that nano‐SalB‐ALA generated higher levels of singlet oxygen, compared to nano‐SalB and nano‐ALA. Cellular uptake assay showed that nano‐SalB‐ALA effectively absorbed by Leuk1 cells. Importantly, cell counting kit‐8 and flow cytometry revealed that PDT with nano‐SalB‐ALA effectively inhibited the viability and induced the apoptosis of Cal27 and Leuk1 cells, respectively. Moreover, the tumor xenograft study revealed that PDT with nano‐SalB‐ALA had a stronger inhibitory effect on tumor growth of nude mice, compared to control groups. Conclusions: The novel photosensitizer nano‐SalB‐ALA remarkably enhanced the efficacy of PDT by improving singlet oxygen production, inhibiting cell proliferation, promoting cell apoptosis, and suppressing tumor growth. These suggest PDT with nano‐SalB‐ALA could be a clinically significant and potent treatment for oral cancer and leukoplakia. [ABSTRACT FROM AUTHOR]

Simple Summary: Head and neck cancer is the seventh most common cancer and most cases of head and neck cancer are squamous cell carcinoma (SCC). Recently, immune checkpoint inhibitors (ICPIs) such as anti-programmed cell death-1 (PD-1) have been developed. The aim of our study was to evaluate the antitumor effect of adenoviral vector carrying B7-1 (AdB7) in a murine SCC model in order to further explore the potential of the B7-1 gene in immunotherapy for head and neck cancers. Results indicated that tumor size was significantly reduced in the mice treated with both AdB7 and anti-mouse PD-1 antibody. Additionally, treatment resulted in significantly increased cell densities of total immune cells and Ki-67+ CD8+ T cells and decreased the number of regulatory T cells. Our findings indicate that adenovirus-mediated B7-1 gene expression may enhance the antitumor effect of antiPD1 against SCC. Background: The goal of this study was to evaluate the antitumor immune effects of B7-1 gene expression in addition to immune checkpoint inhibitor against squamous cell carcinoma. Methods: A murine SCC cell line, KLN205, was infected with adenoviral vector carrying B7-1 (AdB7). Infected cells were injected subcutaneously in the flanks of DBA/2 mice. Three weeks after implantation, anti-mouse PD-1 antibody (antiPD1) was intraperitonially administrated twice a week for a total of six times. Results: CD80 was significantly overexpressed in the AdB7-infected tumors. IFN-gamma in the T cells in the spleen was significantly increased and tumor size was significantly reduced in the mice treated with both AdB7 and antiPD1. Targeted tumors treated with both AdB7 and antiPD1 exhibited significantly increased cell densities of total immune cells as well as Ki-67+ CD8+ T cells and decreased regulatory T cells. Conclusions: These results suggest that the B7-1 gene transfer may enhance the antitumor effect of anti-PD1 antibody against SCC. [ABSTRACT FROM AUTHOR]

Objective: To investigate the in vivo immunomodulatory and anti-tumor mechanisms of the combined treatment of novel Four-Herb formula (4HF) and doxorubicin in triple-negative breast cancer (TNBC). Methods: Murine-derived triple-negative mammary carcinoma cell line, 4T1 cells, was cultured and inoculated into mouse mammary glands. Sixty-six mice were randomly assigned into 6 groups (n=11 in ench): naïve, control, LD 4HF (low dose 4HF), HD 4HF (high dose 4HF), LD 4HF + D (low dose and doxorubicin), and D (doxorubicin). Apart from the naïve group, each mouse received subcutaneous inoculation with 5 × 105 4T1 cells resuspended in 100 µL of normal saline in the mammary fat pads. Starting from the day of tumor cell inoculation, tumors were grown for 6 days. The LD and HD groups received daily oral gavage of 658 and 2,630 mg/kg 4HF, respectively. The LD 4HF+D group received daily oral gavage of 658 mg/kg 4HF and weekly intraperitoneal injection of doxorubicin (5 mg/kg). The D group received weekly intraperitoneal injections of doxorubicin (5 mg/kg). The treatment naïve mice received daily oral gavage of 0.2 mL double distilled water and 0.1 mL normal saline via intraperitoneal injection once a week. The control group received daily oral gavage of 0.2 mL double-distilled water. The treatment period was 30 days. At the end of treatment, mice organs were harvested to analyze immunological activities via immunophenotyping, gene and multiplex analysis, histological staining, and gut microbiota analysis. Results: Mice treated with the combination of 4HF and doxorubicin resulted in significantly reduced tumor and spleen burdens (P<0.05), altered the hypoxia and overall immune lymphocyte landscape, and manipulated gut microbiota to favor the anti-tumor immunological activities. Moreover, immunosuppressive genes, cytokines, and chemokines such as C-C motif chemokine 2 and interleukin-10 of tumors were significantly downregulated (P<0.05). 4HF-doxorubicin combination treatment demonstrated synergetic activities and was most effective in activating the anti-tumor immune response (P<0.05). Conclusion: The above results provide evidence for evaluating the immune regulating mechanisms of 4HF in breast cancer and support its clinical significance in its potential as an adjunctive therapeutic agent or immune supplement. [ABSTRACT FROM AUTHOR]

Background: Energy metabolism disorders and neurogenic inflammation play important roles in the central sensitization to chronic migraine (CM). AMP-activated protein kinase (AMPK) is an intracellular energy sensor, and its activation regulates inflammation and reduces neuropathic pain. However, studies on the involvement of AMPK in the regulation of CM are currently lacking. Therefore, this study aimed to explore the mechanism underlying the involvement of AMPK in the central sensitization to CM. Methods: Mice with recurrent nitroglycerin (NTG)-induced CM were used to detect the expression of AMPK protein in the trigeminal nucleus caudalis (TNC). Following intraperitoneal injection of the AMPK activator 5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR) and inhibitor compound C, the mechanical pain threshold, activity level, and pain-like behaviors in the mice were measured. The expression of calcitonin gene-related peptide (CGRP) and cytokines, M1/M2 microglia, and NF-κB pathway activation were detected after the intervention. Results: Repeated NTG injections resulted in a gradual decrease in AMPK protein expression, and the negative regulation of AMPK by increased ubiquitin-like plant homeodomain and RING finger domain 1 (UHRF1) expression may counteract AMPK activation by increasing ADP/ATP. AICAR can reduce the hyperalgesia and pain-like behaviors of CM mice, improve the activity of mice, reduce the expression of CGRP, IL-1β, IL-6, and TNF-α in the TNC region, and increase the expression of IL-4 and IL-10. Moreover, AMPK in TNC was mainly located in microglia. AICAR could reduce the expression of inducible NO synthase (iNOS) in M1 microglia and increase the expression of Arginase 1 (Arg1) in M2 microglia by inhibiting the activation of NF-κB pathway. Conclusions: AMPK was involved in the central sensitization of CM, and the activation of AMPK reduced neuroinflammation in NTG-induced CM mice. AMPK may provide new insights into interventions for energy metabolism disorders and neurogenic inflammation in migraine. [ABSTRACT FROM AUTHOR]

Simple Summary: Melanoma is an immunogenic tumor, so its outcomes and the efficacy of immunotherapy must be linked to the immune component. In this study, we aimed to find indicators that would allow us to monitor both the prognosis of patients and the efficacy of immunotherapy. We found that complement C2 tended to be upregulated in patients after effective anti-PD-1 therapy, and the high expression group achieved longer survival. Tumor tissues with C2 expression were infiltrated with more M1-type macrophages and tertiary lymphoid structures, and this favorable immune status may be responsible for the favorable outcome. Our findings provide valuable insights into the functional role of C2 in melanoma and its potential implications for clinical therapy. Immunotherapy is an essential therapy for individuals with advanced melanoma. However, not all patients respond to such treatment due to individual differences. We conducted a multidimensional analysis using transcriptome data from our center, as well as publicly available databases. We found that effective nivolumab treatment led to an upregulation of C2 levels, and higher levels following treatment are indicative of a good outcome. Through bioinformatics analyses and immunofluorescence, we identified a correlation between C2 and M1 macrophages. To further investigate the role of C2 in melanoma, we constructed subcutaneous tumorigenic models in C57BL/6 mice. The tumors in the C2 overexpression group exhibited significantly smaller sizes. Flow cytometric analysis of the mouse tumors demonstrated enhanced recruitment of macrophages, particularly of the M1 subtype, in the overexpression group. Moreover, single-cell RNA sequencing analysis revealed that C2-positive tumor cells exhibited enhanced communication with immune cells. We co-cultured tumor cell supernatants with macrophages in vitro and observed the induction of M1 subtype polarization. In addition, we discovered a close correlation between C2 and tertiary lymphoid structures. C2 has been demonstrated to exert a protective effect, mediated by its ability to modulate the tumor microenvironment. C2 serves as a prognostic marker for melanoma and can be employed to monitor the efficacy of immunotherapy. [ABSTRACT FROM AUTHOR]

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Introduction: Cerebrovascular pathologies contribute to cognitive decline during aging, leading to vascular cognitive impairment and dementia (VCID). Levels of circulating insulin-like growth factor 1 (IGF-1), a vasoprotective hormone, decrease during aging. Decreased circulating IGF-1 in animal models leads to the development of VCID-like symptoms, but the cellular mechanisms underlying IGF-1-deficiency associated pathologies in the aged cerebrovasculature remain poorly understood. Here, we test the hypothesis that vascular smooth muscle cells (VSMCs) play an integral part in mediating the vasoprotective effects of IGF-1. Methods: We used a hypertension-based model of cerebrovascular dysfunction in mice with VSMC-specific IGF-1 receptor (Igf1r) deficiency and evaluated the development of cerebrovascular pathologies and cognitive dysfunction. Results: VSMC-specific Igf1r deficiency led to impaired cerebral myogenic autoregulation, independent of blood pressure changes, which was also associated with impaired spatial learning and memory function as measured by radial arm water maze and impaired motor learning measured by rotarod. In contrast, VSMC-specific IGF-1 receptor knockdown did not lead to cerebral microvascular rarefaction. Discussion: These studies suggest that VSMCs are key targets for IGF-1 in the context of cerebrovascular health, playing a role in vessel stability alongside other cells in the neurovascular unit, and that VSMC dysfunction in aging likely contributes to VCID. [ABSTRACT FROM AUTHOR]

Background: The objective of the present study was to evaluate in vitro the cytotoxicity and bioactivity of various endodontic sealers (CeraSeal, BioRoot™ and AH Plus®) in pre-osteoblast mouse cells (MC3T3 cells). Methods: MC3T3 cells (ATCC CRL-2594) were plated in 1 × 104 cells/well in 96-well plates in contact with endodontic sealers at concentrations of 1:10 and 1:100. Cell viability was evaluated by MTT assay after 24 and 48 h. In addition, sealer bioactivity was measured by RT-PCR for mediator of inflammation (Tnf, Ptgs2) and mineralization (Runx2, Msx1, Ssp1 and Dmp1) after 24 h and by Alizarin Red S Assay of mineralization after 28 days. Data were analyzed using one-way ANOVA followed by the Tukey's post-test at a significance level of 5%. Results: BioRoot™ presented 24-hour cytotoxicity (p < 0.05) at 1:10 concentration. In the period of 48 h, no endodontic cement was cytotoxic to the cells compared to the control (p > 0.05). TNF-α gene expression was induced by AH Plus® (p < 0.05), while Ptgs2 was induced by the CeraSeal and BioRoot™ (p < 0.05). The expression of Runx2 was stimulated by BioRoot™ and AH Plus® (p < 0.05). In contrast, the expression of Dmp-1Dmp1 was higher for the CeraSeal and BioRoot™ (p < 0.05). Nonetheless, the sealers did not impact the formation of mineralization nodules (p > 0.05). Conclusion: CeraSeal, BioRoot™ and AH Plus® sealers were not cytotoxic to MC3T3 cells within 48 h, but differentially induced the expression of genes related to inflammation and mineralization without impacting biomineralization by the cells. [ABSTRACT FROM AUTHOR]

Background and objective: The pathogenesis of coronal suture craniosynostosis is often attributed to the dysregulated cellular dynamics, particularly the excessive proliferation and abnormal osteogenic differentiation of suture cells. Despite its clinical significance, the molecular mechanims of this condition remain inadequately understood. This study is dedicated to exploring the influence of the Periostin/Bone Morphogenetic Protein 1 (BMP1) axis on the growth and osteogenic maturation of Suture Mesenchymal Stem Cells (SMSCs), which are pivotal in suture homeostasis. Methods: Neonatal TWIST Basic Helix-Loop-Helix Transcription Factor 1 heterozygous (TWIST1+/−) mice, aged one day, were subjected to adenoviral vector-mediated Periostin upregulation. To modulate Periostin/BMP1 levels in SMSCs, we employed siRNA and pcDNA 3.1 vectors. Histological and molecular characterizations, including hematoxylin and eosin staining, Western blot, and immunohistochemistry were employed to study suture closure phenotypes and protein expression patterns. Cellular assays, encompassing colony formation, 5-ethynyl-2'deoxyuridine, and wound healing tests were conducted to analyze SMSC proliferation and migration. Osteogenic differentiation was quantified using Alkaline Phosphatase (ALP) and Alizarin Red S (ARS) staining, while protein markers of proliferation and differentiation were evaluated by Western blotting. The direct interaction between Periostin and BMP1 was validated through co-immunoprecipitation assays. Results: In the TWIST1+/− model, an upregulation of Periostin coupled with a downregulation of BMP1 was observed. Augmenting Periostin expression mitigated craniosynostosis. In vitro, overexpression of Periostin or BMP1 knockdown suppressed SMSC proliferation, migration, and osteogenic differentiation. Periostin knockdown manifested an inverse biological impact. Notably, the suppressive influence of Periostin overexpression on SMSCs was effectively counteracted by upregulating BMP1. There was a direct interaction between Periostin and BMP1. Conclusion: These findings underscore the significance of the Periostin/BMP1 axis in regulating craniosynostosis and SMSC functions, providing new insights into the molecular mechanisms of craniosynostosis and potential targets for therapeutic intervention. [ABSTRACT FROM AUTHOR]

Simple Summary: Despite remarkable progress in treating early-stage gastric cancer (GC), the clinical outcomes for patients with advanced disease remain very poor. Tissue invasion and metastasis constitute the major causes of cancer-related deaths, including GC. This highlights the urgent need to develop animal models that can recapitulate these processes to develop novel therapeutic strategies. We developed a highly reproducible and cost-effective procedure to establish orthotopic GC xenografts showing high engraftment and metastatic rates via the direct implantation of tumor cell suspensions. Compared with the routine method to establish orthotopic xenograft models by engrafting intact tumor fragments, our approach significantly shortens the experimental timeline and allows for the flexible adjustment of the number of tumor cells implanted to control the rate of tumor progression. Both dose- and time-dependent progressions of tumor invasion and metastasis were nicely recapitulated in our model. Our work provides valuable tools for studying GC progression and metastasis and developing effective therapies. Although the implantation of intact tumor fragments is a common practice to generate orthotopic xenografts to study tumor invasion and metastasis, the direct implantation of tumor cell suspensions is necessary when prior manipulations of tumor cells are required. However, the establishment of orthotopic xenografts using tumor cell suspensions is not mature, and a comparative study directly comparing their engraftment and metastatic capabilities is lacking. It is unclear whether tumor fragments are superior to cell suspensions for successful engraftment and metastasis. In this study, we employed three GC cell lines with varying metastatic capacities to stably express firefly luciferase for monitoring tumor progression in real time. We successfully minimized the risk of cell leakage during the orthotopic injection of tumor cell suspensions without Corning Matrigel by systematically optimizing the surgical procedure, injection volume, and needle size options. Comparable high engraftment and metastatic rates between these two methods were demonstrated using MKN-45 cells with a strong metastatic ability. Importantly, our approach can adjust the rate of tumor progression flexibly and cuts the experimental timeline from 10–12 weeks (for tumor fragments) to 4–5 weeks. Collectively, we provided a highly reproducible procedure with a shortened experimental timeline and low cost for establishing orthotopic GC xenografts via the direct implantation of tumor cell suspensions. [ABSTRACT FROM AUTHOR]

Background: Immune checkpoint inhibitors (ICIs) are a revolutionary paradigm in the treatment of thoracic malignancies and chemoimmunotherapy is a current standard care in this field. Chemotherapeutic agents are known to induce not only direct cytotoxic effects on tumor cells but also immune modulating effects, such as stimulating immunogenic cell death (ICD). Currently, either pemetrexed (PEM) or taxane plus platinum are combined with ICIs for patients with non‐small cell lung cancer (NSCLC); however, it is still unknown whether these agents are immunologically optimal partners for ICIs. Methods: To determine the immunologically optimal chemotherapeutic agent, we first evaluated the ability of several chemotherapeutic agents, including platinum, PEM, taxane, and 5‐fluorouracil (5‐FU) to induce ICD using several thoracic tumor cell lines in vitro. ICD was evaluated by the cell surface expression of calreticulin (CRT) and adenosine‐triphosphate (ATP) secretion. We further performed an antitumor vaccination assay in vivo. Results: 5‐FU induced cell surface expression of CRT and ATP secretion most efficiently among the several chemotherapeutic agents. This effect was enhanced when it was combined with platinum. In the antitumor vaccination assay in vivo, we found that vaccination with dying‐AB1‐HA (a murine malignant mesothelioma cell line) cells treated with 5‐FU, but neither PEM nor PTX, reduced the tumor growth of living‐AB1‐HA cells inoculated 1 week after vaccination by recruiting CD3+CD8+ T cells into the tumor microenvironment. Conclusion: Our findings indicate that fluoropyrimidine can be an immunologically optimal partner of ICIs through the induction of ICD for thoracic malignancies. [ABSTRACT FROM AUTHOR]

Unlike aged somatic cells, which exhibit a decline in molecular fidelity and eventually reach a state of replicative senescence, pluripotent stem cells can indefinitely replenish themselves while retaining full homeostatic capacity. The conferment of beneficial-pluripotency related traits via in vivo partial cellular reprogramming in vivo partial reprogramming significantly extends lifespan and restores aging phenotypes in mouse models. Although the phases of cellular reprogramming are well characterized, details of the rejuvenation processes are poorly defined. To understand whether cellular reprogramming can ameliorate DNA damage, we created a reprogrammable accelerated aging mouse model with an ERCC1 mutation. Importantly, using enhanced partial reprogramming by combining small molecules with the Yamanaka factors, we observed potent reversion of DNA damage, significant upregulation of multiple DNA damage repair processes, and restoration of the epigenetic clock. In addition, we present evidence that pharmacological inhibition of ALK5 and ALK2 receptors in the TGFb pathway are able to phenocopy some benefits including epigenetic clock restoration suggesting a role in the mechanism of rejuvenation by partial reprogramming. [ABSTRACT FROM AUTHOR]

Simple Summary: Breast cancer is a highly heterogeneous disease on the molecular level. The molecular subtype will be altered during metastasis and affect patient outcomes and treatments. Breast cancer is prone to lymph node metastasis. Our study aimed to investigate whether subtype conversion occurs during lymph node metastasis and its underlying mechanism. We confirmed that hormone receptors were down-regulated, while HER2 was up-regulated during lymph node metastasis, and macrophages played a significant role in the process, probably via MNX1. Targeting macrophages or MNX1 may provide new avenues for endocrine therapy and targeted treatment of breast cancer patients with lymph node metastasis. Background: The progression of tumors from less aggressive subtypes to more aggressive states during metastasis poses challenges for treatment strategies. Previous studies have revealed the molecular subtype conversion between primary and metastatic tumors in breast cancer (BC). However, the subtype conversion during lymph node metastasis (LNM) and the underlying mechanism remains unclear. Methods: We compared clinical subtypes in paired primary tumors and positive lymph nodes (PLNs) in BC patients and further validated them in the mouse model. Bioinformatics analysis and macrophage-conditioned medium treatment were performed to investigate the role of macrophages in subtype conversion. Results: During LNM, hormone receptors (HRs) were down-regulated, while HER2 was up-regulated, leading to the transformation of luminal A tumors towards luminal B tumors and from luminal B subtype towards HER2-enriched (HER2-E) subtype. The mouse model demonstrated the elevated levels of HER2 in PLN while retaining luminal characteristics. Among the various cells in the tumor microenvironment (TME), macrophages were the most clinically relevant in terms of prognosis. The treatment of a macrophage-conditioned medium further confirmed the downregulation of HR expression and upregulation of HER2 expression, inducing tamoxifen resistance. Through bioinformatics analysis, MNX1 was identified as a potential transcription factor governing the expression of HR and HER2. Conclusion: Our study revealed the HER2-E subtype conversion during LNM in BC. Macrophages were the crucial cell type in TME, inducing the downregulation of HR and upregulation of HER2, probably via MNX1. Targeting macrophages or MNX1 may provide new avenues for endocrine therapy and targeted treatment of BC patients with LNM. [ABSTRACT FROM AUTHOR]

Simple Summary: In this study, we found that high FTSJ1 expression in triple-negative breast cancer patients was associated with poor prognosis and was associated with reduced infiltration of CD8+T cells in the tumor microenvironment. By knocking down FTSJ1, we observed an inhibitory effect on the proliferation and migration of triple-negative breast cancer, while inducing apoptosis and increasing the sensitivity of TNBC cells to T-cell-mediated cytotoxicity. This finding highlights the importance of FTSJ1 as a potential immunotherapy target in triple-negative breast cancer. FtsJ RNA 2′-O-methyltransferase 1 (FTSJ1) is a member of the methyltransferase superfamily and is involved in the processing and modification of ribosomal RNA. We herein demonstrate that FTSJ1 favors TNBC progression. The knockdown of FTSJ1 inhibits TNBC cell proliferation and development, induces apoptosis of cancer cells, and increases the sensitivity of TNBC cells to T-cell-mediated cytotoxicity. Furthermore, the high expression of FTSJ1 in TNBC attenuates CD8+T cell infiltration in the tumor microenvironment (TME) correlated with poorer prognosis for clinical TNBC patients. In this study, we establish that FTSJ1 acts as a tumor promotor, is involved in cancer immune evasion, and may serve as a potential immunotherapy target in TNBC. [ABSTRACT FROM AUTHOR]

This study investigates the effects of peroxisome proliferator-activated receptor gamma (PPARγ) inhibition on bone and immune cell profiles in aged female mice, as well as in vitro stromal stem cell osteogenic differentiation and inflammation gene expression. The hypothesis was that inhibition of PPARγ would increase bone mass and alter immune and other cellular functions. Our results showed that treatment with PPARγ antagonist GW9662 for 6 weeks reduced bone volume and trabecular number and increased trabecular spacing. However, inhibition of PPARγ had no significant effect on marrow and spleen immune cell composition in aged female mice. In vitro experiments indicated that GW9662 treatment increased the expression of osteogenic genes but did not affect adipogenic genes. Additionally, GW9662 treatment decreased the expression of several inflammation-related genes. Overall, these findings suggest that PPARγ inhibition may have adverse effects on bone in aged female mice. [ABSTRACT FROM AUTHOR]

Simple Summary: Deficiency and targeting of Stabilin-1, which is expressed by liver sinusoidal endothelial cells and subsets of macrophages, has been shown to improve anti-tumor immune responses in preclinical and early clinical studies. This study investigated whether targeting of Stab1 might also influence melanoma liver colonization, as hepatic metastasis of melanoma is a poor prognostic factor. In two models of hepatic melanoma metastasis, no direct influence of anti-Stab1 treatment was found. However, effects on the local microenvironment were observed. We suggest that the organ- and tumor-specific effects of anti-Stab1 therapies should be further considered and analyzed in future human studies. Background: This study analyzed the role of Stabilin-1 on hepatic melanoma metastasis in preclinical mouse models. Methods: In Stabilin-1−/− mice (Stab1 KO), liver colonization of B16F10 luc2 and Wt31 melanoma was investigated. The numbers, morphology, and vascularization of hepatic metastases and the hepatic microenvironment were analyzed by immunofluorescence. Results: While hepatic metastasis of B16F10 luc2 or Wt31 melanoma was unaltered between Stab1 KO and wildtype (Ctrl) mice, metastases of B16F10 luc2 tended to be smaller in Stab1 KO. The endothelial differentiation of both types of liver metastases was similar in Stab1 KO and Ctrl. No differences in initial tumor cell adhesion and retention to the liver vasculature were detected in the B16F10 luc2 model. Analysis of the immune microenvironment revealed a trend towards higher levels of CD45+Gr-1+ cells in Stab1 KO as compared to Ctrl in the B16F10 luc2 model. Interestingly, significantly higher levels of POSTN were found in the matrix of hepatic metastases of Wt31, while liver metastases of B16F10 luc2 showed a trend towards increased deposition of RELN. Conclusions: Hepatic melanoma metastases show resistance to Stabilin-1 targeting approaches. This suggests that anti-Stab1 therapies should be considered with respect to the tumor entity or target organs. [ABSTRACT FROM AUTHOR]