RNA sequencing and proteomic profiling reveal alterations by MPTP in chronic stomach mucosal injury in tree shrew Chinese (Tupaia belangeri chinensis).

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a neurotoxin that can cause gastrointestinal ulcers by affecting dopamine levels. Therefore, MPTP has been considered a toxic substance that causes gastric ulcer disease in experimental animals. In this study, tree shrews were used as the animal model of gastric mucosa injury, and MPTP was intraperitoneally injected at a lower MPTP dosage 2 mg/kg/day for 13 weeks, while tree shrews were not injected as the control group. Under the light microscope, local congestion or diffuse bleeding points of gastric mucosa and multiple redness and swelling bleeding symptoms on the inner wall were observed in the treatment group, as well as immune cell infiltration was found in HE staining, but no such phenomenon was observed in the control group. In order to explore the molecular basis of changes in MPTP induced gastric mucosa injury, the transcriptome and proteome data of gastric mucosa were analyzed. We observed significant differences in mRNA and protein expression levels under the influence of MPTP. The changes in mRNA and proteins are related to increased immune infiltration, cellular processes and angiogenesis. More differentially expressed genes play a role in immune function, especially the candidate genes RPL4 and ANXA1 with significant signal and core role. There are also differentially expressed genes that play a role in mucosal injury and shedding, especially candidate genes GAST and DDC with certain signaling and corresponding functions. Understanding the factors and molecular basis that affect the expression of related genes is crucial for coping with Emotionality gastric mucosa injury disease and developing new treatment methods to establish the ability to resist disease.

Mutant p53-microRNA-200c-ZEB2-Axis-Induced CPT1C Elevation Contributes to Metabolic Reprogramming and Tumor Progression in Basal-Like Breast Cancers.

TP53 is mutated in more than 80% of basal-like breast cancers (BLBCs). BLBCs with TP53 mutation are usually high-grade and have worse responses to chemotherapy, leading to poor clinical outcomes. Wild-type p53 (WTp53) is well-accepted to promote fatty acid oxidation (FAO); however, in this study, we demonstrate that mutant p53 (Mutp53) enhances FAO activity through constitutively upregulating CPT1C via dysregulating the miR-200c-ZEB2 axis. Sustained CPT1C expression contributes to the metabolic preference of FAO, epithelial-mesenchymal transition (EMT) phenotypes, migration, invasion, and cancer stemness in BLBC, which is mediated by modulating the redox status. Furthermore, interference of CPT1C expression impairs tumor growth and pulmonary colonization of BLBC cells in vivo, and even postpones the occurrence of spontaneous metastasis, resulting in a prolonged disease-specific survival (DSS). Consistently, clinical validation reveals that high CPT1C is observed in breast cancer patients with metastasis and is correlated with poor overall, disease-free, progression-free, and disease-specific survival in BLBC patients. Together, unlike WTp53 which transiently transactivates CPT1C, Mutp53 provides long-term benefits through sustaining CPT1C expression by disturbing the miR-200c-ZEB2 axis, which potentiates FAO and facilitates tumor progression in BLBC, suggesting that targeting Mutp53-CPT1C-driven metabolic reprogramming is promising to serve as novel therapeutic strategies for BLBC in the future.

p53-Mediated Indirect Regulation on Cellular Metabolism: From the Mechanism of Pathogenesis to the Development of Cancer Therapeutics.

The transcription factor p53 is the most well-characterized tumor suppressor involved in multiple cellular processes, which has expanded to the regulation of metabolism in recent decades. Accumulating evidence reinforces the link between the disturbance of p53-relevant metabolic activities and tumor development. However, a full-fledged understanding of the metabolic roles of p53 and the underlying detailed molecular mechanisms in human normal and cancer cells remain elusive, and persistent endeavor is required to foster the entry of drugs targeting p53 into clinical use. This mini-review summarizes the indirect regulation of cellular metabolism by wild-type p53 as well as mutant p53, in which mechanisms are categorized into three major groups: through modulating downstream transcriptional targets, protein-protein interaction with other transcription factors, and affecting signaling pathways. Indirect mechanisms expand the p53 regulatory networks of cellular metabolism, making p53 a master regulator of metabolism and a key metabolic sensor. Moreover, we provide a brief overview of recent achievements and potential developments in the therapeutic strategies targeting mutant p53, emphasizing synthetic lethal methods targeting mutant p53 with metabolism. Then, we delineate synthetic lethality targeting mutant p53 with its indirect regulation on metabolism, which expands the synthetic lethal networks of mutant p53 and broadens the horizon of developing novel therapeutic strategies for p53 mutated cancers, providing more opportunities for cancer patients with mutant p53. Finally, the limitations and current research gaps in studies of metabolic networks controlled by p53 and challenges of research on p53-mediated indirect regulation on metabolism are further discussed.

Sumoylation participates in the regulation of YB-1-mediated mismatch repair deficiency and alkylator tolerance.

Numerous reports indicate that enhanced expression of Y-box binding protein-1 (YB-1) in tumor cells is strongly associated with tumorigenesis, aggressiveness, drug resistance, as well as poor prognosis in several types of cancers, and YB-1 is considered to be an oncogene. The molecular mechanism contributing to the regulation of the biological activities of YB-1 remains obscure. Sumoylation, a post-translational modification involving the covalent conjugation of small ubiquitin-like modifier (SUMO) proteins to a target protein, plays key roles in the modulation of protein functions. In this study, our results revealed that YB-1 is sumoylated and that Lys26 is a critical residue for YB-1 sumoylation. Moreover, YB-1 was found to directly interact with SUMO proteins, and disruption of the SUMO-interacting motif (SIM) of YB-1 not only interfered with this interaction but also diminished YB-1 sumoylation. The subcellular localization, protein stability, and transcriptional regulatory activity of YB-1 were not significantly affected by sumoylation. However, decreased sumoylation disrupted the interaction between YB-1 and PCNA as well as YB-1-mediated inhibition of the MutSα/PCNA interaction and MutSα mismatch binding activity, indicating a functional role of YB-1 sumoylation in inducing DNA mismatch repair (MMR) deficiency and spontaneous mutations. The MMR machinery also recognizes alkylator-modified DNA adducts to signal for cell death. We further demonstrated that YB-1 sumoylation is crucial for the inhibition of SN1-type alkylator MNNG-induced cytotoxicity, G2/M-phase arrest, apoptosis, and the MMR-dependent DNA damage response. Collectively, these results provide molecular explanations for the impact of YB-1 sumoylation on MMR deficiency and alkylator tolerance, which may provide insight for designing therapeutic strategies for malignancies and alkylator-resistant cancers associated with YB-1 overexpression.

Process steps for the fractionation of immunoglobulin (Ig) G depleted of IgA, isoagglutinins, and devoid of in vitro thrombogenicity.

BACKGROUND: Plasma-derived immunoglobulins (IgG) are essential medicines that are in worldwide shortage, especially in low- and middle-income countries. Optimised manufacturing processes can increase supply. We evaluated various new process steps for IgG fractionation. MATERIAL AND METHODS: A crude, worst-case, IgG intermediate obtained by caprylic acid fractionation of cryoprecipitate-poor plasma was used as starting experimental material. It was processed inline by Fractogel® (Merck) TMAE anion-exchanger to deplete IgA and IgM, Eshmuno® P (Merck) anti-A and anti-B affinity chromatography to remove anti-A and anti-B isoagglutinins, 0.3% TnBP-1% Triton X-100 (S/D) treatment, C18 chromatography for removal of S/D agents, and single-pass tangential flow filtration (SPTFF) concentration to 20%. Quality, safety, and recovery were evaluated at small and pilot scales to assess purity, removal of IgA, IgM isoagglutinins, S/D agents, thrombogenic factors, and lack of toxicity in a cell model. RESULTS: The starting IgG intermediate contained approximately 90% IgG, IgA, and IgM and 10% albumin. Fractogel® TMAE, equilibrated in 25 mM sodium acetate-pH 6.0 and loaded with up to 225 mg of IgG/mL, could remove IgA and IgM, with over 94% IgG recovery with preserved sub-class distribution in the flow-through. Sequential Eshmuno®-P anti-A and anti-B columns efficiently removed isoagglutinins. The C18 packing, used at up to 17 mL of S/D-IgG solution per mL, removed TnBP and Triton X-100 to less than 1 and 2 ppm, respectively. The 20% purified IgG was devoid of activated factor XI and thrombin generation activity. DISCUSSION: This purification sequence yields a >99% pure, 20% (v/v) IgG product, depleted of IgA, isoagglutinins, and thrombogenic markers, and should be implementable on various IgG intermediates to help improve the supply of immunoglobulins.

Mutant p53 Attenuates Oxidative Phosphorylation and Facilitates Cancer Stemness through Downregulating miR-200c-PCK2 Axis in Basal-Like Breast Cancer.

miR-200c is a tumor suppressor miRNA that plays a critical role in regulating epithelial phenotype and cancer stemness. p53 deficiency downregulates the expression of miR-200c and leads to epithelial-mesenchymal transition (EMT) and stemness phenotype, which contributes to the progression of breast cancers. In this study, we demonstrated that CRISPR-mediated knockout (KO) of miR-200c induces metabolic features similar to the metabolic rewiring caused by p53 hot-spot mutations, and that impairing this metabolic reprogramming interferes with miR-200c deficiency-induced stemness and transformation. Moreover, restoring miR-200c expression compromised EMT, stem-cell properties, and the Warburg effect caused by p53 mutations, suggesting that mutant p53 (MTp53) induces EMT-associated phenotypes and metabolic reprogramming by downregulating miR-200c. Mechanistically, decreased expression of PCK2 was observed in miR-200c- and p53-deficient mammary epithelial cells, and forced expression of miR-200c restored PCK2 in p53 mutant-expressing cells. Reduced PCK2 expression not only led to attenuated oxidative phosphorylation (OXPHOS) and increased stemness in normal mammary epithelial cells but also compromised the enhanced OXPHOS and suppression of cancer stemness exerted by miR-200c in p53 mutation-bearing basal-like breast cancer (BLBC) cells. Clinically, PCK2 expression is negatively associated with EMT markers and is downregulated in basal-like subtype and cases with low miR-200c expression or p53 mutation. Notably, low expression of PCK2 is associated with poor overall survival (OS) in patients with breast cancer. IMPLICATIONS: Together, our results suggest that p53 and miR-200c regulate OXPHOS and stem/cancer stemness through PCK2, and loss of the p53-miR-200c-PCK2 axis might provide metabolic advantages that facilitate cancer stemness, leading to the progression of BLBCs.

Large-Pore Platelet-Rich Fibrin with a Mg Ring to Allow MC3T3-E1 Preosteoblast Migration and to Improve Osteogenic Ability for Bone Defect Repair.

Platelet-rich fibrin (PRF) is a natural fibrin meshwork material with multiple functions that are suitable for tissue engineering applications. PRF provides a suitable scaffold for critical-size bone defect treatment due to its platelet cytokines and rich growth factors. However, the structure of PRF not only promotes cell attachment but also, due to its density, provides a pool for cell migration into the PRF to facilitate regeneration. In our study, we used repeated freeze drying to enlarge the pores of PRF to engineer large-pore PRF (LPPRF), a type of PRF that has expanded pores for cell migration. Moreover, a biodegradable Mg ring was used to provide stability to bone defects and the release of Mg ions during degradation may enhance osteoconduction and osteoinduction. Our results revealed that cell migration was more extensive when LPPRF was used rather than when PRF was used and that LPPRF retained the growth factors present in PRF. Moreover, the Mg ions released from the Mg ring during degradation significantly enhanced the calcium deposition of MC3T3-E1 preosteoblasts. In the present study, a bone substitute comprising LPPRF combined with a Mg ring was demonstrated to have much potential for critical-size bone defect repair.

Inosine induces acute hyperuricaemia in rhesus monkey (Macaca mulatta) as a potential disease animal model.

CONTEXT: The uric acid metabolism pathway is more similar in primates and humans than in rodents. However, there are no reports of using primates to establish animal models of hyperuricaemia (HUA). OBJECTIVES: To establish an animal model highly related to HUA in humans. MATERIALS AND METHODS: Inosine (75, 100 and 200 mg/kg) was intraperitoneally administered to adult male rhesus monkeys (n = 5/group). Blood samples were collected over 8 h, and serum uric acid (SUA) level was determined using commercial assay kits. XO and PNP expression in the liver and URAT1, OAT4 and ABCG2 expression in the kidneys were examined by qPCR and Western blotting to assess the effects of inosine on purine and uric acid metabolism. The validity of the acute HUA model was assessed using ulodesine, allopurinol and febuxostat. RESULTS: Inosine (200 mg/kg) effectively increased the SUA level in rhesus monkeys from 51.77 ± 14.48 at 0 h to 178.32 ± 14.47 µmol/L within 30 min and to peak levels (201.41 ± 42.73 µmol/L) at 1 h. PNP mRNA level was increased, whereas XO mRNA and protein levels in the liver were decreased by the inosine group compared with those in the control group. No changes in mRNA and protein levels of the renal uric acid transporter were observed. Ulodesine, allopurinol and febuxostat eliminated the inosine-induced elevation in SUA in tested monkeys. CONCLUSIONS: An acute HUA animal model with high reproducibility was induced; it can be applied to evaluate new anti-HUA drugs in vivo and explore the disease pathogenesis.

Mixed cryoglobulinaemia in a rhesus macaque (Macaca mulatta).

We report cryoglobulinaemia (CG) in a rhesus macaque whose serum sample was gel-like at <37°C and resolubilised upon warming. Mixed CG was diagnosed using serum protein electrophoresis and serum immunofixation electrophoresis. Renal damage and arthrophyma were observed during necropsy. This is the first report of CG in a non-human primate.

Potassium oxonate induces acute hyperuricemia in the tree shrew (tupaia belangeri chinensis).

Potassium oxonate, a selectively competitive uricase inhibitor, produced hyperuricemia (HUA) in rodents in a previous study. In this study, we employed the tree shrew as an animal model to study potassium oxonate-induced HUA. The effect of allopurinol (ALLO), a uric acid reducer, was also examined in this model. Potassium oxonate at doses of 5, 20, 40, 60, 80, 100, and 1,000 mg/kg was given intraperitoneally to tree shrews. The results showed that potassium oxonate can effectively increase the levels of uric acid in tree shrews at doses ranging from 40 to 100 mg/kg. Semiquantitative RT-PCR showed that the xanthine dehydrogenase/oxidase (XDH/XO) mRNA expression level was significantly higher in the liver tissue of tree shrews with high levels of uric acid. There were no changes in serum urea nitrogen, or serum creatinine values. ALLO can significantly decrease serum uric acid levels (P<0.01) and raise XDH/XO mRNA expression levels in the liver tissue of tree shrews with HUA. XDH/XO mRNA expression levels did not change in untreated tree shrews. Studies on acute toxicity in the tree shrew did not show any significantly abnormal signs. There were no adverse effects at the macroscopic level up to doses ≤100 mg/kg. Potassium oxonate induced acute HUA in tree shrews at lower doses compared with other animal models. Potassium oxonate-treated tree shrews may be a potential animal model for studying pathogenic mechanism and evaluating a new therapeutic agent for treatment of HUA in humans.