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Despite the extensive research conducted on Alzheimer's disease (AD) over the years, no effective drug for AD treatment has been found. Therefore, the development of new drugs for the treatment of AD is of the utmost importance. We recently reported the proteolytic activities of JAL-TA9 (YKGSGFRMI) and ANA-TA9 (SKGQAYRMA), synthetic peptides of nine amino acids each, derived from the Box A region of Tob1 and ANA/BTG3 proteins, respectively. Furthermore, two components of ANA-TA9, ANA-YA4 (YRMI) at the C-terminus end and ANA-SA5 (SKGQA) at the N-terminus end of ANA-TA9, exhibited proteolytic activity against amyloid-β (Aβ) fragment peptides. In this study, we identified the active center of ANA-SA5 using AEBSF, a serine protease inhibitor, and a peptide in which the Ser residue of ANA-SA5 was replaced with Leu. In addition, we demonstrate the proteolytic activity of ANA-SA5 against the soluble form Aβ42 ( a -Aβ42) and solid insoluble form s -Aβ42. Furthermore, ANA-SA5 was not cytotoxic to A549 cells. These results indicate that ANA-SA5 is a promising Catalytide and a potential candidate for the development of new peptide drugs targeting Aβ42 for AD treatment.

Background: Systemic lupus erythematosus is an immunologically dysregulated disease characterized by the presence of multiple autoantibodies. In SLE, B lymphocytes contribute to the dysregulated production of autoantibodies and cytokines. Recently, we discovered that miR-99a-3p binds to both EIF4EBP1 and NCAPG mRNA and that lowering miR-99a-3p can promote B cell autophagy in SLE by increasing EIF4EBP1 expression. However, the functions of miR-99a-3p and NCAPG in SLE have not been extensively investigated., Objective: This work aims to evaluate the levels of miR-99a-3p and NCAPG expression in SLE B cells and to determine whether the aberrant expression of miR-99a-3p and NCAPG contributes to the pathological mechanisms in SLE., Methods: B lymphocytes were obtained through immunomagnetic negative selection. Using RT-qPCR, miR-99a-3p and NCAPG mRNA expressions in B lymphocytes and in the BALL-1 cell line were measured. To determine the relative abundance of NCAPG, PI3K, p-PI3K, AKT, and p-AKT, we normalize them to the level of β-actin using Western blotting. Evaluation of miR-99a-3p and NCAPG's impact on cell proliferation was done utilizing CCK-8 assay. Using flow cytometry, the cell cycle and apoptosis were both measured., Results: Comparing SLE B cells to healthy controls, miR-99a-3p expression was significantly downregulated. Additionally, it was observed that SLE B cells had significantly higher NCAPG mRNA expression. Blocking miR-99a-3p expression in BALL-1 cells with an antagomir elevated NCAPG expression, facilitated PI3K/AKT pathway activation, improved cell proliferation, raised the fraction of S-phase cells, and prevented cell apoptosis. The opposite effects of upregulated miR-99a-3p levels on BALL-1 cells were observed by using an agomir. Furthermore, the effect of decreased miR-99a-3p expression on cell proliferation was partially mediated by elevating NCAPG levels and activating the PI3K/AKT pathway., Conclusion: Our research indicates that lower miR-99a-3p expression in SLE B cells appears to boost B cell number via the NCAPG and PI3K/AKT pathways., Competing Interests: Declaration of conflicting interestsThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Objective: To investigate the regulatory role of Wilms tumor 1-associating protein (WTAP) in hypoxia/reoxygenation (H/R)-induced cardiomyocyte injury and its molecular mechanism., Methods: (1) Experiment I: H9C2 cardiomyocytes were divided into blank control group and H/R model group. H/R was used to induce myocardial ischemia/reperfusion (I/R) injury model in H9C2 cells. The blank control group was not treated. N6-methyladenosine (m6A) RNA methylation assay kit was used to detect the level of m6A. Real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) and Western blotting were used to detect the mRNA and protein expression levels of methyltransferases [WTAP, methyltransferase-like proteins (METTL3, METTL14)], respectively. (2) Experiment II: H9C2 cardiomyocytes were divided into blank control group, H/R+sh-NC group, and H/R+sh-WTAP group. sh-WTAP was transfected to knock down the expression of WTAP in H/R+sh-WTAP group, and the model establishment method in the other groups was the same as experiment I. At 48 hours after transfection, the apoptosis rate of cells was detected by flow cytometry. The protein expressions of WTAP, activated caspase-3, activated poly (ADP-ribose) polymerase (PARP), activating transcription factor 4 (ATF4), proline-rich receptor-like protein kinase (PERK), phosphorylated PERK (p-PERK) and CCAAT/enhancer-binding protein homologous protein (CHOP) were detected by Western blotting. The positive expression of ATF4 was observed by immunofluorescence staining. (3) Experiment III: H9C2 cardiomyocytes were divided into blank control group, H/R+sh-NC group, H/R+sh-WTAP group and H/R+sh-WTAP+ATF4 group. The overexpression plasmid ATF4 was transfected into H9C2 cardiomyocytes, and the modeling method of the other groups were modeled the same as experiment II. The apoptosis rate was detected by flow cytometry. Western blotting was used to detect the protein expressions of ATF4, CHOP, activated caspase-3 and activated PARP., Results: (1) Experiment I: the methylation level of m6A in the H/R group was significantly higher than that in the blank control group. RT-qPCR results showed that the gene expressions of METTL3, METTL14 and WTAP in the H/R model group were significantly higher than those in the blank control group, and WTAP was the most significantly up-regulated. Western blotting results showed the same trend. These results suggested that the expression level of methyltransferase WTAP is significantly up-regulated in H/R-induced cardiomyocytes. (2) Experiment II: the apoptosis level in H/R+sh-WTAP group was significantly lower than that in H/R+sh-NC group [(14.16±1.58)% vs. (24.51±2.38)%, P < 0.05]. Western blotting results showed that the protein expressions of WTAP, activated caspase-3, activated PARP, p-PERK, ATF4 and CHOP in the H/R+sh-WTAP group were significantly lower than those in the H/R+sh-NC group. Fluorescence microscopy results showed that the ATF4 positive signal in the H/R+sh-WTAP group was significantly weaker than that in the H/R+sh-NC group [(19.36±1.81)% vs. (32.83±2.69)%, P < 0.01]. The above results suggested that knockdown of WTAP could inhibit H/R-induced cardiomyocyte apoptosis and endoplasmic reticulum stress. (3) Experiment III: the apoptosis level of H/R+sh-WTAP+ATF4 group was significantly higher than that of H/R+sh-WTAP group [(26.61±2.76)% vs. (17.14±0.87)%, P < 0.05]. Western blotting results showed that the protein expressions of ATF4, CHOP, activated caspase-3 and activated PARP in the H/R+sh-WTAP+ATF4 group were significantly higher than those in the H/R+sh-WTAP group. These results suggested that overexpression of ATF4 reversed the inhibitory effect of sh-WTAP on endoplasmic reticulum stress and apoptosis in H/R-induced cardiomyocytes., Conclusions: Methyltransferase WTAP could regulate ATF4 expression, mediate cell apoptosis and endoplasmic reticulum stress, and promote H/R-induced myocardial cell injury.

Although many efforts have been made to improve management strategies and diagnostic methods in the past several decades, the prevention of anastomotic complications, such as anastomotic leaks and strictures, remain a major clinical challenge. Therefore, new molecular pathways need to be identified that regulate anastomotic healing, and to design new treatments for patients after anastomosis to reduce the occurrence of complications. Rabbits were treated with a MST1/2 inhibitor XMU-XP-1, a Chinese medicine formula Shenhuang plaster (SHP) or a control vehicle immediately after surgery. The anastomotic burst pressure, collagen deposition, and hydroxyproline concentration were evaluated at 3 and 7 days after the surgery, and qRT-PCR and western-blot analyses were used to characterize mRNA and protein expression levels. Both XMU-XP-1 and SHP significantly increased anastomotic burst pressure, collagen deposition, and the concentration of hydroxyproline in intestinal anastomotic tissue at postoperative day 7 (POD 7). Importantly, SHP could induce TGF-β1 expression, which activated its downstream target Smad-2 to activate the TGF-β1 signaling pathway. Moreover, SHP reduced the phosphorylation level of YAP and increased its active form, and treatment with verteporfin, a YAP-TEAD complex inhibitor, significantly suppressed the effects induced by SHP during anastomotic tissue healing. This study demonstrated that activation of the Hippo-YAP pathway enhances anastomotic healing, and that SHP enhances both the TGF-β1/Smad and YAP signaling pathways to promote rabbit anastomotic healing after surgery. These results suggest that SHP could be used to treat patients who underwent anastomosis to prevent the occurrence of anastomotic complications., Competing Interests: The authors report no conflicts of interest in this work.

Dysregulated metal homeostasis is associated with many pathological conditions, including arthritic diseases. Osteoarthritis and rheumatoid arthritis are the two most prevalent disorders that damage the joints and lead to cartilage and bone destruction. Recent studies show that the levels of zinc (Zn) and copper (Cu) are generally altered in the serum of arthritis patients. Therefore, metal dyshomeostasis may reflect the contribution of these trace elements to the disease's pathogenesis and manifestations, suggesting their potential for prognosis and treatment. Carnosine (Car) also emerged as a biomarker in arthritis and exerts protective and osteogenic effects in arthritic joints. Notably, its zinc(II) complex, polaprezinc, has been recently proposed as a drug-repurposing candidate for bone fracture healing. On these bases, this review article aims to provide an overview of the beneficial roles of Cu and Zn in bone and cartilage health and their potential application in tissue engineering. The effects of Car and polaprezinc in promoting cartilage and bone regeneration are also discussed. We hypothesize that polaprezinc could exchange Zn for Cu, present in the culture media, due to its higher sequestering ability towards Cu. However, future studies should unveil the potential contribution of Cu in the beneficial effects of polaprezinc.

Background: Recent studies suggest that cellular senescence is related to the pathogenesis of idiopathic pulmonary fibrosis. However, cellular senescence has yet to be targeted therapeutically in clinical practice. ARV825, a recently developed BRD4 degrader, has been reported as a novel senolytic drug. Conversely, it has also been reported that BRD4 regulates the pro-fibrotic gene expression of fibroblasts. Therefore, this study focuses on the senolytic and anti-fibrotic effects of ARV825 and evaluated these effects on lung fibrosis., Methods: Lung fibroblasts were induced to senescence through serial passage. The expression of senescence markers and pro-fibrotic markers were determined through quantitative PCR or immunoblot analysis. Lung fibrosis was induced in mice through intratracheal administration of bleomycin. Mice treated with ARV825 underwent histological analysis of lung fibrosis using the Ashcroft score. Total lung collagen was quantified through a hydroxyproline assay. Respiratory mechanics analysis was performed using the flexiVent system., Results: For senescent cells, ARV825 induced the expression of an apoptosis marker while reducing the expression of BRD4 and senescence markers. On the other hand, for early passage pre-senescent cells, ARV825 reduced the expression of collagen type 1 and α-smooth muscle actin. In an experimental mouse model of lung fibrosis, ARV825 attenuated lung fibrosis and improved lung function. Immunohistochemical staining revealed a significant decrease in the number of senescent alveolar type 2 cells in lung tissue due to ARV825 treatment., Conclusions: These results suggest that ARV825 may impact the progressive and irreversible course of fibrotic lung diseases., Competing Interests: Conflict of interest The authors have no conflicts of interest., (Copyright © 2023. Published by Elsevier B.V.)

Certain cancers, such as triple-negative breast cancer (TNBC), pose a challenging prognosis due to the absence of identifiable hormone-related receptors and effective targeted therapies. Consequently, novel therapeutics are required for these cancers, offering minimal side effects and reduced drug resistance. Unexpectedly, siRNA-7, initially employed as a control, exhibited significant efficacy in inhibiting cell viability in MDA-MB-231 cells. Through a genome-wide search of seed sequences, the targets of siRNA-7 were identified as cancer-related genes, namely PRKCE, RBPJ, ZNF737, and CDC7 in MDA-MB-231 cells. The mRNA repression analysis confirmed the simultaneous suppression by siRNA-7. Combinatorial administration of single-targeting siRNAs demonstrated a comparable reduction in viability to that achieved by siRNA-7. Importantly, siRNA-7 selectively inhibited cell viability in MDA-MB-231 cells, while normal HDF-n cells remained unaffected. Furthermore, in a xenograft mouse model, siRNA-7 exhibited a remarkable 76% reduction in tumor volume without any loss in body weight. These findings position siRNA-7 as a promising candidate for a novel, safe, specific, and potent TNBC cancer therapeutic. Moreover, the strategy of multiple suppressing small interfering RNA holds potential for the treatment of various diseases associated with gene overexpression., (© 2023 Wiley-VCH GmbH.)

A major active constituent of Moringa oleifera Lam. is 4-[(α-L-rhamnose oxy) benzyl] isothiocyanate (MITC). To broaden MITC's application and improve its biological activity, we synthesized a series of MITC quinazolinone derivatives and evaluated their anticancer activity. The anticancer effects and mechanisms of the compound with the most potent anticancer activity were investigated further. Among 16 MITC quinazolinone derivatives which were analyzed, MITC-12 significantly inhibited the growth of U251, A375, A431, HCT-116, HeLa, and MDA-MB-231 cells. MITC-12 significantly inhibited U251 cell proliferation in a time- and dose-dependent manner and decreased the number of EdU-positive cells, but was not toxic to normal human gastric mucosal cells (GES-1). Further, MITC-12 induced apoptosis of U251 cells, and increased caspase-3 expression levels and the Bax:Bcl-2 ratio. In addition, MITC-12 significantly decreased the proportion of U251 cells in the G1 phase and increased it in S and G2 phases. Transcriptome sequencing showed that MITC-12 had a significant regulatory effect on pathways regulating the cell cycle. Further, MITC-12 significantly decreased the expression levels of the cell cycle-related proteins CDK2, cyclinD1, and cyclinE, and increased those of cyclinA2, as well as the p-JNK:JNK ratio. These results indicate that MITC-12 inhibits U251 cell proliferation by inducing apoptosis and cell cycle arrest, activating JNK, and regulating cell cycle-associated proteins. MITC-12 has potential for use in the prevention and treatment of glioma.

Background: The emergence of chemotherapy resistance usually causes therapeutic failure in advanced cervical cancer. Forkhead box protein M1 (FOXM1) and threonine tyrosine kinase (TTK) are closely associated with cancer drug sensitivity, but the mechanism of FOXM1 on TTK involvement in chemo-treated cervical cancer remains unclear. Here, we aimed to observe the effects of FOXM1 on TTK and on chemotherapy sensitivity in cervical cancer., Methods: The expressions of FOXM1 and TTK in cervical cancer tissues and para-cancerous tissues were analyzed by immunohistochemistry. SiHa and Hela cells were transfected with human lentivirus-FOXM1, small interfering RNA (siRNA) or pcDNA3.1/FOXM1 to analyze the changes in TTK protein expression. Furthermore, the cells were treated with paclitaxel (8 μM) or cisplatin (10 μM) to analyze the effects of FOXM1 on chemotherapy sensitivity. SiHa cells were used to construct a xenograft model to study the effects of FOXM1 expression in response to paclitaxel treatment. The tumor size and weight were observed. The expressions of Ki-67, FOXM1, and TTK protein in tumor tissues were measured by immunohistochemistry., Results: High expression of FOXM1 and TTK were found in the cervical cancer tissues ( p < 0.05). The TTK protein expressions were decreased by FOMX1-siRNA transfection in SiHa and Hela cells ( p < 0.01). The cell viability and cell cycle were also suppressed by FOMX1-siRNA transfection ( p < 0.01) but enhanced by pcDNA3.1/FOXM1 transfection ( p < 0.01). For paclitaxel or cisplatin treatment, the cell viability and cell DNA damage were improved due to the FOXM1 overexpression ( p < 0.01). TTK inhibitor significantly suppressed the effects of FOXM1 overexpression ( p < 0.01)., Conclusions: FOXM1 regulated TTK and affected the therapeutic efficacy of cisplatin and paclitaxel in cervical cancer., Competing Interests: The authors declare no conflict of interest., (© 2023 The Author(s). Published by Discovery Medicine.)

Objective: Atherosclerosis is a chronic cardiovascular disease associated with oxidative stress damage, which is caused by excessive oxidation of low-density lipoprotein (ox-LDL). The role of microRNA miR-34a-5p on oxidative stress in ox-LDL-treated macrophages was investigated in this study., Methods: Flow cytometry was prepared for assessing THP1-derived macrophage apoptosis. The protein and expression levels of miR-34a-5p and MDM4 were examined by Western blot and RT-qPCR, respectively. We also measured the levels of total cholesterol (TC) and triglyceride to determine the lipid accumulation. Subsequently, the activities of superoxide dismutase, malondialdehyde, and reactive oxygen species revealed the level of oxidative stress injury after miR-34a-5p and MDM4 knockdown., Results: After ox-LDL treatment, cell apoptosis of macrophages increased in a dose-dependent and time-dependent manner. With the increase of ox-LDL treatment and the prolongation of treatment time, the expression level of miR-34a-5p was upregulated. Next, interfering with miR-34a-5p inhibited lipid accumulation and oxidative stress injury in ox-LDL-stimulated macrophages. MDM4 was a target gene of miR-34a-5p and was upregulated in ox-LDL-stimulated macrophages. With the increase of ox-LDL treatment and the prolongation of treatment time, the expression level of MDM4 was downregulated. Importantly, MDM4 knockdown partially counteracted the inhibitory effect of miR-34a-5p on oxidative stress injury., Conclusion: MicroRNA miR-34a-5p knockdown suppressed oxidative stress injury via MDM4 in ox-LDL-treated macrophages.

Background: The primary pathogenic factors of Alzheimer's disease (AD) have been identified as oxidative stress, inflammatory damage, and apoptosis. Chrysophanol (CHR) has a good neuroprotective effect on AD, however, the potential mechanism of CHR remains unclear., Purpose: In this study, we focused on the ROS/TXNIP/NLRP3 pathway to determine whether CHR regulates oxidative stress and neuroinflammation., Methods: D-galactose and Aβ 25-35 combination were used to build an in vivo model of AD, and the Y-maze test was used to evaluate the learning and memory function of rats. Morphological changes of neurons in the rat hippocampus were observed using hematoxylin and eosin (HE) staining. AD cell model was established by Aβ 25-35 in PC12 cells. The DCFH-DA test identified reactive oxygen species (ROS). The apoptosis rate was determined using Hoechst33258 and flow cytometry. In addition, the levels of MDA, LDH, T-SOD, CAT, and GSH in serum, cell, and cell culture supernatant were detected by colorimetric method. The protein and mRNA expressions of the targets were detected by Western blot and RT-PCR. Finally, molecular docking was used to further verify the in vivo and in vitro experimental results., Results: CHR could significantly improve learning and memory impairment, reduce hippocampal neuron damage, and reduce ROS production and apoptosis in AD rats. CHR could improve the survival rate, and reduce the oxidative stress and apoptosis in the AD cell model. Moreover, CHR significantly decreased the levels of MDA and LDH, and increased the activities of T-SOD, CAT, and GSH in the AD model. Mechanically, CHR significantly reduced the protein and mRNA expression of TXNIP, NLRP3, Caspase-1, IL-1β, and IL-18, and increase TRX., Conclusions: CHR exerts neuroprotective effects on the Aβ 25-35 -induced AD model mainly by reducing oxidative stress and neuroinflammation, and the mechanism may be related to ROS/TXNIP/NLRP3 signaling pathway., (© 2023. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Evidence suggests that fluoride-induced neurodevelopment damage is linked to mitochondrial disorder, yet the detailed mechanism remains unclear. A cohort of Sprague-Dawley rats developmentally exposed to sodium fluoride (NaF) was established to simulate actual exposure of human beings. Using high-input proteomics and small RNA sequencing technology in rat hippocampus, we found mitochondrial translation as the most striking enriched biological process after NaF treatment, which involves the differentially expressed Required Meiotic Nuclear Division 1 homolog (RMND1) and neural-specific miR-221-3p. Further experiments in vivo and in vitro neuroendocrine pheochromocytoma (PC12) cells demonstrated that NaF impaired mitochondrial translation and function, as shown by declined mitochondrial membrane potential and inhibited expression of mitochondrial translation factors, mitochondrial translation products, and OXPHOS complexes, which was concomitant with decreased RMND1 and transcription factor c-Fos in mRNA and proteins as well as elevated miR-221-3p. Notably, RMND1 overexpression alleviated the NaF-elicited mitochondrial translation impairment by up-regulating translation factors, but not vice versa. Interestingly, ChIP-qPCR confirmed that c-Fos specifically controls the RMND1 transcription through direct binding with Rmnd1 promotor. Interference of gene expression verified c-Fos as an upstream positive regulator of RMND1, implicating in fluoride-caused mitochondrial translation impairment. Furthermore, dual-luciferase reporter assay evidenced that miR-221-3p targets c-Fos by binding its 3' untranslated region. By modulating the miR-221-3p expression, we identified miR-221-3p as a critical negative regulator of c-Fos. More importantly, we proved that miR-221-3p inhibitor improved mitochondrial translation and mitochondrial function to combat NaF neurotoxicity via activating the c-Fos/RMND1 axis, whereas miR-221-3p mimic tended towards opposite effects. Collectively, our data suggest fluoride impairs mitochondrial translation by dysregulating the miR-221-3p/c-Fos/RMND1 axis to trigger mitochondrial dysfunction, leading to neuronal death and neurodevelopment defects., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Objective: The aim of this study is to investigate the effect of let-7c-5p on the malignant behaviors of hepatocellular carcinoma (HCC) and its specific molecular pathway., Methods: Differential expression and survival analysis of let-7c-5p were obtained from The Cancer Genome Atlas database, and then its expression level was preliminarily verified through qPCR. The effect of let-7c-5p on the malignant phenotype of HCC cells was subsequently evaluated using CCK-8, transwell, wound healing, and flow cytometry assays. Downstream mRNA regulated by let-7c-5p was identified and confirmed by ENCORI database, dual-luciferase reporter, and western blot assays. The immunocorrelation of genes was evaluated by Xiantao tool, and TIMER and TISIDB databases., Results: The expression level of let-7c-5p in HCC was obviously reduced, which was found to be closely associated with the short survival time of HCC patients. Cell phenotypic experiments showed that let-7c-5p inhibited proliferation, invasion, and migration and promoted apoptosis of HCC cells. Dual-luciferase reporter and western blot analysis demonstrated that CDCA8 is a downstream mRNA of let-7c-5p and is negatively regulated by it. Rescue experiment revealed that CDCA8 reversed the effect of let-7c-5p on the malignant phenotype of HCC cells. Furthermore, analysis of the public database revealed that CDCA8 is related to some immune cells and immunomodulators, and that it may participate in the regulation of some immune pathways and immune functions., Conclusion: Let-7c-5p has been proved to suppress HCC by down-regulating immune-related CDCA8, which will help understand the pathogenesis of HCC and develop drugs for its treatment., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Osteoarthritis (OA) is a common joint disease, and studies have reported that the endoplasmic reticulum stress (ERS) in chondrocytes caused by the cartilage tissue damage could mediate the activation of Nod-like receptor protein 3 (NLRP3) inflammasomes through inositol-requiring enzyme 1 alpha (IRE1α) and thioredoxin interacting protein (TXNIP). Ginsenoside compound K (CK) has an inhibitory effect on IRE1α activation. However, the role of IRE1α-TXNIP and its interaction with CK are still unclear. In this study, we examined the role and mechanism of action of CK in OA. We found that CK ameliorated OA and ERS in IL-1β-treated chondrocytes and a monoiodoacetate-induced rat OA model. The effect of CK on inflammation, pyroptosis, and ERS was blocked by the ERS inducer tunicamycin. In conclusion, CK hindered OA progression by inhibiting the ERS-IRE1α-TXNIP-NLRP3 axis. Overall, our data indicate that CK could be useful in the treatment of OA and other chronic inflammatory diseases.

Bavachin is a dihydroflavonoid compound isolated from Psoralea corylifolia, and exhibits anti-bacterial, anti-inflammatory, anti-tumor and lipid-lowering activities. Recent attention has gradually drawn on bavachin-induced apoptosis in many human cancer cell lines. However, the anti-cancer effects and related mechanisms in colorectal cancer remain unknown. Here, we investigated the effects of bavachin on colorectal cancer in vivo and in vitro. The results showed that bavachin inhibited the proliferation of human colorectal cancer cells and induce apoptosis. These changes were mediated by activating the MAPK signaling pathway, which significantly up-regulated the expression of Gadd45a. Furthermore, Gadd45a silencing obviously attenuated bavachin-mediated cell apoptosis. Inhibition of the MAPK signaling pathway by JNK/ERK/p38 inhibitors also weakened the up-regulation of Gadd45a by bavachin. The anticancer effect of bavachin was also validated using a mouse xenograft model of human colorectal cancer. In conclusion, these findings suggest that bavachin induces the apoptosis of colorectal cancer cells through activating the MAPK signaling pathway., (Copyright © 2023 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.)

Background: Keloids are "tumor-like" scars that grow beyond the boundary of injury. Its pathogenesis is complex. This paper will discuss the pathogenesis of keloid from the transcriptional regulation mechanism of TRAF3IP2., Methods: IL-17 was utilized to induce human keloid fibroblasts (KFs) and normal dermal fibroblasts. With the application of RT-qPCR and Western blot, TRAF3IP2 expression was detected. Subsequently, the expression of TRAF3IP2 was interfered by cell transfection and the effects of interfering TRAF3IP2 on cell proliferative rate, migration rate, and extracellular matrix were assessed with CCK-8, Wound Healing, immunofluorescence, and Western blot techniques. Proliferation, migration, and (ECM) deposition were detected by JASPAR software predicted the binding sites of transcription factors FOXO4 and TRAF3IP2 promoters. The relationship between FOXO4 and TRAF3IP2 was verified by Dual luciferase activity assay and ChIP. Finally, the expression of TRAF3IP2 and FOXO4 was interfered simultaneously to further explore the mechanism., Results: TRAF3IP2 was enhanced in IL-17 induced KFs. Interference with TRAF3IP2 imparted suppressive effects on the proliferation, migration, and ECM deposition of KFs. FOXO4 could inhibit TRAF3IP2 transcription, and interference with FOXO4 reversed the effect of TRAF3IP2 down-regulation on KFs via TGF-β1/Smad pathway., Conclusion: TRAF3IP2 was regulated by FOXO4 and affected fibroblast proliferation, migration, and ECM deposition in keloid through the TGF-β1/Smad pathway., (© 2022 Wiley Periodicals LLC.)

B-cell progenitor fate determinant interferon regulatory factor 4 (IRF4) exerts key roles in the pathogenesis and progression of multiple myeloma (MM), a currently incurable plasma cell malignancy. Aberrant expression of IRF4 and the establishment of a positive auto-regulatory loop with oncogene MYC, drives a MM specific gene-expression program leading to the abnormal expansion of malignant immature plasma cells. Targeting the IRF4-MYC oncogenic loop has the potential to provide a selective and effective therapy for MM. Here we evaluate the use of bromodomain inhibitors to target the IRF4-MYC axis through combined inhibition of their known epigenetic regulators, BRD4 and CBP/EP300. Although all inhibitors induced cell death, we found no synergistic effect of targeting both of these regulators on the viability of MM cell-lines. Importantly, for all inhibitors over a time period up to 72 h, we detected reduced IRF4 mRNA, but a limited decrease in IRF4 protein expression or mRNA levels of downstream target genes. This indicates that inhibitor-induced loss of cell viability is not mediated through reduced IRF4 protein expression, as previously proposed. Further analysis revealed a long half-life of IRF4 protein in MM cells. In support of our experimental observations, gene network modeling of MM suggests that bromodomain inhibition is exerted primarily through MYC and not IRF4. These findings suggest that despite the autofeedback positive regulatory loop between IRF4 and MYC, bromodomain inhibitors are not effective at targeting IRF4 in MM and that novel therapeutic strategies should focus on the direct inhibition or degradation of IRF4., (© 2022 The Authors. Hematological Oncology published by John Wiley & Sons Ltd.)

B-cell chronic lymphocytic leukemia (CLL) results from intrinsic genetic defects and complex microenvironment stimuli that fuel CLL cell growth through an array of survival signaling pathways. Novel small-molecule agents targeting the B-cell receptor pathway and anti-apoptotic proteins alone or in combination have revolutionized the management of CLL, yet combination therapy carries significant toxicity and CLL remains incurable due to residual disease and relapse. Single-molecule inhibitors that can target multiple disease-driving factors are thus an attractive approach to combat both drug resistance and combination-therapy-related toxicities. We demonstrate that SRX3305, a novel small-molecule BTK/PI3K/BRD4 inhibitor that targets three distinctive facets of CLL biology, attenuates CLL cell proliferation and promotes apoptosis in a dose-dependent fashion. SRX3305 also inhibits the activation-induced proliferation of primary CLL cells in vitro and effectively blocks microenvironment-mediated survival signals, including stromal cell contact. Furthermore, SRX3305 blocks CLL cell migration toward CXCL-12 and CXCL-13, which are major chemokines involved in CLL cell homing and retention in microenvironment niches. Importantly, SRX3305 maintains its anti-tumor effects in ibrutinib-resistant CLL cells. Collectively, this study establishes the preclinical efficacy of SRX3305 in CLL, providing significant rationale for its development as a therapeutic agent for CLL and related disorders.

Circular RNAs (circRNAs) are non-coding RNAs with covalent closed-loop structures that are vital in regulating diverse pathological processes. This work is aimed to investigate the role of circ&#95;0120376 in non-small cell lung cancer (NSCLC). Circ&#95;0120376, microRNA (miR)-148b-3p, and centrosomal protein 55 (CEP55) mRNA expression in NSCLC tissues and cells were determined using qRT-PCR. The influences of circ&#95;0120376 and miR-148b-3p on the proliferation of NSCLC cell lines were analyzed by CCK-8 and colony formation assays. Apoptosis was analyzed by flow cytometry. Cell migration and invasion were analyzed using the Transwell experiment. Binding relationships between circ&#95;0120376 and miR-148b-3p and between miR-148b-3p and CEP55 3'UTR were investigated using the dual-luciferase reporter experiment and the RIP experiment. Western blot was conducted to analyze the regulatory effect of circ&#95;0120376 and miR-148b-3p on CEP55 expression. We found that circ&#95;0120376 was markedly overexpressed in NSCLC, and its overexpression was positively associated with increased T stage and lymph node metastasis of the patients. Functional experiments unveiled that circ&#95;0120376 enhanced the proliferation, migration and invasion of NSCLC cells and impeded apoptosis, while knocking down circ&#95;0120376 remarkably suppressed the malignant features of NSCLC cells mentioned above. Circ&#95;0120376 could adsorb miR-148b-3p to reduce miR-148b-3p expression, and circ&#95;0120376 could increase CEP55 expression via adsorbing miR-148b-3p. In summary, circ&#95;0120376 contributes to the malignancy of NSCLC cells through a ceRNA mechanism via regulating miR-148b-3p/CEP55 axis. Circ&#95;0120376 is likely to be a potential diagnostic biomarker and therapeutic target for NSCLC.

Background: The use of bacteria to synthesize nanoparticles as an environment-friendly method has recently been considered by researchers. Bacteria residing in different mines have shown high potential in the synthesis of metal nanoparticles due to their compatibility with the environment. The aim of this study was to evaluate the ability of Zarshouran gold mine bacteria to synthesize silver nanoparticles and their antibacterial activity., Methods: After isolation of mine bacteria and several screening steps, silver ion tolerant bacteria that were able to synthesize extracellular silver nanoparticles were isolated and the most suitable isolate was selected and sequenced. The characteristics, stability, and production efficiency of silver nanoparticles were evaluated using UV-vis spectrophotometry, DLS, TEM, FTIR, and X-ray diffraction analysis. Finally, the antibacterial effect of silver nanoparticles against pathogenic bacteria was investigated., Results: Among the eight silver-tolerant bacteria, isolate No. 6 had high antibacterial activity and high potential in the synthesis and stabilization of silver nanoparticles. Therefore, this isolate was selected for the next experiments. The results of 16S rDNA sequencing showed that this isolate is related to Bacillus pumilus. We registered in the NCBI Bank called ROM6 with access number MW440543. The DLS and TEM analysis showed that silver nanoparticles produced by this isolate were most spherical with a size of less than 25 nm and were stable for at least 180 days. The efficiency at concentrations less than 0.9 g/l silver nitrate was over 90% and the minimum inhibition concentration of nanoparticles was determined against S. aureus, E. coli, P. aeruginosa, and A. baumannii ranging from 1.4 to 5.6 µg/ml., Conclusion: We found that the bacteria residing in the gold mine have a high capacity for the synthesis of spherical and high stable silver nanoparticles with a strong antibacterial effect., (© 2022. The Author(s).)

Objectives: Adipokine resistin is highly expressed in the serum and synovial uid (SF) of patients with knee osteoarthritis (KOA) but its pathogenic role in KOA remains unclear. We aimed to explore the mechanism of resistin/CAP1 in human KOA chondrocytes., Methods: We enrolled 103 patients with radiographic KOA and 86 healthy participants as controls. Resistin levels in serum and SF were determined by enzyme-linked immunosorbent assay (ELISA). CAP1 expression was measured in cartilage tissues using immunohistochemistry, quantitative real-time polymerase chain reaction (qRT-PCR), and western blot. Effects of resistin on chondrocytes and CAP1 were evaluated via qRT-PCR and co-immunoprecipitation. The roles of CAP1, p38-MAPK, and NF-κB signalling pathways in KOA development were evaluated using adenovirus-mediated CAP1 short hairpin RNA, qRT-PCR, western blot, and ELISA., Results: Resistin expression in serum and SF was elevated in severe radiographic KOA. CAP1 levels were higher in KOA cartilage and were positively correlated with resistin expression. Resistin promoted CCL3, CCL4, MMP13, and ADAMTS-4 expression through the CAP1 receptor. Resistin also directly bound to CAP1, as confirmed by co-immunoprecipitation. CAP1 knockdown in chondrocytes attenuated resistin-induced expression of CCL3, CCL4, MMP13, and ADAMTS-4 and activated p38-MAPK and NF-κB signalling pathways., Conclusions: Resistin binds CAP1 and upregulates the expression of proinflammatory cytokines and matrix-degrading enzymes via p38-MAPK and NF-κB signalling in human chondrocytes.

Circular RNAs (CircRNAs) have attracted increasing attention in the diagnosis and treatment of human cancers. CircUBAP2 has been identified to promote the progression of triple-negative breast cancer (TNBC), but the function of circUBAP2 in the cisplatin (DDP) resistance of TNBC remains obscure. Our investigation showed that circUBAP2 was significantly upregulated in DDP-resistant TNBC and TNBC sensitivity to DDP could be enhanced by silencing of circUBAP2. Moreover, circUBAP2 was revealed to be a ceRNA for miR-300 to upregulate the expression of anti-silencing function 1B histone chaperone (ASF1B). The effect of circUBAP2/miR-300/ASF1B axis on DDP resistance of TNBC was evaluated by rescue experiments, which demonstrated that circUBAP2 inhibited TNBC sensitivity to DDP through miR-300/ASF1B axis. Furthermore, it was discovered that ASF1B activated PI3K/AKT/mTOR signaling to facilitate the DDP resistance of TNBC cells. In summary, this research revealed a novel regulatory mechanism that circUBAP2 functioned as ceRNA of miR-300 to upregulate ASF1B, which further triggered the PI3K/AKT/mTOR (PAM) signaling to enhance the DDP resistance of TNBC.

Globally, hepatocellular carcinoma (HCC) is one of the most common causes of cancer-associated mortalities. It has a high rate of metastasis and recurrence, which predict a poor prognosis. G-protein-coupled receptor (GPCR)-kinase interacting protein-1 (GIT1) is a multifunctional scaffold protein that mediates the progression of various tumors. Studies have correlated GIT1 with HCC, however, these correlations have not been fully elucidated. Therefore, we aimed at evaluating the expression of GIT1 in HCC tissues and cells, and to investigate its role and potential mechanisms in HCC progression. The expression levels of GIT1 in HCC tissues and other cancers was determined by using the Oncomine and TCGA databases. Functional analysis of GIT1 in HCC was evaluated through in vitro and in vivo experiments, whereby, HCC cells were transfected with synthetically overexpressed and short hairpin RNA (shRNA) lentivirus-mediated plasmids. Kaplan-Meier and Cox regression methods were used to establish the associations between GIT1 and clinical outcomes of 158 HCC patients. GIT1 was found to be elevated in HCC tissues where it promoted the invasion, migration, and proliferation of HCC cells. Moreover, the overexpression of GIT1 prompted epithelial-mesenchymal transition (EMT) by activating extracellular regulated kinase 1/2 (ERK1/2) pathway, which was shown to be reversed by SCH772984, a specific ERK1/2 inhibitor. GIT1 was also found to be associated with malignant features of HCC, leading to a poorer prognosis. In conclusion, GIT1 promotes HCC progression by inducing EMT and may reflect the course of HCC patients.

The trace element zinc is essential for many aspects of physiology. The mitochondrion is a major Zn 2+ store, and excessive mitochondrial Zn 2+ is linked to neurodegeneration. How mitochondria maintain their Zn 2+ homeostasis is unknown. Here, we find that the SLC-30A9 transporter localizes on mitochondria and is required for export of Zn 2+ from mitochondria in both Caenorhabditis elegans and human cells. Loss of slc-30a9 leads to elevated Zn 2+ levels in mitochondria, a severely swollen mitochondrial matrix in many tissues, compromised mitochondrial metabolic function, reductive stress, and induction of the mitochondrial stress response. SLC-30A9 is also essential for organismal fertility and sperm activation in C. elegans , during which Zn 2+ exits from mitochondria and acts as an activation signal. In slc-30a9 -deficient neurons, misshapen mitochondria show reduced distribution in axons and dendrites, providing a potential mechanism for the Birk-Landau-Perez cerebrorenal syndrome where an SLC30A9 mutation was found., Competing Interests: The authors declare no competing interest.

Background: Gliomas are highly aggressive and lack of efficient targeted therapy. YAP, as a Hippo pathway downstream effector, plays a key role in promoting tumor development through the interaction with transcription factor TEAD on the NH3-terminal proline-rich domain. Therefore, targeting TEAD-interacting domain of YAP may provide a novel approach for the treatment of gliomas., Materials and Methods: We generated a truncated YAP protein which includes the TEAD-binding domain (YAPBD), and supposed YAPBD can interact with endogenous TEAD but lost the function to activate YAP target gene expressions. The association of YAP expression with the malignant characters of glioma tissues were determined by immunohistochemistry. TEAD-binding capacity of YAPBD was determined by co-immunoprecipitation. The cell proliferation and migration were determined by MTT assay, xenograft assay, wound healing assay and transwell assay, respectively. YAP target genes were detected by Western blot., Results: YAP was highly expressed in glioma tissues and associated with tumor malignancy. YAPBD could block the TEAD-YAP complex formation by competing with YAP binding to TEAD. YAPBD could inhibit glioma cell growth both in vitro and in vivo, through the induction of cell cycle arrest and apoptosis. The cell cycle-related gene cyclin D1 and c-myc, and anti-apoptotic gene Bcl-2, Bcl-xL and survivin were inhibited after YAPBD overexpression. Furthermore, YAPBD also decreased cell migration and invasion, and repressed epithelial-mesenchymal transition., Conclusion: YAPBD can block glioma cell survival and repress YAP-dependent gene expressions, indicating gene therapy which targets TEAD-YAP complex would be a potential and significant novel approach for human malignant gliomas.

Background: ALMS1-IT1, a recently identified lncRNA, has been proven to play a crucial role in regulating tumor progression and predicting the survival time of tumor patients. Data analysis from the Human Body Map (HBM) revealed that ALMS1-IT1 is expressed mainly in brain tissues., Methods: In this study, the role of ALMS1-IT in regulating neuro-inflammation and functional recovery was investigated after ischemic cerebral damage. To this end, the rat model of transient middle cerebral artery occlusion (tMCAO) was constructed, the cell model of oxygen-glucose deprivation (OGD) was established using BV2 microglial cells, and the aberrant expression of ALMS1-IT1 was assessed in brain tissues. After ALMS1- IT1 knockdown through intrathecal injection of Lv-shALMS1-IT1, neuro-inflammatory response and functional tests including a modified neurological severity score (mNSS) and a foot-fault test were assessed., Results: The level of ALMS1-IT1 was promptly enhanced at 12 hours (h) following MCAO, peaking at 48 h, and remaining high at day 14 compared to the sham group. Pro-inflammatory cytokines (IL-1β, IL-6, and TNF- α) were increased after MCAO, whereas ALMS1-IT1 inhibition suppressed the expression of IL-1β, IL-6 and TNF-α in MCAO rats. The results from mNSS and foot-fault test showed that ALMS1-IT1 knockdown significantly improved spatial learning and sensorimotor function of MCAO rats. Mechanistically, ALMS1-IT1 knockdown suppressed the activation of NF-κB signaling in vitro and in vivo, as evidenced by decreased p65 expression and p65 nuclear translocation. ALMS1-IT1 overexpression facilitated pro-inflammatory cytokines expression in microglia, whereas the effect was blocked by treatment with JSH-23 (a specific NF-κB inhibitor)., Conclusion: These data demonstrated that ALMS1-IT1 inhibition improved neurological function of MCAO rats, at least in part by repressing NF-κB-dependent neuro-inflammation., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

The Ca 2+ -mediated S100 family protein S100A6 has a crucial task in various intracellular and extracellular activities thereby demonstrating a possible involvement in the advancement and development of malignant tumors. S100A6 has been found to associate with receptor for advanced glycation end products, RAGE, through its extracellular extension. This extension is famously identified as a prominent receptor for many S100 family associates. Additionally, S100A6 binds to S100B protein and forms a heterodimer. Thus, we consider the S100B protein to be a prospective drug molecule to obstruct the interacting regions amongst S100A6 and RAGE V domain. We applied the NMR spectroscopy method to locate the binding area amid the S100A6m (mutant S100A6, cysteine at 3rd position of S100A6 is replaced with serine, C3S) and S100B proteins. The 1 H- 15 N HSQC NMR titrations revealed the probable requisite dynamics of S100A6m and S100B interfaces. Utilizing data from the NMR titrations as input parameters, we ran the HADDOCK program and created a S100A6m-S100B heterodimer complex. The obtained complex was then superimposed with the reported complex of S100A6m-RAGE V domain. This superimposition displayed the possibility of S100B to be a potential antagonist that can block the interface area of the S100A6m and the RAGE V domain. Moreover, an in vitro cancer model using SW480 cells in water-soluble tetrazolium-1 assay (WST-1) showed a noticeable change in the cell proliferation as an effect of these proteins. Our study indicates the possibility to develop a S100B-like competitor that could play a key role in the treatment of S100- and RAGE-mediated human diseases., (Copyright © 2020 Elsevier Inc. All rights reserved.)

While hundreds of genes are induced by type I interferons, their roles in restricting the influenza virus life cycle remain mostly unknown. Using a loss-of-function CRISPR screen in cells prestimulated with interferon beta (IFN-β), we identified a small number of factors required for restricting influenza A virus replication. In addition to known components of the interferon signaling pathway, we found that replication termination factor 2 (RTF2) restricts influenza virus at the nuclear stage (and perhaps other stages) of the viral life cycle, based on several lines of evidence. First, a deficiency in RTF2 leads to higher levels of viral primary transcription, even in the presence of cycloheximide to block genome replication and secondary transcription. Second, cells that lack RTF2 have enhanced activity of a viral reporter that depends solely on four viral proteins that carry out replication and transcription in the nucleus. Third, when the RTF2 protein is mislocalized outside the nucleus, it is not able to restrict replication. Finally, the absence of RTF2 leads not only to enhanced viral transcription but also to reduced expression of antiviral factors in response to interferon. RTF2 thus inhibits primary influenza virus transcription, likely acts in the nucleus, and contributes to the upregulation of antiviral effectors in response to type I interferons. IMPORTANCE Viral infection triggers the secretion of type I interferons, which in turn induce the expression of hundreds of antiviral genes. However, the roles of these induced genes in controlling viral infections remain largely unknown, limiting our ability to develop host-based antiviral therapeutics against pathogenic viruses, such as influenza virus. Here, we performed a loss-of-function genetic CRISPR screen in cells prestimulated with type I interferon to identify antiviral genes that restrict influenza A virus replication. Besides finding key components of the interferon signaling pathway, we discovered a new restriction factor, RTF2, which acts in the nucleus, restricts influenza virus transcription, and contributes to the interferon-induced upregulation of known restriction factors. Our work contributes to the field of antiviral immunology by discovering and characterizing a novel restriction factor of influenza virus and may ultimately be useful for understanding how to control a virus that causes significant morbidity and mortality worldwide., (Copyright © 2020 Chia et al.)

Malignant mesothelioma (MM) is a very aggressive asbestos-related cancer, for which no therapy proves to be effective. We have recently shown that the oncolytic adenovirus dl922-947 had antitumor effects in MM cell lines and murine xenografts. Previous studies demonstrated that dl922-947 -induced host cell cycle checkpoint deregulation and consequent DNA lesions associated with the virus efficacy. However, the cellular DNA damage response (DDR) can counteract this virus action. Therefore, we assessed whether AZD1775, an inhibitor of the G2/M DNA damage checkpoint kinase WEE1, could enhance MM cell sensitivity to dl922-947 . Through cell viability assays, we found that AZD1775 synergized with dl922-947 selectively in MM cell lines and increased dl922-947 -induced cell death, which showed hallmarks of apoptosis (annexinV-positivity, caspase-dependency, BCL-XL decrease, chromatin condensation). Predictably, dl922-947 and/or AZD1775 activated the DDR, as indicated by increased levels of three main DDR players: phosphorylated histone H2AX (γ-H2AX), phospho-replication protein A (RPA)32, phospho-checkpoint kinase 1 (CHK1). Dl922-947 also increased inactive Tyr-15-phosphorylated cyclin-dependent kinase 1 (CDK1), a key WEE1 substrate, which is indicative of G2/M checkpoint activation. This increase in phospho-CDK1 was effectively suppressed by AZD1775, thus suggesting that this compound could, indeed, abrogate the dl922-947 -induced DNA damage checkpoint in MM cells. Overall, our data suggest that the dl922-947 -AZD1775 combination could be a feasible strategy against MM.

Lung adenocarcinoma (LUAD) is the top prevalent histological kind of lung cancer worldwide. Recent evidences have demonstrated that Sauchinone plays an anticancer role in tumor cell invasion and migration. Therefore, we performed this investigation to explain the potential role of Sauchinone in LUAD as well as the potential mechanism involved. Cell counting kit 8 (CCK-8) and transwell experiments were implemented to measure the proliferative, invasive and migratory abilities of LUAD cells. qRT-PCR and Western blot were performed to detect the transfection efficiency of si-EIF4EBP1s. Additionally, Western blot was also implemented to evaluate the effect of Sauchinone on EIF4EBP1 expression level as well as cell cycle-related proteins. Our findings showed that Sauchinone remarkably suppressed the proliferative ability of LUAD cells in a dose-dependent and time-dependent manner. EIF4EBP1 was a candidate target gene of Sauchinone. EIF4EBP1 expression was increased in LUAD tissues, and its high expression induced a poorer prognosis of LUAD patients. EIF4EBP1 expression was positively associated with cell cycle in LUAD. Sauchinone treatment attenuated EIF4EBP1 expression and cell cycle-related protein levels. Knockdown of EIF4EBP1 repressed the proliferation, invasion and migration of LUAD cells; furthermore, Sauchinone stimulation enforced its inhibitory effect. Meanwhile, the treatment of Sauchinone intensified the arrest of cell cycle induced by EIF4EBP1 knockdown. To sum up, our discovery indicated that Sauchinone exerts an anticancer role through down-regulating EIF4EBP1 and mediating cell cycle in LUAD.

Proper chromatin function and maintenance of genomic stability depends on spatiotemporal coordination between the transcription and replication machinery. Loss of this coordination can lead to DNA damage from increased transcription-replication collision events. We report that deregulated transcription following BRD4 loss in cancer cells leads to the accumulation of RNA:DNA hybrids (R-loops) and collisions with the replication machinery causing replication stress and DNA damage. Whole genome BRD4 and γH2AX ChIP-Seq with R-loop IP qPCR reveals that BRD4 inhibition leads to accumulation of R-loops and DNA damage at a subset of known BDR4, JMJD6, and CHD4 co-regulated genes. Interference with BRD4 function causes transcriptional downregulation of the DNA damage response protein TopBP1, resulting in failure to activate the ATR-Chk1 pathway despite increased replication stress, leading to apoptotic cell death in S-phase and mitotic catastrophe. These findings demonstrate that inhibition of BRD4 induces transcription-replication conflicts, DNA damage, and cell death in oncogenic cells.

To accomplish the remarkable task of lifelong infection, the Epstein-Barr virus (EBV) switches between four viral genome latency and lytic programmes to navigate the B-cell compartment and evade immune responses. The transforming programme, consisting of highly immunogenic EBV nuclear antigen (EBNA) and latent membrane proteins (LMPs), is expressed in newly infected B lymphocytes and in post-transplant lymphomas. On memory cell differentiation and in most EBV-associated Burkitt's lymphomas, all but one viral antigen are repressed for immunoevasion. To gain insights into the epigenetic mechanisms that restrict immunogenic oncoprotein expression, a genome-scale CRISPR-Cas9 screen was performed in EBV and Burkitt's lymphoma cells. Here, we show that the ubiquitin ligase ubiquitin-like PHD and RING finger domain-containing protein 1 (UHRF1) and its DNA methyltransferase partner DNA methyltransferase I (DNMT1) are critical for the restriction of EBNA and LMP expression. All UHRF1 reader and writer domains were necessary for silencing and DNMT3B was identified as an upstream viral genome CpG methylation initiator. Polycomb repressive complex I exerted a further layer of control over LMP expression, suggesting a second mechanism for latency programme switching. UHRF1, DNMT1 and DNMT3B are upregulated in germinal centre B cells, the Burkitt's lymphoma cell of origin, providing a molecular link between B-cell state and the EBV latency programme. These results suggest rational therapeutic targets to manipulate EBV oncoprotein expression.

Background: Vasohibins (VASH), which are angiogenesis regulators, consist of Vasohibin-1 (VASH1) and Vasohibin-2 (VASH2). VASH1 is an angiogenesis inhibitor, while VASH2 is a proangiogenic factor. Patients with esophageal squamous cell carcinoma (ESCC) with high tumor expression levels of VASH1 and VASH2 have been reported to show a poor prognosis. The clinical significance of VASH concentrations in the blood of patients with ESCC has not yet been investigated., Methods: Plasma samples from 89 patients with ESCC were analyzed, and the relationships between the plasma VASH concentrations and the clinicopathological factors of the patients were evaluated. Immunohistochemical examination (IHC) of the resected tumor specimens for VASH was performed in 56 patients, and the correlation between the plasma VASH concentrations and tumor expression levels of VASH was analyzed., Results: The patient group with high plasma concentrations of VASH1 showed a higher frequency of lymph node metastasis (P = 0.01) and an invasive growth pattern (P = 0.05). Furthermore, poorly differentiated cancer occurred at a higher frequency in the patient group with high plasma concentrations of VASH2 (P < 0.01). High tumor expression levels of VASH1 were encountered more frequently in the patient group with high plasma concentrations of VASH1 (P = 0.03), and high tumor expression levels of VASH2 were encountered more frequently in the patient group with high plasma concentrations of VASH2 (P = 0.04)., Conclusions: In patients with ESCC, high plasma concentrations were associated with poor clinical outcomes for both VASH1 and VASH2. We propose that results indicate that plasma VASH1 and VASH2 are useful biomarkers in patients with ESCC.

Molecular genetic aberrations in the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway are common in human cancers including glioblastoma, yet, novel therapeutic approaches targeting this pathway in glioblastoma have not been successful. We hypothesized that molecular profiling in combination with in vitro drug sensitivity testing allows to identify signatures associated with sensitivity or resistance to PI3K/mTOR pathway inhibition. We analyzed the molecular mechanisms determining sensitivity to PI3K/mTOR inhibition using gene silencing or pharmacological target inhibition and proliferation, clonogenicity, or spherogenicity as readouts, in human long-term glioma cell (LTC) lines and glioma-initiating cells (GIC). Cultured glioma cells were universally sensitive to growth inhibition induced by PQR309, a novel, dual pan-PI3K/mTOR antagonist. Cells exhibited profound growth arrest, but little apoptotic or necrotic cell death as confirmed by electron microscopy; yet, there was evidence of senescence. Cell lines with high basal levels of phosphorylated (active) AKT, low levels of phosphorylated (inactive) protein translation repressor eukaryotic initiation factor (eIF) 4E-binding protein 1 (p4E-BP1), and high levels of Ser9-phosphorylated (inactive) glycogen synthase kinase 3 beta (pGSK3β) were more sensitive to PQR309. Accordingly, the activity of PQR309 was synergistically enhanced by AKT gene silencing or direct pharmacological AKT inhibition. In vivo studies confirmed the anti-glioma activity of PQR309 alone or in combination with AKT inhibition in the orthotopic LN-229 glioma xenograft model in nude mice. These data justify to explore combined targeted therapy approaches in glioblastoma that aim at down-regulating AKT function to enhance the therapeutic potential of dual PI3K/mTOR inhibitors., (© 2019 International Society for Neurochemistry.)

Maresin Conjugates in Tissue Regeneration 1 (MCTR1) is a newly identified macrophage-derived sulfido-conjugated mediator that stimulates the resolution of inflammation. This study assessed the role of MCTR1 in alveolar fluid clearance (AFC) in a rat model of acute lung injury (ALI) induced by lipopolysaccharide (LPS). Rats were intravenously injected with MCTR1 at a dose of 200 ng/rat, 8 hours after administration of 14 mg/kg LPS. The level of AFC was then determined in live rats. Primary rat ATII (Alveolar Type II) epithelial cells were also treated with MCTR1 (100 nmol/L) in a culture medium containing LPS for 8 hours. MCTR1 treatment improved AFC (18.85 ± 2.07 vs 10.11 ± 1.08, P < .0001) and ameliorated ALI in rats. MCTR1 also significantly promoted AFC by up-regulating epithelial sodium channel (ENaC) and Na + -K + -adenosine triphosphatase (Na, K-ATPase) expressions in vivo. MCTR1 also activated Na, K-ATPase and elevated phosphorylated-Akt (P-Akt) by up-regulating the expression of phosphorylated Nedd4-2 (P-Nedd4-2) in vivo and in vitro. However, BOC-2 (ALX inhibitor), KH7 (cAMP inhibitor) and LY294002 (PI3K inhibitor) abrogated the improved AFC induced by MCTR1. Based on the findings of this study, MCTR1 may be a novel therapeutic approach to improve reabsorption of pulmonary oedema during ALI/acute respiratory distress syndrome (ARDS)., (© 2020 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Background: Vasoinhibin, a protein derived from prolactin, regulates various vascular functions including endothelial cell survival. Of note, vasoinhibin is present in the central nervous system, where it triggers neuroendocrine and behavioral responses to stress. Moreover, vasoinhibin compromises nerve growth factor (NGF)-induced neurite outgrowth in primary sensory neurons of the peripheral nervous system. Nonetheless, information on the functions of vasoinhibin in developing neurons remains limited. The present study explored whether vasoinhibin affects the neurotrophic actions of NGF by measuring the cell differentiation and survival of PC12 pheochromocytoma cells., Methods: The effects of recombinant or lentiviral vector-transduced human vasoinhibin were tested on differentiating PC12 cells. Neurite outgrowth was quantified by measuring their length and density. The MTT assay was employed to assess cell viability, and ELISA was used to quantify DNA fragmentation as an index of apoptosis. Phosphorylated Akt and ERK1/2 were analyzed by Western blotting., Results: The addition of a human recombinant vasoinhibin, and the transduction of a lentiviral vector carrying a human vasoinhibin sequence, significantly reduced NGF-induced neurite outgrowth, cell survival, and phosphorylation of Akt and ERK1/2, and increased DNA fragmentation and caspase 3 activation in PC12 cells., Conclusions: Vasoinhibin downregulates NGF-induced differentiation and survival of PC12 cells, blocking tropomyosin receptor kinase A-triggered signaling pathways and increasing apoptosis. These results establish that vasoinhibin interaction with NGF and other neurotrophins may be critical in mediating pathways involved in neuronal survival and differentiation., (© 2019 S. Karger AG, Basel.)

Poly(ADP-ribosyl)ation, which is catalyzed by PARP family proteins, is one of the main reactions in the cell response to genomic DNA damage. Massive impact of DNA-damaging agents (such as oxidative stress and ionizing radiation) causes numerous breaks in DNA. In this case, the development of a fast cell response, which allows the genomic DNA integrity to be retained, may be more important than the repair by more accurate but long-term restoration of the DNA structure. This is the first study to show the possibility of eliminating DNA breaks through their PARP3-dependent mono(ADP-ribosyl)ation followed by ligation and repair of the formed ribo-AP sites by the base excision repair (BER) enzyme complex. Taken together, the results of the studies on ADP-ribosylation of DNA and the data obtained in this study suggest that PARP3 may be a component of the DNA break repair system involving the BER enzyme complex.

Hyperglycemia is a highly dangerous factor to various diseases, even resulting in death of people. Inflammation plays a key role in this process. The aim of this study was to explore the role of malignant fibrous histiocytoma amplified sequence 1 (MFHAS1) in high-glucose induced inflammation. Our research showed that high glucose stimulated the expression of MFHAS1, and overexpression of MFHAS1 can attenuate high-glucose induced inflammation in endothelial cells by decreasing the secretion of cytokines interleukin-1β (IL-1β), interleukin-1α (IL-1α), adhesion molecule intercellular adhesion molecule-1 (ICAM), interleukin-6 (IL-6), interleukin-8 (IL-8), and chemokine ligand 1 (CXCL-1). Furthermore, we found that MFHAS1 promoted the phosphorylation of Akt and the expression of heme oxygenase-1 (HO-1). Our results indicated that MFHAS1 deadened high-glucose induced inflammation by activating AKT/HO-1 pathway, suggesting that MFHAS1 may act as a new therapeutic target of diabetes mellitus.

Epithelial-mesenchymal transition (EMT) and its reverse process mesenchymal-epithelial transition (MET) programs are involced in the metastatic process. More and more evidence confirms that EMT is vital for the initiation and dissemination of cancer cells whereas MET is critical for successful metastatic colonization of a secondary organ. The regulating mechanism of EMT mediated cancer progression and metastasis has been deeply investigated. However, what processes are dependent on MET in metastatic cascades remains unclear. Here, we created a cell based high-content siRNA screen using the breast cancer cell line 4TO7 to search for kinases that were involved in Git2-induced MET. Our results revealed that 58 kinases including transferase, phosphorylation regulators, ATP/nucleotide partners potentially participate in Git2-induced MET. Our preliminary data is expected to facilitate elucidation of the mechanism on how MET is initiated during cancer metastasis.

The human cytomegalovirus major immediate early proteins IE1 and IE2 are critical drivers of virus replication and are considered pivotal in determining the balance between productive and latent infection. IE1 and IE2 are derived from the same primary transcript by alternative splicing and regulation of their expression likely involves a complex interplay between cellular and viral factors. Here we show that knockdown of the host ubiquitin-dependent segregase VCP/p97, results in loss of IE2 expression, subsequent suppression of early and late gene expression and, ultimately, failure in virus replication. RNAseq analysis showed increased levels of IE1 splicing, with a corresponding decrease in IE2 splicing following VCP knockdown. Global analysis of viral transcription showed the expression of a subset of viral genes is not reduced despite the loss of IE2 expression, including UL112/113. Furthermore, Immunofluorescence studies demonstrated that VCP strongly colocalised with the viral replication compartments in the nucleus. Finally, we show that NMS-873, a small molecule inhibitor of VCP, is a potent HCMV antiviral with potential as a novel host targeting therapeutic for HCMV infection.

Activation of Ataxia Telangiectasia Mediated protein kinase (ATM) by its phosphorylation on serine 1981 and phosphorylation of histone H2AX on serine 139 (γH2AX) are the key events reporting DNA damage, primarily formation of DNA double strand breaks. These events are detected immunocytochemically in individual cells using phospho-specific Abs. The protocols are presented that describe the methodology of immunofluorescent labeling of cells in conjunction with specific staining of cellular DNA. Flow- and imaging-cytometry, the latter exemplified as laser scanning cytometry, is used to quantify intensity of cellular fluorescence reporting activation of ATM and induction of γH2AX with respect to cellular DNA content, which in turn reports the cell cycle phase. Different protocols are presented for analysis of cells either grown in suspension or attached to surface of culture vessels. Examples of ATM activation and H2AX phosphorylation in response to DNA damage in leukemic HL-60 cells by DNA topoisomerase I inhibitor topotecan, and in lung carcinoma A549 cells by hydrogen peroxide, are presented.

Radiotherapy is one of the important treatment strategies for patients with advanced hepatocellular carcinomas. Developing novel sensitizers for radiotherapy is a key issue due to the low intrinsic radiosensitivity of hepatocellular carcinomas. It was reported the wild-type NBS1 inhibitory peptide (wtNIP) can increase radiosensitivity in several cancer cell lines by abrogating ATM-NBS1 interaction and interrupting cellular DNA damage response. Here, we developed a novel NGRconjugated peptide (NGR-sR9-wtNIP) through coupling the CNGRC angiogenic vessel-homing peptide NGR with the wtNIP peptide. Fusion peptide was tested for internalization, cytotoxicity in Hep3B cells and for tumor localization, and for toxicity in nude mice bearing human hepatocellular carcinomas xenografts. The radiosensitizing activity of NGR-sR9-wtNIP was investigated as well. We found that NGR-sR9-wtNIP can inhibit irradiation induced NBS1 phosphorylation and induce radiosensitization in Hep3B cells. When combined with IR, NGR-sR9-wtNIP suppressed tumor growth obviously in xenograft mice. In addition, the fusion peptide localized in tumor tissue specifically and barely led to any side effects on mice. Taken together, our data strongly suggest that NGRsR9- wtNIP has radiosensitizing potential for radiotherapy of hepatocellular carcinomas.

Multisite modifications are widely recognized as an essential feature of many switch-like responses in signal transduction. It is usually assumed that the modification of one site directly or indirectly increases the rate of modification of neighboring sites. In this paper we provide a new set of assumptions for a multisite system to become highly ultrasensitive even in the absence of cooperativity or allostery. We assume that the individual sites are modified independently of each other, and that protein activity is an ultrasensitive function of the fraction of modified sites. These assumptions are particularly useful in the context of multisite systems with a large (8+) number of sites. We estimate the apparent Hill coefficient of the dose responses in the sequential and nonsequential cases, highlight their different qualitative properties, and discuss a formula to approximate dose responses in the nonsequential case. As an example we describe a model of bacterial chemotaxis that features robust ultrasensitivity and perfect adaptation over a wide range of ligand concentrations, based on non-allosteric multisite behavior at the level of receptors and flagella. We also include a model of the inactivation of the yeast pheromone protein Ste5 by cell cycle proteins.

S100A6 is a calcium binding protein belonging to the S100 family. In this work we examined the function of extracellular S100A6. Using mesenchymal stem cells isolated from Wharton's jelly of the umbilical cord (WJMS cells) we have shown that S100A6 is secreted by these cells, and when added to the medium, increases their adhesion and inhibits proliferation. The search for a potential target/receptor of S100A6 in the membrane fraction of WJMS cells allowed us to identify some proteins, among them integrin β1, which interacts with S100A6 in a calcium dependent manner. The interaction between S100A6 and integrin β1, was then confirmed by ELISA using purified proteins. Applying specific antibodies against integrin β1 reversed the effect on cell adhesion and proliferation observed in the presence of S100A6 which indicates that S100A6 exerts its function due to interaction with integrin β1. Since the data show the influence of extracellular S100A6 on cells isolated from Wharton's jelly, our results might help to establish molecular mechanisms leading to some pathologies characteristic for this tissue., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The hormone prolactin (PRL) regulates neuroendocrine and emotional stress responses. It is found in the hypothalamus, where the protein is partially cleaved to vasoinhibins, a family of N-terminal antiangiogenic PRL fragments ranging from 14 to 18kDa molecular masses, with unknown effects on the stress response. Here, we show that the intracerebroventricular administration of a recombinant vasoinhibin, containing the first 123 amino acids of human PRL that correspond to a 14kDa PRL, exerts anxiogenic and depressive-like effects detected in the elevated plus-maze, the open field, and the forced swimming tests. To investigate whether stressor exposure affects the generation of vasoinhibins in the hypothalamus, the concentrations of PRL mRNA, PRL, and vasoinhibins were evaluated in hypothalamic extracts of virgin female rats immobilized for 30min at different time points after stress onset. The hypothalamic levels of PRL mRNA and protein were higher at 60min but declined at 360min to levels seen in non-stressed animals. The elevation of hypothalamic PRL did not correlate with the stress-induced increase in circulating PRL levels, nor was it modified by blocking adenohypophyseal PRL secretion with bromocriptine. A vasoinhibin having an electrophoretic migration rate corresponding to 17kDa was detected in the hypothalamus. Despite the elevation in hypothalamic PRL, the levels of this hypothalamic vasoinhibin were similar in stressed and non-stressed rats. Stress reduced the rate of cleavage of PRL to this vasoinhibin as shown by the incubation of recombinant PRL with hypothalamic extracts from stressed rats. These results suggest that vasoinhibins are potent anxiogenic and depressive factors and that stress increases PRL levels in the hypothalamus partly by reducing its conversion to vasoinhibins. The reciprocal interplay between PRL and vasoinhibins may represent an effective mechanism to regulate anxiety and depression., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The synthetic ~5 kDa ABP (amyloid-ß binding peptide) consists of a region of the 228 kDa human pericentrioloar material-1 (PCM-1) protein that selectively and avidly binds in vitro Aβ1-42 oligomers, believed to be key co-drivers of Alzheimer's disease (AD), but not monomers (Chakravarthy et al., (2013) [3]). ABP also prevents Aß1-42 from triggering the apoptotic death of cultured human SHSY5Y neuroblasts, likely by sequestering Aß oligomers, suggesting that it might be a potential AD therapeutic. Here we support this possibility by showing that ABP also recognizes and binds Aβ1-42 aggregates in sections of cortices and hippocampi from brains of AD transgenic mice and human AD patients. More importantly, ABP targets Aβ1-42 aggregates when microinjected into the hippocampi of the brains of live AD transgenic mice., (Crown Copyright © 2014. Published by Elsevier Inc. All rights reserved.)

Endothelial nitric oxide synthase (eNOS)-derived nitric oxide is a major vasorelaxing factor and a mediator of vasopermeability and angiogenesis. Vasoinhibins, a family of antiangiogenic prolactin fragments that include 16 K prolactin, block most eNOS-mediated vascular effects. Vasoinhibins activate protein phosphatase 2A, causing eNOS inactivation through dephosphorylation of eNOS at serine residue 1179 in bovine endothelial cells and thereby blocking vascular permeability. In this study, we examined whether human eNOS phosphorylation at S1177 (analogous to bovine S1179) influences other actions of vasoinhibins. Bovine umbilical vein endothelial cells were stably transfected with human wild-type eNOS (WT) or with phospho-mimetic (S1177D) or non-phosphorylatable (S1177A) eNOS mutants. Vasoinhibins inhibited the increases in eNOS activity, migration, and proliferation following the overexpression of WT eNOS but did not affect these responses in cells expressing S1177D and S1177A eNOS mutants. We conclude that eNOS inhibition by dephosphorylation of S1177 is fundamental for the inhibition of endothelial cell migration and proliferation by vasoinhibins.

Sister chromatid cohesion depends on Sororin, a protein that stabilizes acetylated cohesin complexes on DNA by antagonizing the cohesin release factor Wings-apart like protein (Wapl). Cohesion is essential for chromosome biorientation but has to be dissolved to enable sister chromatid separation. To achieve this, the majority of cohesin is removed from chromosome arms in prophase and prometaphase in a manner that depends on Wapl and phosphorylation of cohesin's subunit stromal antigen 2 (SA2), whereas centromeric cohesin is cleaved in metaphase by the protease separase. Here we show that the mitotic kinases Aurora B and Cyclin-dependent kinase 1 (Cdk1) destabilize interactions between Sororin and the cohesin subunit precocious dissociation of sisters protein 5 (Pds5) by phosphorylating Sororin, leading to release of acetylated cohesin from chromosome arms and loss of cohesion. At centromeres, the cohesin protector shugoshin (Sgo1)-protein phosphatase 2A (PP2A) antagonizes Aurora B and Cdk1 partly by dephosphorylating Sororin and thus maintains cohesion until metaphase. We propose that the stepwise loss of cohesion between chromosome arms and centromeres is caused by local regulation of Wapl activity, which is controlled by the phosphorylation state of Sororin.

The anaphase-promoting complex/cyclosome (APC/C) is a ~1.5-MDa multiprotein E3 ligase enzyme that regulates cell division by promoting timely ubiquitin-mediated proteolysis of key cell-cycle regulatory proteins. Inhibition of human APC/C(CDH1) during interphase by early mitotic inhibitor 1 (EMI1) is essential for accurate coordination of DNA synthesis and mitosis. Here, we report a hybrid structural approach involving NMR, electron microscopy and enzymology, which reveal that EMI1's 143-residue C-terminal domain inhibits multiple APC/C(CDH1) functions. The intrinsically disordered D-box, linker and tail elements, together with a structured zinc-binding domain, bind distinct regions of APC/C(CDH1) to synergistically both block the substrate-binding site and inhibit ubiquitin-chain elongation. The functional importance of intrinsic structural disorder is explained by enabling a small inhibitory domain to bind multiple sites to shut down various functions of a 'molecular machine' nearly 100 times its size.