Transcription-replication conflicts (TRCs) are a key source of replication stress in cancer, with pancreatic ductal adenocarcinoma (PDAC) showing uniquely high levels. This study investigated the mechanism, oncogene dependency, subtype specificity, and preclinical activity of the TRC-targeting molecule AOH1996 in PDAC models. Initial clinical evidence of AOH1996 activity in patients with PDAC is also provided.

The oncogene-dependent toxicity of AOH1996 was studied in KRAS(G12D)-inducible systems. Its effects on replication fork progression, TRCs, DNA damage, cell cycle, and apoptosis were assessed in PDAC cell lines. Subtype-specific responses were tested in organoids, and in vivo efficacy was evaluated using murine and patient-derived xenografts. Clinical activity was measured through radiographic response and progression-free survival in patients.

AOH1996 exhibited dose-dependent cytotoxicity reliant on KRAS(G12D) induction (average half maximal inhibitory concentration: 0.93 μM). It inhibited replication fork progression and induced TRCs by enhancing interactions between RNA Polymerase II and proliferating cell nuclear antigen, causing transcription-dependent DNA damage and transcription shutdown. Organoids with high replication stress were most sensitive (half maximal inhibitory concentration: 406 nM-2 μM). In mouse models, AOH1996 reduced tumor growth, induced tumor-selective DNA damage, and prolonged survival (median 14 vs 21 days, P = .04) without toxicity. Two patients with chemotherapy-refractory PDAC treated with AOH1996 showed up to 49% tumor shrinkage in hepatic metastases.

AOH1996 safely and effectively targets TRCs in preclinical PDAC models, with initial clinical evidence supporting its potential for treating chemotherapy-refractory PDAC. Further clinical development is warranted.

Perfluorooctanesulfonic acid (PFOS) and sodium nitrite may have complex adverse effects on aquatic animals. This study assessed the interactive effects of PFOS and sodium nitrite on Chinese mitten crab (Eriocheir sinensis). A 2 × 3 factorial experiment with 0, 0.1, and 5 mg/L PFOS and 0, 3.50 mg/L sodium nitrite evaluated impacts on growth, behavior, oxygen consumption, health, energy metabolism, and hepatopancreas transcriptome. PFOS <0.1 mg/L with 3.50 mg/L nitrite significantly decreased PFOS accumulation in the hepatopancreas and improved feeding rates and hepatopancreas structure (P < 0.05). Under 5 mg/L PFOS and nitrite, survival, weight gain, hepatosomatic index, and feeding rate significantly decreased (P < 0.05). PFOS (0.1 mg/L) with nitrite significantly prolonged righting response time and increased locomotion speed (P < 0.05). PFOS (5 mg/L) significantly decreased serum triglyceride and lactate levels, while PFOS and nitrite decreased glucose, triglyceride, and glycogen levels and increased lactate in hepatopancreas (P < 0.05). Transcriptomic analysis indicated that PFOS affects p53 signaling, cell cycle and neurotransmission pathways, with notable changes in cell proliferation genes (pcna, ccna, cdk1, cdk2, rbx1) primarily downregulated by PFOS. Overall, PFOS disrupts neurotransmitter regulation and causes hepatopancreatic damage, while nitrite can reduce the toxicity of PFOS by decreasing its hepatopancreas accumulation. However, high levels of PFOS combined with sodium nitrite exacerbate toxicity, emphasizing the need for comprehensive assessment of environmental pollutant interactions.

Modern management of chicks hinders the vertical transmission of intestinal microbiota, which is closely related to immunity. Inulin is a substrate that can be utilized by the microbiota. This study aimed to determine whether fecal microbiota transplantation (FMT) combined with inulin played a "1 + 1 > 2" role in enhancing the development and function of immune organs. Chicks were treated with 1 % inulin and/or fecal microbiota suspension on days 1-6. The growth performance, immune organ development, and immune indicators were evaluated on days 7, 14, and 21. Results showed that the combination of FMT and inulin significantly increased the immune organ index on day 7 and promoted the morphological structure and the expression of proliferating cell nuclear antigen (PCNA) in immune organs on days 7, 14, and 21. Each treatment increased the gene expression of interferon-gamma (IFN-γ), interleukin-4 (IL-4), interleukin-2 (IL-2), B cell-activating factor receptor (BAFFR), B cell linker (BLNK), C-X-C Motif Chemokine Ligand 12 (CXCL12), C-X-C Motif Chemokine Receptor 4 (CXCR4), and Biotin (Bu-1) to varying degrees. FMT combined with inulin significantly increased the expression of IgA-positive cells on days 7 and 14. In conclusion, the synergistic effect of FMT and inulin had beneficial impacts on the development and function of immune organs.

PCNA plays multiple roles in cancer development, including cell proliferation regulation, DNA repair, replication, and serving as a widely used biomarker and therapeutic target. Despite its significant role in oncology, PCNA has historically been considered "undruggable" due to the absence of known endogenous small molecule modulators and identifiable ligand binding sites. Unlike other protein-protein interfaces, PCNA lacks explicit binding grooves, featuring a relatively small and shallow surface pocket, which hinders the discovery of traditional small molecule targets. Recent breakthroughs have introduced promising PCNA-targeting candidates, with ATX-101 and AOH1996 entering phase I clinical trials for cancer therapy, garnering academic and industry interest. These achievements provide new evidence for PCNA as a drug target. This article provides insight and perspective on the application of small-molecule PCNA inhibitors in cancer treatment, covering PCNA function, its relationship with cancer, structural modification of small molecule inhibitors, and discovery strategies.

Electrospun nanocarrier systems, widely employed in the medical field, exhibit the capability to encapsulate multiple drugs and mitigate complications. Doxorubicin hydrochloride (DOX) represents a frequently utilized chemotherapeutic agent for liver cancer patients. Sodium bicarbonate (SB) serves to neutralize the acidic tumor microenvironment, while ibuprofen (IBU) attenuates inflammatory factor production. The combination of these three commonly used drugs facilitates antitumor efficacy and relapse prevention. Composite fibrous membranes were prepared by incorporating the antitumor drug DOX into MSN, which was then codispersed with IBU in a poly l-lactic acid (PLLA) electrospinning solution after acid sensitization using SB. The resulting membrane was characterized using transmission electron microscopy and scanning electron microscopy. The toxic effect of this fibrous membrane and its pro-apoptotic effect on tumor cells were evaluated, along with the expression of cell proliferation-related factors, immune/inflammatory factors, and apoptosis-related factors. Immunohistochemistry and HE staining confirmed its ability to inhibit recurrence of postoperative residual cancer without causing toxicity to vital organs. The PLLA-MSN@DOX-SB-IBU nanofibrous membrane not only mitigates the cardiotoxicity associated with DOX but also inhibits tumor cell proliferation and enhances the tumor microenvironment, demonstrating significant antitumor efficacy. Furthermore, it effectively prevents the recurrence of residual cancer postsurgery while exhibiting excellent biocompatibility. The PLLA-MSN@DOX-SB-IBU nanofibrous membrane demonstrates significant potential in impeding the progression of hepatocellular carcinoma and mitigating the recurrence of residual cancer following surgical intervention for hepatocellular carcinoma.

Nasal polyp (NP) is a pathological benign mass that affects the nasal cavity and paranasal sinuses. This lesion is occasionally associated with chronic rhinosinusitis (CRS), which is named chronic rhinosinusitis with nasal polyps (CRSwNP). Proliferating cell nuclear antigen (PCNA) indicates abnormal cell proliferation which may help assess the nasopharyngeal lesions and upper airway cancers. This study aimed to examine the PCNA expression in NP tissue. This study was conducted under the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines. A thorough search using relevant keywords was performed on the electronic database to identify studies examining the PCNA expression in NP. Meta-analysis on pooled single group proportion and means of PCNA expression was conducted by the metamean and metaprop functions in R software (version 4.4.0). Quality assessment was done according to the Joanna Briggs Institute (JBI) checklist. Ten articles were included in this study. The PCNA expression in NP was higher than in normal nasal mucosa (NNM), while its expression in NP was lower than in inverted papilloma (IP). The mean percentage of PCNA expression in NP was 15.73% (95%-CI[2.87-28.58]) with considerable heterogeneity (I 2  = 97%[96-98%],p-value < 0.01). Based on a cutoff staining (> 5%), 64% of NP samples were PCNA-positive (95%-CI[42-81%]) with high heterogeneity (I 2  = 76%[47-89%], p-value < 0.01). NP tissue has a relatively high expression of the PCNA oncoprotein based on the immunohistochemistry (IHC) staining. Although more than half of NP samples were positive for PCNA, still the mean expression of this marker in NP was lower than in IP tissue.

The online version contains supplementary material available at 10.1007/s12070-024-05172-y.

Postoperative therapies for osteosarcoma present substantial challenges due to tumor recurrence and extensive bone defects. To tackle these challenges, laser powder bed fusion is utilized to fabricate biodegradable Zn-Li porous scaffolds that supress tumors and promote osteogenesis. After the structure design and composition selection, the Zn-0.8Li porous scaffold with Gyroid unit optimally balances the co-release of Zn2+ and Li+ during degradation, resulting in favorable antitumor and osteogenic effects. In vitro, the Zn-0.8Li scaffold significantly inhibits osteosarcoma progression by suppressing tumor cell proliferation, promoting apoptosis, alleviating migration, and simultaneously promotes osteogenic differentiation through the enhanced expression of osteogenic markers. In vivo, the Zn-0.8Li scaffold inhibits the malignant osteosarcoma behavior and facilitates bone regeneration in areas with bone defects. Transcriptomic analysis further reveals that the simultaneous release of Zn2+ and Li+ from the biodegradable Zn-0.8Li scaffold contributes to anti-osteosarcoma activity by downregulating PI3K/Akt signaling pathways. Taken together, the Zn-0.8Li porous scaffold fabricated using laser powder bed fusion with enhanced antitumor and osteogenic properties is a promising alternative for the postoperative management of osteosarcoma.

Harmine is present in a variety of medicinal plants, and its effects on colon cancer cells remain unclear. Here, we found that harmine exhibited significant inhibitory effects on the proliferation of colon cancer cells by inhibiting the phosphorylation levels of the FAK/AKT and ERK1/2/CREB. Furthermore, harmine also inhibited the migration of colon cancer cells and suppressed the expression levels of MMP-2, MMP-9, and VEGF. Additionally, harmine-induced apoptosis in colon cancer cells by regulating the expression of Bcl-2 and Bax. In conclusion, our findings suggest that harmine exerts a significant inhibitory effect on the development of colon cancer cells.

Proliferating cell nuclear antigen (PCNA) is essential for DNA replication and repair, cell growth, and survival. PCNA also enhances androgen receptor (AR) signaling in prostate cancer (PC) cells. We identified a PCNA interaction protein (PIP) box at the N-terminal domain of AR and developed a small peptide PCNA inhibitor R9-AR-PIP containing AR PIP-box. We also identified a series of small molecule PCNA inhibitors (PCNA-Is) that bind directly to PCNA and interrupt PCNA functions. The present study investigated the effects of the PCNA inhibitors on the sensitivity of PC cells to X-ray radiation.

The effects of targeting PCNA on radio sensitivity of PC cells were investigated in four lines of castration-resistant PC (CRPC) cells with different AR expression statuses. The cells were treated with the PCNA inhibitors and X-ray radiation alone or in combination. The effects of the treatment on expression of AR target genes, DNA damage response, DNA damage, homologous recombination repair (HRR), and cytotoxicity were evaluated.

We found that the androgen response element (ARE) occupancy of the DNA damage response gene PARP1 by AR is significantly attenuated by PCNA-I1S or R9-AR-PIP combined with X-ray radiation, while X-ray radiation alone does not enhance the ARE occupancy. PCNA-I1S or R9-AR-PIP alone significantly inhibits occupancy of the AR-occupied regions (AROR) in PRKDC and XRCC2 genes. R9-AR-PIP and PCNA-I1S inhibit expression of AR-Vs target gene cyclin A2 and show the additive effects with radiation in AR-positive CRPC cells. Targeting PCNA by PCNA-I1S and R9-AR-PIP downregulates expression of DNA damage response genes EXO1, Rad54L, Rad51, and/or PARP1 and shows the additive effects with radiation as compared with their respective controls in AR-positive CRPC LNCaP-AI, 22Rv1, and R1-D567 cells, but not in AR-negative PC-3 cells. R9-AR-PIP and PCNA-I1S elevate the levels of phospho-DNA-PKcs(S2056) and γH2AX, indicating DNA damage in response to radiation in AR-positive cells. The HRR is significantly attenuated by PCNA inhibitors PCNA-I1S, R9-AR-PIP, and T2AA in all four CRPC cells examined, and inhibited by Enzalutamide (Enz) only in 22RV1 cells. The cytotoxicity induced by X-ray radiation in androgen-dependent LNCaP cells is enhanced by Enz and a lower concentration of R9-AR-PIP in the colony formation assay. R9-AR-PIP at higher concentration reduces the colony formation and has an additive effect with X-ray radiation in all AR expressing cells, regardless of AR-FL and AR-Vs, but does not significantly alter the colony formation in AR-negative PC-3 cells. PCNA-I1S attenuates colony formation and has an additive effect with ionizing radiation in all four CRPC cells, regardless of AR expression status.

These data provide a strong rationale for the therapy studies using PCNA-I1S or R9-AR-PIP in combination with X-ray radiation against CRPC tumors in preclinical models.

Dihydroquercetin (DHQ), also known as Taxifolin (TA), is a flavanonol with various biological activities, such as anticancer, anti-inflammatory, and antioxidative properties. It has been found to effectively increase the viability of porcine intestinal epithelial cells (IPEC-J2). However, the precise mechanism by which DHQ increases the proliferation of IPEC-J2 cells is not entirely understood. This study aimed to explore the potential pathways through which DHQ encourages the proliferation of IPEC-J2 cells. The findings indicated that DHQ significantly improved the protein expression of tight junction proteins (ZO-1, Occludin, and Claudin1) and a molecular biomarker of proliferation (PCNA) in IPEC-J2 cells. Furthermore, DHQ was found to increase the Wnt/β-catenin pathway-associated β-catenin, c-Myc, and cyclin D1 mRNA expression, and promote the protein expression of β-catenin and TCF4. To confirm the involvement of the Wnt/β-catenin signaling pathway in the DHQ-promoted proliferation of IPEC-J2 cells, the inhibitor LF3, which targets β-catenin/TCF4 interaction, was used. It was found that LF3 inhibited the protein expressions upregulated by DHQ and blocked the promotion of cell proliferation. These results indicate that DHQ positively regulates IPEC-J2 cell proliferation through the Wnt/β-catenin pathway, providing constructive insights into the role of DHQ in regulating intestine development.

Pulmonary arterial hypertension (PAH) is a worldwide challenging disease characterized by progressive elevation of pulmonary artery pressure. The proliferation, migration and phenotypic transformation of pulmonary smooth muscle cells are the key steps of pulmonary vascular remodeling. Quercetin (3,3', 4', 5, 6-pentahydroxyflavone, Que) is a natural flavonol compound that has antioxidant, anti-inflammatory, anti-tumor and other biological activities. Studies have shown that Que has therapeutic effects on PAH. However, the effect of quercetin on pulmonary vascular remodeling in PAH and its mechanism remain unclear.

In vivo, PAH rats were constructed by intraperitoneal injection of monocrotaline (MCT) at 60 mg/kg. Human pulmonary artery smooth muscle cells (HPASMCs) were treated with platelet-derived growth factor BB (PDGF-BB) 20 ng/mL to construct PAH cell model in vitro. The results showed that in vivo studies, MCT could induce right ventricular wall hyperplasia, narrow the small and medium pulmonary artery cavity, up-regulate the expression of proliferating and migration-related proteins proliferating cell nuclear antigen (PCNA) and osteopontin (OPN), and down-regulate the expression of alpha-smooth muscle actin (α-SMA). Que reversed the MCT-induced results. This process works by down-regulating the transforming growth factor-β1 (TGF-β1)/ Smad2/3 signaling pathway. In vitro studies, Que had the same effect on PDGF-BB-induced proliferation and migration cell models.

Que inhibits the proliferation, migration and phenotypic transformation of HPASMCs by down-regulating TGF-β1/Smad2/Smad3 pathway, thereby reducing right ventricular hyperplasia (RVH) and pulmonary vascular remodeling, providing potential pharmacological and molecular explanations for the treatment of PAH.

Meat quality in goats is partly determined by the intramuscular fat (IMF) content, which is associated with the proliferation and differentiation of intramuscular preadipocytes. Emerging studies have suggested that miRNA plays a crucial role in adipocyte proliferation and differentiation. In our recent study, we observed the expression variations in miR-196a in the longissimus dorsi muscle of Jianzhou goats at different ages. However, the specific function and underlying mechanism of miR-196a in IMF deposition are still unclear. This study demonstrated that miR-196a significantly enhanced adipogenesis and apoptosis and reduced the proliferation of preadipocytes. Subsequently, RNA-seq was employed to determine genes regulated by miR-196a, and 677 differentially expressed genes were detected after miR-196a overexpression. The PI3K-Akt pathway was identified as activated in miR-196a regulating intramuscular adipogenesis via Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and further verified via Western blot and rescue assays. Lastly, using RT-qPCR, Western blot, dual-luciferase, and rescue assays, we found that miR-196a promoted adipogenesis and suppressed the proliferation of intramuscular preadipocytes by the downregulation of MAP3K1. In summary, these results suggest that miR-196a regulates IMF deposition by targeting MAP3K1 and activating the PI3K-Akt pathway and provide a theoretical foundation for improving goat meat quality through molecular breeding.

An increasing number of circRNAs have been found to be involved in the development of gastric cancer. However, the function of circ_0003789 in regulating gastric cancer progression is unclear. Here, we aimed to investigate the expression, function and molecular mechanism of circ_0003789 in gastric cancer pathogenesis. Circ_0003789, miR-429 and ZFP36 ring finger protein like 2 (ZFP36L2) mRNA were quantified by quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation was illustrated by 5-Ethynyl-2'-deoxyuridine (Edu), cell counting kit-8 (CCK-8) and colony formation assays. Apoptosis was determined by flow cytometry. Protein level was detected by Western blotting assay. Xenograft assays were used for functional analysis of circ_0003789 in vivo. The relationship between miR-429 and circ_0003789 or ZFP36L2 was predicted by starbase3.0 online database and identified by dual luciferase reporter assay. The expression levels of circ_0003789 and ZFP36L2 were significantly upregulated in gastric cancer tissues and cells, while the expression of miR-429 was downregulated. Downregulation of circ_0003789 inhibited gastric cancer cell growth and invasion and promoted apoptosis in vitro. Circ_0003789 acted as a sponge of miR-429. Moreover, miR-429 silencing by miR-429 inhibitors attenuated the effects of circ_0003789 interference on cell growth, apoptosis and invasion. ZFP36L2 was targeted by miR-429, and the effects of miR-429 on cell growth, invasion and apoptosis were attenuated by ZFP36L2 overexpression. Circ_0003789 could enhance ZFP36L2 expression by interacting with miR-429. Silencing of circ_0003789 inhibited tumor growth in vivo. Circ_0003789 regulates tumor progression in gastric cancer through miR-429/ZFP36L2 axis. This finding implies that circ_0003789 may be a therapeutic target for gastric cancer.

This study was conducted to investigate the protective effects of chlorogenic acid (CGA) on inflammatory responses and intestinal health of lipopolysaccharide (LPS)-challenged broilers. One hundred and forty-four 1-day-old male broiler chicks were divided into 3 groups with 6 replicates of 8 birds each. The groups were as follows: 1) Control group: birds fed a basal diet; 2) LPS group: LPS-challenged birds fed a basal diet; 3) CGA group: LPS-challenged birds fed a CGA-supplemented diet. The LPS was intraperitoneally administered at a dose of 1 mg/kg of body weight. CGA increased the weight gain and feed intake of LPS-challenged birds by 37.05% and 24.29%, respectively (P < 0.05). CGA also alleviated LPS-induced inflammation, as evidenced by lower levels of pro-inflammatory cytokines in the serum and jejunum (tumor necrosis factor-α, interferon-γ, interleukin-1β, and interleukin-6), and the decreased myeloperoxidase activity in the jejunum (P < 0.05). These effects were accompanied by a decrease in the mRNA abundance of toll-like receptor 4 and myeloid differentiation factor 88 and an inhibition of nuclear factor kappa-B translocation in the jejunum (P < 0.05). CGA reduced circulating diamine oxidase activity and levels of D-lactate and endotoxin, and positively regulated the expression of jejunal claudin-3 and zonula occludens-1 in LPS-challenged broilers (P < 0.05). Compared to the LPS group, CGA reduced the apoptotic rate of epithelial cells and cytochrome c concentration in the jejunum, and normalized the expression of genes responsible for proliferation and apoptosis in jejunal epithelial cells, including cysteine aspartate-specific protease-9, B cell lymphoma-2, and proliferating cell nuclear antigen (P < 0.05). Furthermore, CGA normalized the altered phosphorylation of protein kinase B and glycogen synthase kinase-3β, as well as the translocation of nuclear β-catenin in the jejunum of LPS-challenged broilers (P < 0.05). These results suggested that CGA supplementation improved growth performance, alleviated inflammation, and helped maintain intestinal integrity and barrier function in LPS-challenged broilers, possibly through the regulation of the toll-like receptor 4/nuclear factor kappa-B and protein kinase B/Wnt/β-catenin pathways.

The activation of pregnane X receptor (PXR) or peroxisome proliferator-activated receptor α (PPARα) can induce liver enlargement. Recently, we reported that PXR or PPARα activation-induced hepatomegaly depends on yes-associated protein (YAP) signaling and is characterized by hepatocyte hypertrophy around the central vein area and hepatocyte proliferation around the portal vein area. However, it remains unclear whether PXR or PPARα activation-induced hepatomegaly can be reversed after the withdrawal of their agonists. In this study, we investigated the regression of enlarged liver to normal size following the withdrawal of PCN or WY-14643 (typical agonists of mouse PXR or PPARα) in C57BL/6 mice. The immunohistochemistry analysis of CTNNB1 and KI67 showed a reversal of hepatocyte size and a decrease in hepatocyte proliferation after the withdrawal of agonists. In details, the expression of PXR or PPARα downstream proteins (CYP3A11, CYP2B10, ACOX1, and CYP4A) and the expression of proliferation-related proteins (CCNA1, CCND1, and PCNA) returned to the normal levels. Furthermore, YAP and its downstream proteins (CTGF, CYR61, and ANKRD1) also restored to the normal states, which was consistent with the change in liver size. These findings demonstrate the reversibility of PXR or PPARα activation-induced hepatomegaly and provide new data for the safety of PXR and PPARα as drug targets.

The efficacy of Shaoyao Decoction (SYD), a traditional Chinese medicine prescription, in treating damp-heat colitis is established, but its underlying mechanism remains to be elucidated.

Our study aims to investigate the effect and mechanism of action of SYD in treating damp-heat colitis.

A mouse model of damp-heat colitis was induced and treated with SYD via gavage for seven days. The therapeutic efficacy of SYD was assessed through clinical indicators and histopathological examinations. The inflammatory factors and oxidative stress parameters were detected by ELISA and biochemical kits. We also analyzed alterations in the gut microbiome via 16 S rRNA gene sequencing and quantified serum indole derivatives using targeted tryptophan metabolomics. Western blotting and immunofluorescence were used to detect the expressions of AHR, CYP1A1, STAT3 and tight junction (TJ) proteins. The ELISA kit was utilized to detect the content of antibacterial peptides (Reg3β and Reg3γ) in colon. The immunohistochemistry was employed to detect the expressions of proliferating cell nuclear antigen (PCNA) protein.

SYD effectively alleviated symptoms in mice with damp-heat colitis, including body weight loss, shortened colon, elevated DAI, enlarged spleen, and damage to the intestinal mucosa. SYD notably reduced IL-6, TNF-α, IL-1β and MDA levels in colon tissues, while increasing IL-10 and T-AOC levels. Furthermore, SYD mitigated gut microbiota disturbance, restored microbial tryptophan metabolite production (such as IA, IAA, and IAld), notably increased the protein levels of AHR, CYP1A1 and p-STAT3 in colon tissue, and elevated the IL-22 level. Moreover, the expression levels of Reg3β, Reg3γ, occludin, ZO-1 and PCNA were increased in SYD group.

Our study showed that SYD ameliorates damp-heat colitis by restructuring gut microbiota structure, enhancing the metabolism of tryptophan associated with gut microbiota to activate the AHR/IL-22/STAT3 pathway, thereby recovering damaged intestinal mucosa. This research offers novel insights into the therapeutic mechanisms of SYD on damp-heat colitis.

Proliferating cell nuclear antigen (PCNA) is an essential scaffold protein in many cellular processes. It is best known for its role as a DNA sliding clamp and processivity factor during DNA replication, which has been extensively reviewed by others. However, the importance of PCNA extends beyond its DNA-associated functions in DNA replication, chromatin remodelling, DNA repair and DNA damage tolerance (DDT), as new non-canonical roles of PCNA in the cytosol have recently been identified. These include roles in the regulation of immune evasion, apoptosis, metabolism, and cellular signalling. The diverse roles of PCNA are largely mediated by its myriad protein interactions, and its centrality to cellular processes makes PCNA a valid therapeutic anticancer target. PCNA is expressed in all cells and plays an essential role in normal cellular homeostasis; therefore, the main challenge in targeting PCNA is to selectively kill cancer cells while avoiding unacceptable toxicity to healthy cells. This chapter focuses on the stress-related roles of PCNA, and how targeting these PCNA roles can be exploited in cancer therapy.

Deoxynivalenol (DON) is a common food contaminant that can impair male reproductive function. This study investigated the effects and mechanisms of DON exposure on progenitor Leydig cell (PLC) development in prepubertal male rats. Rats were orally administrated DON (0-4 mg/kg) from postnatal days 21-28. DON increased PLC proliferation but inhibited PLC maturation and function, including reducing testosterone levels and downregulating biomarkers like HSD11B1 and INSL3 at ≥2 mg/kg. DON also stimulated mitochondrial fission via upregulating DRP1 and FIS1 protein levels and increased oxidative stress by reducing antioxidant capacity (including NRF2, SOD1, SOD2, and CAT) in PLCs in vivo. In vitro, DON (2-4 μM) inhibited PLC androgen biosynthesis, increased reactive oxygen species production and protein levels of DRP1, FIS1, MFF, and pAMPK, decreased mitochondrial membrane potential and MFN1 protein levels, and caused mitochondrial fragmentation. The mitochondrial fission inhibitor mdivi-1 attenuated DON-induced impairments in PLCs. DON inhibited PLC steroidogenesis, increased oxidative stress, perturbed mitochondrial homeostasis, and impaired maturation. In conclusion, DON disrupts PLC development in prepubertal rats by stimulating mitochondrial fission.

Immunological liver injury (ILI) is a common liver disease associated with the microbiota-gut-liver axis. Jian Gan powder (JGP) exhibits both protective and therapeutic effects on hepatitis virus-induced ILI in the clinic. However, the underlying mechanisms remain elusive. The aim of this study is to investigate the hepatoprotective effects and associated mechanisms of JGP in the context of gut microbiota, utilizing a mouse model of ILI.

The mouse model was established employing Bacillus Calmette-Guérin (BCG) plus lipopolysaccharide (LPS). Following treatment with JGP (7.5, 15, or 30 g/kg), serum, liver, and fresh fecal samples were analyzed. 16S rRNA gene sequencing and untargeted metabolomics profiling were performed to assess the role of JGP on the gut microbiota and its metabolites.

JGP treatment markedly reduced serum IFN-γ, IL-6, IL-22, and hepatic p-STAT3 (phosphorylated transducer and activator of transcription-3) expression. In contrast, JGP increased the percentage of proliferating cell nuclear antigen-positive liver cells in treated mice. Fecal 16S rRNA gene sequencing revealed that JGP treatment restored the levels of Alloprevotella, Burkholderia-Caballeronia-Paraburkholderia, Muribaculum, Streptococcus, and Stenotrophomonas. Additionally, metabolomics analysis of fecal samples showed that JGP restored the levels of allylestrenol, eplerenone, phosphatidylethanolamine (PE) (P-20:0/0:0), sphingomyelin (SM) d27:1, soyasapogenol C, chrysin, and soyasaponin I.

JGP intervention improves ILI by restoring gut microbiota and modifying its metabolic profiles. These results provide a novel insight into the mechanism of JGP in treating ILI and the scientific basis to support its clinical application.

Proliferating cell nuclear antigen (PCNA) is an essential factor for DNA metabolism. The influence of PCNA on DNA replication and repair, combined with the high expression rate of PCNA in various tumours renders PCNA a promising target for cancer therapy. In this context, an autodisplay-based screening method was developed to identify peptidic PCNA interaction inhibitors. A 12-mer randomized peptide library consisting of 2.54 × 106 colony-forming units was constructed and displayed at the surface of Escherichia coli BL21 (DE3) cells by autodisplay. Cells exhibiting an enhanced binding to fluorescent mScarlet-I-PCNA were enriched in four sorting rounds by flow cytometry. This led to the discovery of five peptide variants with affinity to mScarlet-I-PCNA. Among these, P3 (TCPLRWITHDHP) exhibited the highest binding signal. Subsequent flow cytometric analysis revealed a dissociation constant of 0.62 μM for PCNA-P3 interaction. Furthermore, the inhibition of PCNA interactions was investigated using p15, a PIP-box containing protein involved in DNA replication and repair. P3 inhibited the PCNA-p1551-70 interaction with a half maximal inhibitory activity of 16.2 μM, characterizing P3 as a potent inhibitor of the PCNA-p15 interaction.

PRPF19 seems to play either tumor-promoting or anti-tumor roles depending on cancer types. This study aimed to clarify the potential role and mechanism of PRPF19 in bladder cancer. PRPF19 expression and its correlation with patients' overall survival were analyzed in bladder cancer. The effects of PRPF19 on the viability, apoptosis, DNA damage repair, and gemcitabine sensitivity in human bladder cancer cells (T24 and 5637) were analyzed through loss- or gain-of-function methods. Moreover, the influences of DDB1 small interfering RNA on these indexes were evaluated in bladder cancer cells. At last, rescue experiment using DDB1 overexpression was carried out to confirm whether PRPF19 functioned via regulating DDB1. PRPF19 was highly expressed in bladder cancer tissues and cells. Elevated PRPF19 expression was related to shorter overall survival of bladder cancer patients. Downregulation of PRPF19 inhibited cell proliferation, promoted cell apoptosis, increased the number of γ-H2AX-positive cells, and reduced the mRNA and protein levels of DDB1 and BRCA1. Meanwhile, knockdown of PRPF19 decreased the IC50 of gemcitabine and promoted gemcitabine-induced cell apoptosis. Whereas, PRPF19 overexpression significantly decreased gemcitabine-induced apoptosis in bladder cancer cells. DDB1 downregulation suppressed cell proliferation and BRCA1 expression, but elevated the number of γ-H2AX-positive cells and gemcitabine sensitivity. Upregulation of DDB1 attenuated γ-H2AX-positive cell number, BRCA1 expression and IC50 of gemcitabine that were affected by PRPF19 silencing. In conclusion, PRPF19 expression was upregulated in bladder cancer. It promoted cell growth and DNA damage repair, and decreased gemcitabine sensitivity via positively regulating DDB1 expression.

The online version contains supplementary material available at 10.1007/s10616-023-00599-7.

Cytosolic proliferating cell nuclear antigen (PCNA) is involved in neutrophil survival and function, in which it acts as a scaffold and associates with proteins involved in apoptosis, NADPH oxidase activation, cytoskeletal dynamics, and metabolism. While the PCNA interactome has been characterized in neutrophils under homeostatic conditions, less is known about neutrophil PCNA in pathophysiological contexts. Granulocyte colony-stimulating factor (G-CSF) is a cytokine produced in response to inflammatory stimuli that regulates many aspects of neutrophil biology. Here, we used isolated normal-density neutrophils from G-CSF-treated haemopoietic stem cell donors (GDs) as a model to understand the role of PCNA during inflammation. Proteomic analysis of the neutrophil cytosol revealed significant differences between GDs and healthy donors (HDs). PCNA was one of the most upregulated proteins in GDs, and the PCNA interactome was significantly different in GDs compared with HDs. Importantly, while PCNA associated with almost all enzymes involved in glycolysis in HDs, these associations were decreased in GDs. Functionally, neutrophils from GDs had a significant increase in glycolysis compared with HDs. Using p21 competitor peptides, we showed that PCNA negatively regulates neutrophil glycolysis in HDs but had no effect on GD neutrophils. These data demonstrate that G-CSF alters the PCNA scaffold, affecting interactions with key glycolytic enzymes, and thus regulates glycolysis, the main energy pathway utilized by neutrophils. By this selective control of glycolysis, PCNA can organize neutrophils functionality in parallel with other PCNA mechanisms of prolonged survival. PCNA may therefore be instrumental in the reprogramming that neutrophils undergo in inflammatory or tumoral settings.

Boron is an essential trace element with roles in growth, development, and physiological functions; however, its mechanism of action is still unclear. In this study, the regulatory roles of the PI3K/Akt signaling pathway on boron-induced changes in barrier function, proliferation, and apoptosis in rat intestinal epithelial cells were evaluated. Occludin levels, the proportion of cells in the G2/M phase, cell proliferation rate, and mRNA and protein expression levels of PCNA were higher, while the proportions of cells in the G0/G1 and S phases, apoptosis rate, and caspase-3 mRNA and protein expression levels were lower in cells treated with 0.8 mmol/L boron than in control IEC-6 cells (P < 0.01 or P < 0.05). However, 40 mmol/L boron decreased ZO-1 and Occludin levels, the proportion of cells in the G2/M phase, cell proliferation rate, and mRNA and protein levels of PCNA and increased the apoptosis rate and caspase-3 mRNA expression (P < 0.01 or P < 0.05). After specifically blocking PI3K and Akt signals (using LY294002 and MK-2206 2HCL), 0.8 mmol/L boron had no effects on Occludin, PCNA level, apoptosis rates, and caspase-3 levels (P < 0.05); however, the proliferation rate and PCNA levels decreased significantly (P < 0.01 or P < 0.05). The addition of 40 mmol/L boron did not affect ZO-1 and Occludin levels and did not affect the apoptosis rate or PCNA and caspase-3 levels. These results suggested that the PI3K/Akt signaling pathway mediates the effects of low-dose boron on IEC-6 cells.

The role of SMU1 in DNA replication and RNA splicing is well-established, yet its specific function and dysregulated mechanisms in gastric cancer (GC) remain inadequately elucidated. This study seeks to investigate the potential oncogenic and progression-promoting effects of SMU1 in GC, with the ultimate goal of informing novel approaches for treatment and diagnosis.

The study investigated the expression levels of SMU1 in GC and adjacent normal tissues by analyzing data from the TCGA (27 tissue pairs) and GEO (47 tissue pairs) databases. Immunohistochemistry was used to examine 277 tumor tissue and adjacent non-tumor tissue spots from GC tissue chips, along with relevant follow-up information. The study further assessed the proliferation, invasion, and migration capabilities of cells by manipulating SMU1 expression levels and conducting various assays, including CCK-8, EdU incorporation, colony formation, transwells, flow cytometry, and subcutaneous tumorigenesis assays.

Our study revealed a significant upregulation of SMU1 mRNA and protein levels in GC tissues compared to adjacent tissues. Univariate and multivariate Cox analysis demonstrated that elevated levels of SMU1 were independent prognostic factors for GC prognosis (P = 0.036). Additionally, median survival analysis indicated a significant association between high SMU1 expression and poor prognosis in GC patients (P = 0.0002). In experiments conducted both in vivo and in vitro, it was determined that elevated levels of SMU1 can enhance the proliferation, invasion, and migration of GC cells, whereas suppression of SMU1 can impede the progression of GC by modulating the G1/S checkpoint of the cell cycle.

Our research introduces the novel idea that SMU1 could serve as a prognostic marker for GC progression, influencing cell proliferation through cell cycle activation. These results offer valuable insights into the understanding, diagnosis, and management of gastric carcinoma.

Considering the therapeutic efficacy of Stachydrine on breast cancer (BC), this study aims to decipher the relevant mechanism. The effects of Stachydrine on BC cell viability, proliferation and apoptosis were firstly investigated. Then, Bioinformatics was applied to sort out the candidate interacting with Stachydrine as well as its expression and downstream target in BC. Relative expressions of genes of interest as well as proliferation- and apoptosis-related factors in BC cells were quantified through quantitative reverse-transcription PCR and western blot as appropriate. As a result, Stachydrine inhibited the proliferation, down-regulated the expressions of proliferating cell nuclear antigen and CyclinD1, enhanced cell cycle arrest and apoptosis, and up-regulated the levels of Cleaved caspase-3 and Cleaved caspase-9 in BC cells. Phospholipase A2 Group IIA (PLA2G2A) was predicted as the candidate interacting with Stachydrine and to be lowly expressed in BC. PLA2G2A silencing reversed while PLA2G2A overexpression reinforced the effects of Stachydrine. Decorin (DCN) was the downstream target of PLA2G2A and also lowly expressed in BC. PLA2G2A silencing counteracted yet overexpressed PLA2G2A strengthened the promoting effects of Stachydrine on DCN level. Collectively, Stachydrine inhibits the growth of BC cells to promote cell cycle arrest and apoptosis via PLA2G2A/DCN axis.

Considering the extensive and widespread impact on individuals, cancer can presently be categorized as a pandemic. In many instances, the development of tumors has been linked to endemic microbe infections. Among parasitic infections, Trypanosoma cruzi stands out as one of the most extensively discussed protozoans in the literature that explores the association between diseases of parasite origin and cancer. However, the effective association remains an unsolved paradox. Both the parasite, along with protozoan-derived molecules, and the associated antiparasitic immune response can induce alterations in various host cell pathways, leading to modifications in cell cycle, metabolism, glycosylation, DNA mutations, or changes in neuronal signaling. Furthermore, the presence of the parasite can trigger cell death or a senescent phenotype and modulate the immune system, the metastatic cascade, and the formation of new blood vessels. The interaction among the parasite (and its molecules), the host, and cancer undoubtedly encompasses various mechanisms that operate differentially depending on the context. Remarkably, contrary to expectations, the evidence tilts the balance toward inhibiting tumor growth or resisting tumor development. This effect is primarily observed in malignant cells, rather than normal cells, indicating a selective or specific component. Nevertheless, nonspecific bystander mechanisms, such as T. cruzi's adjuvancy or the presence of proinflammatory cytokines, may also play a significant role in this phenomenon. This work aims to elucidate this complex scenario by synthesizing the main findings presented in the literature and by proposing new questions and answers, thereby adding pieces to this challenging puzzle.

Breast cancer is the most common cancer in women, the so-called "Triple-Negative Breast Cancer" (TNBC) subtype remaining the most challenging to treat, with low tumor-free survival and poor clinical evolution. Therefore, there is a clear medical need for innovative and more efficient treatment options for TNBC. The aim of the present study was to evaluate the potential therapeutic interest of the association of the tumor-penetrating BR2 peptide with monophosphoester 2-aminoethyl dihydrogen phosphate (2-AEH2P), a monophosphoester involved in cell membrane turnover, in TNBC. For that purpose, viability, migration, proliferative capacity, and gene expression analysis of proteins involved in the control of proliferation and apoptosis were evaluated upon treatment of an array of TNBC cells with the BR2 peptide and 2-AEH2P, either separately or combined. Our data showed that, while possessing limited single-agent activity, the 2-AEH2P+BR2 association promoted significant cytotoxicity in TNBC cells but not in normal cells, with reduced proliferative potential and inhibition of cell migration. Mechanically, the 2-AEH2P+BR2 combination promoted an increase in cells expressing p53 caspase 3 and caspase 8, a reduction in cells expressing tumor progression and metastasis markers such as VEGF and PCNA, as well as a reduction in mitochondrial electrical potential. Our results indicate that the combination of the BR2 peptide with 2-AEH2P+BR2 may represent a promising therapeutic strategy in TNBC with potential use in clinical settings.

The action of buckwheat, rooibos and vitex on healthy female reproductive systems, as well as their ability to mitigate the reproductive toxicity of environmental contaminant toluene have not yet been examined. We analysed the influence of toluene (0, 10, 100 or 1000 ng/mL) with and without these plant extracts (10 μg/mL) on cultured porcine ovarian granulosa cells. Cell viability, proliferation (PCNA accumulation), apoptosis (accumulation of bax) and release of progesterone (P) and oestradiol (E) were measured. Toluene reduced ovarian cell viability and proliferation, increased apoptosis and suppressed E but not P release. Plant extracts, given alone, were also able to directly suppress some ovarian cell functions. The addition of buckwheat promoted toluene action on cell viability, proliferation and P release, but it did not modify other toluene effects. Rooibos mitigated toluene action on cell viability, proliferation and apoptosis but promoted its action on P and E. The addition of vitex mitigated all the tested toluene effects. These observations: (1) demonstrate the direct toxic influence of toluene on ovarian cells, (2) demonstrate the ability of food/medicinal plants to either promote or mitigate toluene effects and (3) suggest that vitex could be a natural protector against the suppressive effect of toluene on female reproduction.

The rare earth element (REE) pollution in the hydrosphere has become a matter of serious concern lately. In this study, using inductively coupled plasma mass spectrometry, the REE contents in soft tissue, byssus and shell liquor of the mussel Mytilus galloprovincialis, as a potential REE pollution bioindicator, in the Black Sea were determined for the first time. The highest REE levels were observed in mussels from the seabed. The REE contents in byssus and shell liquor were higher than in soft tissue. In byssus, the contents of Y, La, Ce, Nd and Dy were the highest, whereas in shell liquor, the heavier REE and Sc were the most concentrated. No likely REE-associated risks from the mussel meat consumption were detected. In soft tissue and byssus, REE contents significantly correlated with that of silicon.

Hepatocellular carcinoma (HCC) is the most common primary malignant tumor. Although sorafenib and regorafenib have been approved for first-line and second-line treatment, respectively, of patients with advanced HCC, long-term treatment often results in acquired resistance. Given that glycolysis-mediated lactate production can contribute to drug resistance and impair HCC treatment efficacy, we investigated the effects of ketone body treatment on the metabolic shift in sorafenib-resistant HCC cells. We discovered differential expression of 3-hydroxymethyl glutaryl-CoA synthase 2 (HMGCS2) and the ketone body D-β-hydroxybutyrate (β-HB) in four sorafenib-resistant HCC cell lines. In sorafenib-resistant HCC cells, lower HMGCS2 and β-HB levels were correlated with more glycolytic alterations and higher lactate production. β-HB treatment enhanced pyruvate dehydrogenase (PDH) expression and decreased lactate dehydrogenase (LDHA) expression and lactate production in sorafenib-resistant HCC cells. Additionally, β-HB combined with sorafenib or regorafenib promoted the antiproliferative and antimigratory abilities of sorafenib-resistant HCC cells by inhibiting the B-raf/mitogen-activated protein kinase pathway and mesenchymal N-cadherin-vimentin axis. Although the in vivo β-HB administration did not affect tumor growth, the expression of proliferative and glycolytic proteins was inhibited in subcutaneous sorafenib-resistant tumors. In conclusion, exogenous β-HB treatment can reduce lactate production and reverse sorafenib resistance by inducing a glycolytic shift; it can also synergize with regorafenib for treating sorafenib-resistant HCC.

Accumulating evidence indicates that circular RNAs (circRNAs) are inextricably linked to cancer development. However, the function and mechanism of nucleus-localized circRNAs in hepatocellular carcinoma (HCC) still require investigation. Here, qRT-PCR and receiver-operating characteristic curve were used to detect the expression and diagnostic potential of circSLC39A5 for HCC. The biological function of circSLC39A5 in HCC was investigated in vitro and in vivo. Nucleoplasmic separation assay, fluorescence in situ hybridization, RNA pulldown, RNA immunoprecipitation, the HDOCK Server, the NucleicNet Webserver, crosslinking-immunoprecipitation, MG132 treatment, and chromatin immunoprecipitation were utilized to explore the potential molecular mechanism of circSLC39A5 in HCC. The results showed that circSLC39A5 was downregulated in both HCC tissues and plasma and was associated with satellite nodules and lymph node metastasis/vascular invasion. CircSLC39A5 was stably expressed in plasma samples under different storage conditions, showing good diagnostic potential for HCC (AUC = 0.915). CircSLC39A5 inhibited proliferation, migration, and invasion, facilitated the apoptosis of HCC cells, and was associated with low expression of Ki67 and CD34. Remarkably, circSLC39A5 is mainly localized in the nucleus and binds to the transcription factor signal transducer and activator of transcription 1 (STAT1), affecting its stabilization and expression. STAT1 binds to the promoter of thymine DNA glycosylase (TDG). Overexpression of circSLC39A5 elevates TDG expression and reverses the increase of proliferating cell nuclear antigen (PCNA) expression and the overactive cell proliferation caused by TDG silencing. Our findings uncovered a novel plasma circRNA, circSLC39A5, which may be a potential circulating diagnostic marker for HCC, and the mechanism by which nucleus-localized circSLC39A5 exerts a transcriptional regulatory role in HCC by affecting STAT1/TDG/PCNA provides new insights into the mechanism of circRNAs.

Invasive micropapillary carcinoma (IMPC) of the breast is a special histopathologic type of cancer with a high recurrence rate and the biological features of invasion and metastasis. Previous spatial transcriptome studies indicated extensive metabolic reprogramming in IMPC, which contributes to tumor cell heterogeneity. However, the impact of metabolome alterations on IMPC biological behavior is unclear. Herein, endogenous metabolite-targeted metabolomic analysis was done on frozen tumor tissue samples from 25 patients with breast IMPC and 34 patients with invasive ductal carcinoma not otherwise specified (IDC-NOS) by liquid chromatography-mass spectrometry. An IMPC-like state, which is an intermediate transitional morphologic phenotype between IMPC and IDC-NOS, was observed. The metabolic type of IMPC and IDC-NOS was related to breast cancer molecular type. Arginine methylation modification and 4-hydroxy-phenylpyruvate metabolic changes play a major role in the metabolic reprogramming of IMPC. High protein arginine-N-methyltransferase (PRMT) 1 expression was an independent factor related to the poor prognosis of patients with IMPC in terms of disease-free survival. PRMT1 promoted H4R3me2a, which induced tumor cell proliferation via cell cycle regulation and facilitated tumor cell metastasis via the tumor necrosis factor signaling pathway. This study identified the metabolic type-related features and intermediate transition morphology of IMPC. The identification of potential targets of PRMT1 has the potential to provide a basis for the precise diagnosis and treatment of breast IMPC.

Mentha arvensis has been utilized in diverse traditional medicines as an antidiabetic, anticarcinogenic, antiallergic, antifungal, and antibacterial agent. In this work, we have explored the phytochemical analyses and pharmacological potential of Mentha arvensis using both in silico and in vitro approaches for drug discovery.

To determine the extract with the highest potential for powerful bioactivity, ethanol was used as the solvent. The phytochemical components of the extracts were quantified using liquid chromatography-mass spectrometry analysis. The potential bioactivities of extracts and lead phytocompounds, including their antibacterial, cytotoxic, and anti-diabetic effects, were evaluated.

The compounds oleanolic acid, rosmarinic acid, luteolin, isoorientin, and ursolic acid have been identified through liquid chromatography mass spectrometry analysis. Based on antimicrobial research, it has been found that the Mentha arvensis extract shows potential activity against K. pneumoniae which was 13.39 ± 0.16. Mentha arvensis has demonstrated a greater degree of efficacy in inhibiting α-glucosidase, with an inhibition rate of 58.36 ± 0.12, and in inhibiting α-amylase, with an inhibition rate of 42.18 ± 0.83. The growth of HepG2 cells was observed to be significantly suppressed upon treatment with extracts obtained from Mentha arvensis. Finally, In-silico methods demonstrated that the Luteolin and Rosmarinic acid exhibit acceptable drug-like characteristics. Furthermore, Molecular docking studies further demonstrated that both compounds have strong potential to inhibit the active sites of therapeutically relevant enzymes involved in Diabetes, Bacterial infections, and Cancer.

The results of this study suggest that the Mentha arvensis extract possesses potent pharmacological potentials, particularly in terms of antibacterial, anti-diabetic, and cytotoxic effects. Particularly, Luteolin and Rosmarinic acid were identified as the top contenders for potential bioactivity with acceptable drug-like properties.

Proliferating cell nuclear antigen (PCNA) is the key regulator of human DNA metabolism. One important interaction partner is p15, involved in DNA replication and repair. Targeting the PCNA-p15 interaction is a promising therapeutic strategy against cancer. Here, a Förster resonance energy transfer (FRET)-based assay for the analysis of the PCNA-p15 interaction was developed. Next to the application as screening tool for the identification and characterization of PCNA-p15 interaction inhibitors, the assay is also suitable for the investigation of mutation-induced changes in their affinity. This is particularly useful for analyzing disease associated PCNA or p15 variants at the molecular level. Recently, the PCNA variant C148S has been associated with Ataxia-telangiectasia-like disorder type 2 (ATLD2). ATLD2 is a neurodegenerative disease based on defects in DNA repair due to an impaired PCNA. Incubation time dependent FRET measurements indicated no effect on PCNA^C148S-p15 affinity, but on PCNA stability. The impaired stability and increased aggregation behavior of PCNA^C148S was confirmed by intrinsic tryptophan fluorescence, differential scanning fluorimetry (DSF) and asymmetrical flow field-flow fractionation (AF4) measurements. The analysis of the disease associated PCNA variant demonstrated the versatility of the interaction assay as developed.

Transient transfection of foreign DNA is the most widely used laboratory technique to study gene function and product. However, the transfection efficiency depends on many parameters, including DNA quantity and quality, transfection methods and target cell lines. Here, we describe the considerable advantage of mRNA electroporation compared to conventional DNA-based systems. Indeed, our methodology offers extremely high transfection efficiency up to 98% regardless of the cell line tested. Protein expression takes place a few hours post-transfection and lasts over 72 h, but overall, the electrotransfer of mRNAs enables the monitoring of the level of protein expressed by simply modulating the amount of mRNAs used. As a result, we successfully conducted cell imaging by matching the levels of expressed V_HHs and the antigen present in the cell, preventing the necessity to remove the excess unbound V_HHs. Altogether, our results demonstrate that mRNA electrotransfer could easily supplant the conventional DNA-based transient expression system.

Spermatogenesis is temperature-dependent, and the increase in testicular temperature seriously affects mammalian spermatogenesis and semen quality. In this study, the testicular heat stress model of mice was made with a 43 °C water bath for 25 min, and the effects of heat stress on semen quality and spermatogenesis-related regulators were analyzed. On the 7th day after heat stress, testis weight shrank to 68.45% and sperm density dropped to 33.20%. High-throughput sequencing analysis showed that 98 microRNAs (miRNAs) and 369 mRNAs were down-regulated, while 77 miRNAs and 1424 mRNAs were up-regulated after heat stress. Through gene ontology (GO) analysis of differentially expressed genes and miRNA-mRNA co-expression networks, it was found that heat stress may be involved in the regulation of testicular atrophy and spermatogenesis disorders by affecting cell meiosis process and cell cycle. In addition, through functional enrichment analysis, co-expression regulatory network, correlation analysis and in vitro experiment, it was found that miR-143-3p may be a representative potential key regulatory factor affecting spermatogenesis under heat stress. In summary, our results enrich the understanding of miRNAs in testicular heat stress and provide a reference for the prevention and treatment of heat-stress-induced spermatogenesis disorders.

Pulmonary arterial hypertension (PAH) is a chronic disease characterized by pulmonary vascular remodeling. It refers to the abnormal proliferation of pulmonary artery smooth muscle cells (PASMCs), and hypoxia is an important risk factor for this progression. The present study aims to investigate the role of YTHDF1 in the regulation of hypoxic PASMC proliferation and the underlying mechanism. Human PASMCs were transfected with si-YTHDF1/2/3 followed by treatment of hypoxia, and the PASMC proliferation and Foxm1 expression were detected. Through RNA pull-down, RNA immunoprecipitation, and protein synthesis assay, the mechanism of YTHDF1 regulating Foxm1 was explored. Next, Foxm1 was inhibited by thiostrepton, and cell proliferation was detected. In vivo, mice received a tail vein injection of adenovirus containing si-YTHDF1 and were exposed to hypoxia treatment. Pulmonary vascular changes, right ventricular systolic pressure (RVSP), and genes involving proliferation were analyzed. YTHDF1 silencing reduced more hypoxic PASMC proliferation and Foxm1 protein level than YTHDF2/3 silencing. Mechanical results showed that YTHDF1 interacted with Foxm1 mRNA and up-regulated Foxm1 protein level by enhancing the translation efficiency in an m6A-dependent manner. Furthermore, YTHDF1 facilitated hypoxic PASMC proliferation and proliferation marker expressions through up-regulation of Foxm1 in an m6A-dependent manner. In vivo, the YTHDF1 silencing alleviated pulmonary vascular changes and fibrosis, reduced RVSP, inhibited the interaction of YTHDF1 and Foxm1, and reduced proliferation marker levels, as compared to the PAH group. In conclusion, YTHDF1 silencing inhibits hypoxic PASMC proliferation by regulating Foxm1 translation in an m6A-dependent manner.

Citrinin, a mycotoxin existing in fruits, has nephrotoxicity, hepatotoxicity and embryotoxicity. The effects of citrinin on Leydig cell development in prepuberty remains unclear. Male Sprague-Dawley rats were gavaged with 0, 1, 2.5, and 5 mg/kg citrinin from postnatal days 21-28. Citrinin at 5 mg/kg significantly decreased serum testosterone levels, while increasing serum LH and FSH levels. Citrinin at 1-5 mg/kg markedly downregulated Hsd17b3 and HSD17B3 expression, while upregulating Srd5a1 (SRD5A1) and Akr1c14 (AKR1C14) expression at 2.5 and/or 5 mg/kg. Citrinin at 5 mg/kg also significantly increased PCNA-labeling index in Leydig cells. Citrinin at 5 mg/kg significantly raised testicular MDA amount, whiling at 2.5 and 5 mg/kg downregulating SOD1 and SOD2 expression. Citrinin at 5 mg/kg markedly decreased the ratio of Bcl2 to Bax, in consistent with the increased apoptosis in Leydig cells judged by TUNEL assay. Enzymatic assay revealed that citrinin inhibited rat testicular HSD3B1 activity at 100 µM and HSD17B3 activity at 10-100 μM. Citrinin at 50 μM and higher also induced reactive oxygen species (ROS) and apoptosis of R2C cell line. In conclusion, citrinin inhibits Leydig cell development at multiple levels via different mechanisms and oxidative stress partially plays a role.