African swine fever (ASF) caused by ASF virus (ASFV) is a fatal disease in pigs and results in great economic losses. Due to the lack of available vaccines and treatments, serological diagnosis of ASF plays a key role in the surveillance program, but due to the lack of knowledge and the complexity of the ASFV genome, the candidate target viral proteins are still being researched. False negativity is still a big obstacle during the diagnostic process. In this study, the high antigenic viral proteins p30, p54 and p72 were screened to find the antigenic dominant domains and the tandem His-p30-54-72 was derived. An indirect enzyme-linked immunosorbent assay (iELISA) coated with His-p30-54-72 was developed with a cut-off value of 0.371. A total of 192 clinical samples were detected by His-p30-54-72-coated indirect ELISA (iELISA) and commercial ASFV antibody kits. The results showed that the positive rate of His-p30-54-72-coated iELISA was increased by 4.7% and 14.6% compared with a single viral protein-based commercial ASFV antibody kits. These results provide a platform for future ASFV clinical diagnosis and vaccine immune effect evaluation.

Inhibition of histone deacetylases (HDACs) has been extensively studied in the development of anticancer drugs. In the discovery of potent HDAC inhibitors with novel structures, the 2-substituted phenylquinoline-4-carboxylic acid group was introduced to the cap moiety of HDAC inhibitors. In total, 30 compounds were synthesized with hydroxamic acid or hydrazide zinc-binding groups. In the enzyme inhibitory test, active compound D28 and its analog D29 exhibited significant HDAC3 selectivity against HDAC1, 2, 3, and 6. However, compared with D28, the hydrazide-bearing compounds (D29 and D30) with remarkably improved enzyme inhibitory activities did not exhibit significant antiproliferative potency in the in vitro anticancer study. Further K562 cell-based mechanistic results revealed that induction of G2/M cell cycle arrest and promotion of apoptosis make important contributions to the anticancer effects of molecule D28. Collectively, an HDAC3 selective inhibitor (D28) with potent in vitro anticancer activity was developed as a lead compound for the treatment of cancer.

In discovery of novel HDAC inhibitory with anticancer potency, pharmacophores of phenanthridine were introduced to the structure of HDAC inhibitors. Fatty and aromatic linkers were evaluated for their solubility and activity. Both enzyme inhibitory and in vitro antiproliferative (against U937 cells) screening results revealed better activities of compounds with aromatic linker than molecules with fatty linker. Compared with SAHA (IC50 values of 1.34, 0.14, 2.58, 0.67 and 18.17 µM), molecule Fb-4 exhibited 0.87, 0.09, 0.32, 0.34 and 17.37 µM of IC50 values against K562, U266, MCF-7, U937 and HEPG2 cells, respectively. As revealed by cell cycle and apoptotic analysis, induction of G2/M phase arrest and apoptosis plays an important role in the inhibition of MCF-7 cells by Fb-4. Generally, a potent HDAC inhibitor was developed in the present study which could be utilised as a lead compound for further anticancer drug design.

In discovery of HDAC inhibitors (HDACIs) with improved anticancer potency, structural modification was performed on the previous derived indole-3-butyric acid derivative. Among all the synthesised compounds, molecule I13 exhibited high HDAC inhibitory and antiproliferative potencies in the in vitro investigations. The IC50 values of I13 against HDAC1, HDAC3, and HDAC6 were 13.9, 12.1, and 7.71 nM, respectively. In the cancer cell based screening, molecule I13 showed increased antiproliferative activities in the inhibition of U937, U266, HepG2, A2780, and PNAC-1 cells compared with SAHA. In the HepG2 xenograft model, 50 mg/kg/d of I13 could inhibit tumour growth in athymic mice compared with 100 mg/kg/d of SAHA. Induction of apoptosis was revealed to play an important role in the anticancer potency of molecule I13. Collectively, a HDACI (I13) with high anticancer activity was discovered which can be utilised as a lead compound for further HDACI design.

Inhibition of histone deacetylases (HDACs) has been an important emerging therapy for the treatment of multiple cancers. However, the application of HDAC inhibitors is restricted by the limited potency against solid tumors. In order to discover novel HDAC inhibitors with potent antitumor activities, nitrogen mustard group was introduced to the structure of CI994. The derived molecule N-(2-aminophenyl)-4-(bis(2-chloroethyl)amino)benzamide (NA) exhibited enzyme inhibitory pattern of class I selectivity with IC50 values of 95.2, 260.7, and 255.7 nM against HDAC1, HDAC2, and HDAC3, respectively. In the antiproliferative assay, NA exhibited 10.3-fold (2.66 μM) and 11.3-fold (1.73 μM) higher potency than did suberoylanilide hydroxamic acid (SAHA) (27.3 and 19.5 μM) in inhibition of A2780 and HepG2 cell growth, respectively. Further HepG2 cell-based cell cycle and apoptosis studies revealed that induction of the G2/M phase arrest and cell apoptosis contributes to the antitumor effects of NA. It is suggested that NA could be utilized as a lead compound in the development of bifunctional HDAC inhibitors for the treatment of solid tumors.

Triple-negative breast cancer is a high-risk breast cancer with poor survival rate. To date, there is a lack of targeted therapy for this type of cancer. One unique phenomenon is that inflammatory breast cancer is frequently triple negative. However, it is still ambiguous how inflammation influences triple-negative breast cancer growth and responding to chemotherapy. Herein, we investigated the levels of inflammation-associated enzyme, iNOS, in 20 triple-negative breast cancer patients' tumors, and examined its correlation with patients' responses to platinum-based neoadjuvant chemotherapy. Our studies showed that triple-negative breast cancer patients with attenuated iNOS levels in tumor cells after treatment showed better responses to platinum-based neoadjuvant chemotherapy than other triple-negative breast cancer patients. Our further in vitro studies confirmed that induction of proper levels of NO increased the resistance to cisplatin in triple-negative MDA-MB-231 cells. Our data suggest that aberrant high level of iNOS/NO are associated with less effectiveness of platinum-based neoadjuvant chemotherapy in triple-negative breast cancer. Therefore, we propose to monitor iNOS levels as a new predictor for triple-negative breast cancer patient's response to platinum-based neoadjuvant chemotherapy. Moreover, iNOS/NO is considered as a potential target for combination therapy with platinum drugs for triple-negative breast cancer.

Introduction: Development of Poly (ADP-ribose) polymerase (PARP) inhibitors has been extensively studied in cancer treatment. Olaparib, the first approved PARP inhibitor, showed potency in the inhibition of both BRCA (breast cancer associated)-mutated and BRCA-unmutated cancers. Methods: Aiming to the discovery of olaparib analogs for the treatment of cancer, structural modifications were performed based on the scaffold of olaparib. In the first series, reduction of carbonyl group to CH2 led to decrease of PARP1 inhibitory activity. Preserving the original carbonyl group, molecules with potent PARP1 inhibitory activities were derived by introduction of hydrazide and aromatic nitrogen mustard groups. The synthesized compounds were evaluated in the in the PARP1 enzyme inhibitory screening, cancer cell based antiproliferative assay, cell cycle arrest and apoptosis studies. Results: It is remarkable that, molecule C2 with chlorambucil substitution, exhibited potent PARP1 inhibitory activity and a broad-spectrum of anticancer potency in the in vitro antiproliferative assay. Compared with olaparib and chlorambucil, molecule C2 also showed significant potency in inhibition of a variety of BRCA-unmutated cell lines. Further analysis revealed the effects of C2 in induction of G2/M phase cell cycle arrest and promotion of apoptosis. Discussion: Collectively, the olaparib-chlorambucil hybrid molecule (C2) could be utilized as a lead compound for further drug design.

Aminopeptidase N (APN, CD13) is closely associated with the development and progression of cancer. Previous studies suggested APN as a biomarker for cancer stem cells. APN inhibitors have been intensively evaluated as chemosensitizers for cancer treatments. In the present study, tetrahydro-β-carboline scaffold was introduced to the structure of APN inhibitors. The synthesized compounds showed potent enzyme inhibitory activities compared with Bestatin, an approved APN inhibitor, in cell-based enzymatic assay. In combination with chemotherapeutic drugs, representative APN inhibitor molecules D12, D14 and D16 significantly improved the antiproliferative potency of anticancer drugs in the in vitro tests. Further mechanistic studies revealed that the anticancer effects of these drug combinations are correlated with decreased APN expression, increased ROS level, and induction of cell apoptosis. The spheroid-formation assay and colony-formation assay results showed effectiveness of Paclitaxel-APN inhibitor combination against breast cancer stem cell growth. The combined drug treatment led to reduced mRNA expression of OCT-4, SOX-2 and Nanog in the cancer stem cells tested, suggesting the reduced stemness of the cells. In the in vivo study, the selected APN inhibitors, especially D12, exhibited improved anticancer activity in combination with Paclitaxel compared with Bestatin. Collectively, potent APN inhibitors were discovered, which could be used as lead compounds for tumor chemo-sensitization and cancer stem cell-based therapies.

DNA methylation has been implicated in the pathogenesis of diabetic kidney disease (DKD), but the underlying mechanisms remain unclear. In this study, we tested the hypothesis that aberrant DNA methylation in peripheral immune cells contributes to DKD progression. We showed that levels of DNA methyltransferase 1 (DNMT1), a key enzyme for DNA methylation, were increased along with inflammatory activity of peripheral blood mononuclear cells in DKD patients. Inhibition of DNMT1 with 5-aza-2'-deoxycytidine (5-Aza) markedly increased the proportion of CD4+CD25+ regulatory T cells in peripheral blood mononuclear cells in culture and in diabetic animals. Adoptive transfer of immune cells from 5-Aza-treated animals showed beneficial effects on the host immune system, resulting in a significant improvement of DKD. Using genome-wide DNA methylation assays, we identified the differentially methylated cytosines in the promoter regions of mammalian target of rapamycin (mTOR) regulators in peripheral blood mononuclear cells of diabetic patients. Further, mRNA arrays confirmed the consistent induction of genes expressed in the mTOR pathway. Importantly, down-regulation of DNMT1 expression via RNA interference resulted in prominent cytosine demethylation of mTOR negative regulators and subsequent decrease of mTOR activity. Lastly, modulation of mTOR resulted in changes in the effect of 5-aza on diabetic immune cells. Thus, up-regulation of DNMT1 in diabetic immune cells induces aberrant cytosine methylation of the upstream regulators of mTOR, leading to pathogenic activation of the mTOR pathway and consequent inflammation in diabetic kidneys. Hence, this study highlights therapeutic potential of targeting epigenetic events in immune system for treating DKD.

Osteoarthritis (OA) is a degenerative joint disease. Dysregulated microRNA (miRNA) expressions are implicated in OA progression. Consequently, the current study set out to investigate the mechanism of miR-140-5p in OA cartilage injury. Firstly, the murine and cell models of OA were established, and cartilage tissues of OA mice were observed using hematoxylin and eosin staining and safranin O staining. Chondrocyte pyroptosis was further assessed using immunohistochemical and Calcein-AM/PI staining. The levels of gasdermin-D (GSDMD)-N, cleaved caspase-1, interleukin (IL)-1β, and IL-18 in cartilage tissues and cells were determined using Western blot and enzyme-linked immunosorbent assay kits. The targeting relationship between miR-140-5p and cathepsin B (CTSB) was verified using a dual-luciferase assay. Moreover, the binding of CTSB and Nod-like receptor protein 3 (NLRP3) was detected using co-immunoprecipitation assay. Lastly, the effects of NLRP3 activation and CTSB overexpression on chondrocyte pyroptosis were documented. It was found that OA induction aggravated cartilage tissue injury and enhanced chondrocyte pyroptosis. miR-140-5p was poorly-expressed in OA models, and miR-140-5p over-expression alleviated chondrocyte pyroptosis, as evidenced by decreased GSDMD-N, cleaved caspase-1, IL-1β, and IL-18 levels. miR-140-5p targeted the CTSB gene, whereas CTSB further bound to NLRP3 and activated the NLRP3 inflammasome. Additionally, CTSB over-expression or NLRP3 activation reversed the inhibitory effect of miR-140-5p on chondrocyte pyroptosis. Collectively, our findings revealed that miR-140-5p repressed chondrocyte pyroptosis and alleviated OA cartilage injury via inhibition of the CTSB/NLRP3. This study may confer a theoretical basis for the treatment of OA cartilage injury.