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The prognosis of rhegmatogenous retinal detachment (RRD) with choroidal detachment (RRDCD) is often poor and complicated. This study focused on the identification of the characteristic proteins and signal pathways associated with the etiology of RRDCD and to provide guidance for diagnosis and treatment of RRDCD. In this study, vitreous humor samples were obtained from 16 RRDCD patients, 14 with RRD, 12 with idiopathic epiretinal macular membrane (IEMM), and 5 healthy controls from donated corpse eyes. Data-independent acquisition mass spectrometry and bioinformatics analysis were employed to identify differentially expressed proteins (DEPs). In the vitreous humor, 14,842 peptides were identified. Patients with RRDCD had 249 DEPs (93 upregulated and 156 downregulated), with 89 in patients with RRD and 61 in patients with IEMM. Enrichment analysis of the GO and Kyoto Encyclopedia of Genes and Genomes DEP databases indicated functional clusters related to inflammation and immunity, protein degradation and absorption, cell adhesion molecules (CAMs), the hedgehog signaling pathway, and lipid metabolism. Weighted gene co-expression network analysis showed that DEPs with positive co-expression of RRDCD participated in immune-related pathways led by the complement and coagulation cascade, whereas DEPs with negative co-expression of RRDCD participated in protein degradation and absorption, CAMs, and the hedgehog signaling pathway. In summary, our study provides important clues and the theoretical basis for exploring the pathogenesis, progression, and prognosis of ocular fundus disease.

H9N2 avian influenza viruses are widely prevalent in birds and pose an increasing threat to humans because of their enhanced virulence and transmissibility in mammals. Active surveillance on the prevalence and evolution of H9N2 viruses in different avian hosts will help develop eradication measures. We isolated 16 H9N2 viruses from chickens, green peafowls, and wild birds in eastern China from 2017 to 2019 and characterized their comparative genetic evolution, receptor-binding specificity, antigenic diversity, replication, and transmission in chickens and mice. The phylogenetic analysis indicated that the green peafowl viruses and swan reassortant shared the same ancestor with the poultry H9N2 viruses prevalent in eastern China, while the seven wild bird viruses belonged to wild bird lineage. The chicken, peafowl, and swan H9N2 viruses that belonged to the poultry lineage preferentially recognized α-2, 6-linked sialic acids (human-like receptor), but the wild bird lineage viruses can bind both α-2, 3 (avian-like receptor) and human-like receptor similarly. Interestingly, the H9N2 viruses of poultry lineage replicated well and transmitted efficiently, but the viruses of wild bird lineage replicated and transmitted with low efficiency. Importantly, the H9N2 viruses of poultry lineage replicated in higher titer in mammal cells and mice than the viruses of wild birds lineage. Altogether, our study indicates that co-circulation of the H9N2 viruses in poultry, wild birds, and ornamental birds increased their cross-transmission risk in different birds because of their widespread dissemination.

Retinal detachment (RD) and its special form of rhegmatogenous RD associated with choroidal detachment (RRDCD) feature similar pathological alterations, including enhanced retinal cell inflammation. Although the importance of the complement components C3a and C5a and their corresponding receptors in retinal maintenance has been demonstrated, the relevance of these molecules to the pathogenesis of RD or RRDCD remains to be investigated. The contents of C3a, C5a and inflammatory factors, such as TNF-α, IL-1β, IL-6 and prostaglandin (PG)E2, in related clinical samples were examined by ELISA. Subsequently, human retinal pigment epithelial (HRPE) cells were subjected to challenge with the C3a and C5a recombinant proteins with or without C3a and C5a antagonists and NF-κB inhibitor, and the cell viability and inflammatory cytokines were then determined by a Cell Counting Kit-8 assay and ELISA, respectively. In addition, reverse transcription-quantitative PCR and western blot analyses were utilized to examine the mRNA or/and protein levels of C3a and its receptor C3aR, as well as C5a and its receptor C5aR, and NF-κB. In addition, the correlation of C3a and C5a with the aforementioned inflammatory factors was analyzed. The inflammatory factor levels of C3a and C5a were considerably elevated in patients with RRDCD compared to those in the controls. Consistently, C3a and C5a treatment led to increased cell viability and aggravated inflammation in HRPE cells. Accordingly, C3a and C5a induced upregulation of their corresponding receptors C3aR and C5aR, which was in turn observed to be linked to the activation of the NF-κB signaling pathway. Furthermore, there was a positive correlation of the complements C3a and C5a with individual TNF-α, IL-1β, IL-6 and PGE2. Taken together, the C3a-C3aR and C5a-C5aR pathways were indicated to promote cell viability and inflammation of HRPE cells by targeting the NF-κB signaling pathway.

Acute myeloid leukemia (AML) is a heterogeneous group of fast growing cancers of myeloid progenitor cells, for which effective treatments are still lacking. Identification of signaling inhibitors that block their proliferation could reveal the proliferative mechanism of a given leukemia cell, and provide small molecule drugs for targeted therapy for AML. In this study, kinase inhibitors that block the majority of cancer signaling pathways are evaluated for their inhibition of two AML cell lines of the M5 subtypes, CTV-1 and THP-1. While THP-1 cells do not respond to any of these inhibitors, CTV-1 cells are potently inhibited by dasatinib, bosutinib, crizotinib, A-770041, and WH-4-23, all potent inhibitors for Lck, a Src family kinase. CTV-1 cells contain a kinase activity that phosphorylates an Lck-specific peptide substrate in an Lck inhibitor-sensitive manner. Furthermore, the Lck gene is over-expressed in CTV-1, and it contains four mutations, two of which are located in regions critical for Lck negative regulation, and are confirmed to activate Lck. Collectively, these results provide strong evidence that mutated and overexpressed Lck is driving CTV-1 proliferation. While Lck activation and overexpression is rare in AML, this study provides a potential therapeutic strategy for treating patients with a similar oncogenic mechanism.

Triple negative breast cancer is a collection of heterogeneous breast cancers that are immunohistochemically negative for estrogen receptor, progesterone receptor, and ErbB2 (due to deletion or lack of amplification). No dominant proliferative driver has been identified for this type of cancer, and effective targeted therapy is lacking. In this study, we hypothesized that triple negative breast cancer cells are multi-driver cancer cells, and evaluated a biphasic mathematical model for identifying potent and synergistic drug combinations for multi-driver cancer cells. The responses of two triple negative breast cancer cell lines, MDA-MB-231 and MDA-MB-468, to a panel of targeted therapy drugs were determined over a broad range of concentrations. The analyses of the drug responses by the biphasic mathematical model revealed that both cell lines were indeed dependent on multiple drivers, and inhibitors of individual drivers caused a biphasic response: a target-specific partial inhibition at low nM concentrations, and an off-target toxicity at &mu, M concentrations. We further demonstrated that combinations of drugs, targeting each driver, cause potent, synergistic, and cell-specific cell killing. Immunoblotting analysis of the effects of the individual drugs and drug combinations on the signaling pathways supports the above conclusion. These results support a multi-driver proliferation hypothesis for these triple negative breast cancer cells, and demonstrate the applicability of the biphasic mathematical model for identifying effective and synergistic targeted drug combinations for triple negative breast cancer cells.

Treatment of colorectal cancer mostly relies on traditional therapeutic approaches, such as surgery and chemotherapy. Limited options of targeted therapy for colorectal cancer narrowly focus on blocking cancer-generic targets VEGFR and EGFR. Identifying the oncogenic drivers, understanding their contribution to proliferation, and finding inhibitors to block such drivers are the keys to developing targeted therapy for colorectal cancer. In this study, ten colorectal cancer cell lines were screened against a panel of protein kinase inhibitors blocking key oncogenic signaling pathways. The results show that four of the 10 cell lines did not respond to any kinase inhibitors significantly, the other six were mildly inhibited by AZD-6244, BMS-754807, and/or dasatinib. Mechanistic analyses demonstrate that these inhibitors independently block the MAP kinase pathway, IR/IGF-1R/AKT pathway, and Src kinases, suggesting a multi-driver nature of proliferative signaling in these cells. Most of these cell lines were potently and synergistically inhibited by pair-wise combinations of these drugs. Furthermore, seven of the 10 cell lines were inhibited by the triple combination of AZD-6244/BMS-754807/dasatinib with IC50&rsquo, s between 10 and 84 nM. These results suggest that combination targeted therapy may be an effective strategy against colorectal cancer.

Quantifying the response of cancer cells to a drug, and understanding the mechanistic basis of the response, are the cornerstones for anti-cancer drug discovery. Classical single target-based IC50 measurements are inadequate at describing cancer cell responses to targeted drugs. In this study, based on an analysis of targeted inhibition of colorectal cancer cell lines, we develop a new biphasic mathematical model that accurately describes the cell&ndash, drug response. The model describes the drug response using three kinetic parameters: ratio of target-specific inhibition, F1, potency of target-specific inhibition, Kd1, and potency of off-target toxicity, Kd2. Determination of these kinetic parameters also provides a mechanistic basis for predicting effective combination targeted therapy for multi-driver cancer cells. The experiments confirmed that a combination of inhibitors, each blocking a driver pathway and having a distinct target-specific effect, resulted in a potent and synergistic blockade of cell viability, improving potency over mono-agent treatment by one to two orders of magnitude. We further demonstrate that mono-driver cancer cells represent a special scenario in which F1 becomes nearly 100%, and the drug response becomes monophasic. Application of this model to the responses of &gt, 400 cell lines to kinase inhibitor dasatinib revealed that the ratio of biphasic versus monophasic responses is about 4:1. This study develops a new mathematical model of quantifying cancer cell response to targeted therapy, and suggests a new framework for developing rational combination targeted therapy for colorectal and other multi-driver cancers.

To construct a prokaryotic expression plasmid of DNA G-quadruplex antibody, express it in E.coli BL21 (DE3) bacterial expression system, purify and identify the antibody.Chemically synthesized BG4 gene of DNA G-quadruplex antibodies was inserted into pSANG10 plasmid to construct DNA G-quadruplex antibody expression vector pSANG10-BG4. BL21 (DE3) as the host strain was utilized for self-induced expression of the protein. Osmotic lysis method was used for collecting this protein. Thereafter, the protein was purified by histidine tag affinity chromatography and identified by SDS-PAGE and Western blotting. The function of this protein was verified in SW480 colon cancer cells.Double enzyme digestion and gene sequencing confirmed that DNA G-quadruplex antibody expression vector was successfully constructed. The relative molecular mass (Mr) of this protein was 30 000 to 37 000. The protein in a soluble form was expressed in the periplasm of BL21. The protein was of the same size as expected.The DNA G-quadruplex antibody has been successfully prepared.