Housefly Phormicin inhibits Staphylococcus aureus and MRSA by disrupting biofilm formation and altering gene expression in vitro and in vivo.

The increasing drug resistance of pathogenic bacteria is a crisis that threatens public health. Antimicrobial peptides (AMPs) have been suggested to be potentially effective alternatives to solve this problem. Here, we tested housefly Phormicin-derived peptides for effects on Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA) infections in vitro and in vivo. A decreased bacterial load of MRSA was observed in the mouse scald model after treatment with Phormicin and in the positive control group (vancomycin). A mouse scrape model indicated that Phormicin helps the host fight drug-resistant MRSA infections. The protective effect of Phormicin on MRSA was confirmed in the Hermetia illucens larvae model. Phormicin also disrupted the formation of S. aureus and MRSA biofilms. Furthermore, this effect coincided with the downregulation of biofilm formation-related gene expression (agrC, sigB, RNAIII, altA, rbf, hla, hld, geh and psmɑ). Notably, virulence genes and several regulatory factors were also altered by Phormicin treatment. Based on these findings, housefly Phormicin helps the host inhibit MRSA infection through effects on biofilm formation and related gene networks. Therefore, housefly Phormicin potential represents a candidate agent for clinical MRSA chemotherapy.

Clinical Implications of Tumor-Infiltrating Immune Cells in Breast Cancer.

The immune infiltration of tumors is closely related to clinical outcomes. The composition of tumor-infiltrating immune cells (TIICs) can serve as biomarkers for predicting response to treatment and survival in different patient subgroups in terms of chemotherapy and immunotherapy. This study is focused on investigating the clinical implications of TIICs in breast cancer patients. We performed several in silico analyses of gene expression profiles in 2976 nonmetastatic tumor samples. CIBERSORT was used to estimate the proportion of 22 immune cell types to analyze their correlation with overall survival (OS) and disease-free survival (DFS) in different breast cancer subtypes and stages. Our results showed that a higher fraction of plasma cells in estrogen receptor (ER)-positive breast cancer patients indicated an increase in DFS (hazard ratio [HR]=0.66, 95% confidence interval [CI] 0.54~0.82, p<0.01), while a decreased OS was correlated with a greater number of M0 macrophages (HR=2.02, 95% CI 1.27~3.30, p=0.01) and regulatory T cells (HR=1.90, 95% CI 1.20~3.02, p=0.02). In ER-negative or progesterone receptor (PR)-negative subtypes or in a combined subtype, the increase in activated memory CD4+ T cells was correlated with increased DFS (HR=0.46, 95% CI 0.33~0.63, p<0.01). In all breast cancer patients, a higher proportion of M0 macrophages indicated a decreased DFS (HR=1.67, 95% CI 1.22~2.27, p<0.01), while increased OS was associated with relatively larger fractions of resting memory CD4+ T cells (HR=0.70, 95% CI 0.55~0.90, p=0.02) and γδ T cells (HR=0.66, 95% CI 0.51~0.85, p<0.01). Therefore, this study revealed that the composition of TIICs is different in patients with various subtypes of breast cancer and is directly related to prognosis, suggesting that TIICs are important participants in tumor progression and may, potentially be used for future diagnosis and treatment.

Caveolin-1 plays a key role in the oleanolic acid-induced apoptosis of HL-60 cells.

Our previous study found that caveolin-1 (CAV-1) protein expression is upregulated during oleanolic acid (OA)-induced inhibition of proliferation and promotion of apoptosis in HL-60 cells. CAV-1 is the main structural protein component of caveolae, playing important roles in tumorigenesis and tumor development. It has been shown that cav-1 expression is lower in leukemia cancer cell lines SUP-B15, HL-60, THP-1 and K562 and in chronic lymphocytic leukemia primary (CLP) cells when compared with normal white blood cells, with the lowest cav-1 expression level found in HL-60 cells. To study the effects of cav-1 in HL-60 cells and the effects of cav-1 overexpression on OA drug efficacy, cav-1 was overexpressed in HL-60 cells using lentiviral-mediated transfection combined with OA treatment. The results showed that cav-1 overexpression inhibited HL-60 cell proliferation, promoted apoptosis, arrested the cell cycle in the G1 phase and inhibited activation of the PI3K/AKT/mTOR signaling pathway. Overexpression of CAV-1 also increased HL-60 cell sensitivity to OA. To further verify whether OA affects HL-60 cells via the activation of downstream signaling pathways by CAV-1, cav-1 gene expression was silenced using RNAi, and the cells were treated with OA to examine its efficacy. The results showed that after cav-1 silencing, OA had little effect on cell activity, apoptosis, the cell cycle and phosphorylation of HL-60 cells. This study is the first to show that CAV-1 plays a crucial role in the effects of OA on HL-60 cells.

[Effect of overexpression of CAV1 mediated by lentivirus on proliferation and apoptosis of HL-60 cells].

This study was purposed to explore the effect of lentivirus-mediated CAV1 overexpression on proliferation and apoptosis in HL-60 cells. Recombinant lentiviral expression vector pcDNA-EF1-CAV1 was constructed, and cotransfected the 293TN cells with a mixture of pPACK packaging plasmids. Then collecting virus suspension infects the HL-60 cells, which make CAV1 gene stable transfection and high expression in the cells. The CAV1 protein expression status in HL-60 cells transfected was evaluated through Western blot method. Proliferative activity and apoptosis of HL-60 cells before and after transfection were detected by CCK-8 method and flow cytometry, respectively. The results showed that the PCR-positive clone screening and results of nucleotide sequencing confirmed that the CAV1 gene inserted into the expression vector pcDNA-EF1-GFP correctly, recombinant lentiviral particles Lv-CAV1 transfected HL-60 cells successfully and with transfection rate up to 90%. The result of Western blot showed that CAV1 protein expression in HL-60 cells significantly increased at 48 hours after transfection. CCK-8 result indicated that cell proliferation activity increased at 48 h after transfection (P < 0.05), flow cytometry testing results displayed that apoptosis rate of HL-60 cells obviously decreased after transfection (P < 0.01). It is concluded that the overexpression of CAV1 in HL-60 cells can inhibit cell proliferation activity and promote cell apoptosis.

[The synthesis and function analysis of omega-3 fatty acid desaturase gene from Caenorhabditis briggssae].

Omega-3 polyunsaturated fatty acids (PUFAs) have been broadly investigated and shown to exert many preventive and therapeutic actions besides their important role in maintenances human health and normal development. In mammals, the level of omega-3 PUFAs is relatively too low compared with omega-6 PUFAs, which metabolically and functionally distinct from omega-3 PUFAs and often have important opposing physiological functions. Either the inefficiency of omega-3 PUFAs or the excess of omega-6 PUFAs will cause many healthy problems. So methods have been sought to increase the amount of omega-3 PUFAs and to improve the omega-6/omega-3 ratio in body. In this study, the sFat-1 gene, which putatively encodes a omega-3 fatty acid desaturase, was chemically synthesized according to the sequence from Caenorhabditis briggssae (with codon usage modified), and constructed into a mammal expression vector pcDNA3. 1-sFat1-EGFP. This vector was introduced into CHO cells by lipid-mediated transfection, and it's expression quickly and effectively elevated the cellular omega-3 PUFAs (from 18-carbon to 22-carbon) contents and dramatically improved the ratio of omega-6/omega-3 PUFAs. Cellular lipids extracts from stably selected cells were analyzed with GC-MS and the results showed that amount of total omega-6 PUFAs dropped from 48.97% (in GFP cells)to 35.29% (in sFat-1 cells), whereas the amount of total omega-3 PUFAs increased from 7.86% to 24.02%, respectively. The omega-6/omega-3 ratio also dropped from 6.23 to 1.47. These data demonstrates the Caenorhabditis briggssae omega-3 Fatty Acid Desaturase gene, sFat-1, was synthesized successfully and can produce omega-3 PUFAs by using the corresponding omega-6 PUFAs as substrates, which shows its potential for use in the production of omega-3 PUFAs in transgenic animals.

[Synthesis of total sFat-1 gene by PCR-restrict enzyme ligation method].

Gene synthesis is very important in life science research, and it becomes a technique in common use. It is difficult for long gene synthesis, because the mismatches and mutations of DNA sequence in nucleotide fragments assembling. This study established a new method for long gene synthesis, which was referred to as PCR-restrict enzyme ligation method. With this method, a omega-3 fatty acid desaturase gene, sFat-1, from Caenorhabditis briggssae, was successfully assembled from 27 synthesized nucleotide fragments (60 ~ 68 bp for each fragment ) following 3 rounds of PCR (7 reactions) and 2 rounds of restrict enzyme ligation (3 reactions). This shows that the PCR-restrict enzyme ligation method is an effective method for long gene synthesis.