Clinical outcomes of coronavirus disease in patients with breast cancer treated with granulocyte colony-stimulating factor following chemotherapy: Triangulation of evidence using population-based cohort and Mendelian randomization analyses.

Recombinant human granulocyte colony-stimulating factor (G-CSF) administration in patients with cancer and coronavirus disease (COVID-19) remains controversial. Concerns exist that it may worsen COVID-19 outcomes by triggering an inflammatory cytokine storm, despite its common use for managing chemotherapy-induced neutropenia (CIN) or febrile neutropenia post-chemotherapy. Here, we determined whether prophylactic or therapeutic G-CSF administration following chemotherapy exacerbates COVID-19 progression to severe/critical conditions in breast cancer patients with COVID-19. Between December 2022 and February 2023, all 503 enrolled breast cancer patients had concurrent COVID-19 and received G-CSF post-chemotherapy, with most being vaccinated pre-chemotherapy. We prospectively observed COVID-19-related adverse outcomes, conducted association analyses, and subsequently performed Mendelian randomization (MR) analyses to validate the causal effect of genetically predicted G-CSF or its associated granulocyte traits on COVID-19 adverse outcomes. Only 0.99% (5/503) of breast cancer patients experienced COVID-19-related hospitalization following prophylactic or therapeutic G-CSF administration after chemotherapy. No mortality or progression to severe/critical COVID-19 occurred after G-CSF administration. Notably, no significant associations were observed between the application, dosage, or response to G-CSF and COVID-19-related hospitalization (all p >.05). Similarly, the MR analyses showed no evidence of causality of genetically predicted G-CSF or related granulocyte traits on COVID-19-related hospitalization or COVID-19 severity (all p >.05). There is insufficient evidence to substantiate the notion that the prophylactic or therapeutic administration of G-CSF after chemotherapy for managing CIN in patients with breast cancer and COVID-19 would worsen COVID-19 outcomes, leading to severe or critical conditions, or even death, especially considering the context of COVID-19 vaccination.

Chloromycetin resistance of clinically isolated E coli is conversed by using EGS technique to repress the chloromycetin acetyl transferase.

AIM: To explore the possibility of repression of chloromycetin (Cm) acyl transferase by using external guided sequence (EGS) in order to converse the clinical E coli isolates from Cm- resistant to Cm- sensitive. METHODS: EGS directed against chloromycetin acetyl transferase gene (cat) was cloned to vector pEGFP-C1 which contains the kanamycin (Km) resistance gene. The recombinant plasmid pEGFP-C1+EGScat1+cat2 was constructed and the blank vector without EGS fragment was used as control plasmids. By using the CaCl(2) transformation method, the recombinant plasmids were introduced into the clinically isolated Cm resistant but Km sensitive E coli strains. Transformants were screened on LB agar plates containing Km. Extraction of plasmids and PCR were applied to identify the positive clones. The growth curve of EGS transformed bacteria cultured in broth with Cm resistance was determined by using spectrophotometer at A(600). Drug sensitivity was tested in solid culture containing Cm by using KB method. RESULTS: Transformation studies were carried out on 16 clinically isolated Cm-resistant (250 microg/mL of Cm) E coli strains by using pEGFP-C1-EGScat1cat2 recombinant plasmid. Transformants were screened on LB-agar plates containing Km after the transformation using EGS. Of the 16 tested strains, 4 strains were transformed successfully. Transformants with EGS plasmid showed growth inhibition when grown in liquid broth culture containing 200 microg/mL of Cm. In drug sensitivity test, these strains were sensitive to Cm on LB-agar plates containing 200 microg/mL of Cm. Extraction of plasmids and PCR amplification showed the existence of EGS plasmids in these four transformed strains. These results indicated that the Cat of the four clinical isolates had been suppressed and the four strains were converted to Cm sensitive ones. CONCLUSION: The EGS directed against Cat is able to inhibit the expression of Cat, and hence convert Cm-resistant bacteria to Cm-sensitive ones. Thus, the EGS has the capability of converting the phenotype of clinical drug-resistant isolates strains to drug-sensitive ones.

[Experimental study on phenotypic conversion of clinical chloromycetin-resistant strains of E. coli to drug-sensitive strains by using EGS technique in vitro].

OBJECTIVE: To explore the possibility of phenotypic conversion of clinical chloromycetin (Cm)-resistant isolates of E.coli to drug-sensitive ones with external guide sequences (EGS) in vitro. METHODS: Recombinant EGS plasmids directed against Cm acetyl transferase (cat) and containing kanamycin (Km) drug-resistance gene and control plasmids only containing kanamycin-resistance gene without EGS were constructed. By using CaCl(2) method, the recombinant plasmids were introduced into the clinically isolated Cm-resistant E.coli strains. Extraction of plasmids and PCR were applied to identify the EGS positive clones; The growth rate in liquid broth culture of Cm-resistant bacteria after EGS containing plasmid transformation was determined by spectrophotometer A(600). Drug sensitivity was tested in solid culture by using KB method. RESULTS: Transformation studies were carried out on 16 clinically isolated Cm-resistant E.coli strains with pEGFP-C1-EGS + cat1 + cat2 recombinant plasmid. Transformants were screened on LB-agar plates containing Km after transformation using EGS. In 4 tested strains of them, transformants with specific EGS plasmid showed growth inhibition when grown in liquid broth culture containing 100 approximately 200 micro g/ml of Cm. They were sensitive to Cm on LB-agar plates containing 100 approximately 200 micro g/ml of Cm in drug-sensitivity test. Extraction of plasmids showed the existence of EGS bands. PCR amplified products of EGS. The above facts indicated that the 4 strains out of the 16 clinical isolates had been converted to drug-sensitive phenotype, and Cm-resistant clinically isolated E. coli resumed sensitivity to Cm. CONCLUSION: EGS has the capability of converting the phenotype of clinical drug-resistant isolates to drug sensitivity.

Interfering with CXCR4 expression inhibits proliferation, adhesion and migration of breast cancer MDA-MB-231 cells.

To investigate the effect and mechanism of the CXC chemokine receptor 4 (CXCR4) in the proliferation and migration of breast cancer, a short-hairpin RNA (shRNA) eukaryotic expression vector targeting CXCR4 was constructed, and the impact of such on the proliferation, adhesion and migration of human breast cancer MDA-MB-231 cells was observed. The fragments of CXCR4-shRNA were synthesized and cloned into a pGCsi-U6-Neo-green fluorescent protein vector. The recombinant plasmids were transfected into 293T cells and the most efficacious interfering vector was selected. MDA-MB-231 cells were transfected by liposome assay. The effects of silencing CXCR4 expression by shRNA on the growth, adhesion and migration of MDA-MB-231 cells were determined by Cell Counting Kit-8, cell-matrix adhesion and wound-healing assays. The shRNA eukaryotic expression vectors targeting CXCR4 (CXCR4-shRNA) were successfully constructed and transfected into 293T cells. Quantitative polymerase chain reaction and western blot analysis revealed that the maximum inhibitory rate of CXCR4 expression was 81.3%. CXCR4-shRNA transfection significantly inhibited the proliferation of MDA-MB-231 cells (P<0.05), as well as the adhesion between MDA-MB-231 cells and the extracellular matrix (P<0.05). Furthermore, wound-healing assays demonstrated that the migration distance of MDA-MB-231 cells in the CXCR4-shRNA transfection group was significantly smaller than that in the control plasmid and blank control groups (P<0.01). The CXCR4-shRNA interfering vector specifically inhibited CXCR4 expression, as well as the proliferation, adhesion and migration of MDA-MB-231 cells.

RNAi silencing of the MEKK3 gene promotes TRAIL-induced apoptosis in MCF-7 cells and suppresses the transcriptional activity of NF-κB.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family of cytokines, which can induce apoptotic cell death in a variety of tumor cells or transformed cells, yet, it is relatively non-toxic to most normal cells. Consequently, TRAIL was thought to be a promising agent for cancer therapy. However, recent research reports revealed that many tumors are unresponsive to TRAIL treatment. Apoptotic agents were identified that when used in combination with TRAIL can sensitize tumor cells to TRAIL-mediated apoptosis. It was demonstrated that MEKK3-siRNA sensitized MCF-7 cells to TRAIL cytoxicity. In addition, we investigated the discrepancy of the expression of MEKK3 in breast cancers. It was concluded that elevated MEKK3 expression is found at high frequencies in breast cancer compared to normal breast tissue. Further experiments on the signal machinery showed that MEKK3-siRNA increased the sensitivity of MCF-7 cells to TRAIL by suppressing the transcription activity of NF-κB, and enhancing the caspase-processing to generate executive apoptotic signals. These findings indicate that down-regulation of MEKK3 by siRNA approaches will lead to successful treatment of human breast cancer with TRAIL.