Two Major Medicinal Honeys Have Different Mechanisms of Bactericidal Activity

Honey is increasingly valued for its antibacterial activity, but knowledge regarding the mechanism of action is still incomplete. We assessed the bactericidal activity and mechanism of action of Revamil® source (RS) honey and manuka honey, the sources of two major medical-grade honeys. RS honey killed Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa within 2 hours, whereas manuka honey had such rapid activity only against B. subtilis. After 24 hours of incubation, both honeys killed all tested bacteria, including methicillin-resistant Staphylococcus aureus, but manuka honey retained activity up to higher dilutions than RS honey. Bee defensin-1 and H2O2 were the major factors involved in rapid bactericidal activity of RS honey. These factors were absent in manuka honey, but this honey contained 44-fold higher concentrations of methylglyoxal than RS honey. Methylglyoxal was a major bactericidal factor in manuka honey, but after neutralization of this compound manuka honey retained bactericidal activity due to several unknown factors. RS and manuka honey have highly distinct compositions of bactericidal factors, resulting in large differences in bactericidal activity

Structural and functional studies of enzymes in nucleotide metabolism

Enzymes in nucleotide metabolism serve as the producers of the building blocks for DNA and RNA. From a medical perspective, nucleotide metabolism, and in particular salvage pathway enzymes, have attracted special interest, as nucleoside prodrugs given in the treatment of cancer and HIV are converted into their active metabolite forms by these enzymes. In this thesis, two enzymes; uridine monophosphate kinase (UMPK) from Ureaplasma parvum (Up) and human phosphoribosyltransferase domain containing protein 1 (PRTFDC1), have been investigated. Furthermore, a nucleoside analog library (NAL) consisting of 45 FDA-approved nucleoside analogs has been developed. The structure of Up-UMPK revealed that it was a hexamer. Kinetic constants were determined for UMP and ATP. UTP was a competitive inhibitor of UMP, and a non-competitive inhibitor of ATP. In contrast to other bacterial UMPKs, Up-UMPK was not activated by GTP. PRTFDC1 is a homolog of hypoxanthine-guanine phosphoribosyltransferase (HPRT). Mutations in HPRT are associated with Lesch-Nyhan syndrome. The three-dimensional structures of PRTFDC1 and HRPT are very similar. Even though PRTFDC1 recognizes guanine and hypoxanthine as substrates, the functional turnover rates are less than 1% of the activity of HPRT. NAL was screened using the high-throughput method, differential static light scattering (DSLS). An interaction profile of 23 enzymes involved in nucleotide metabolism and NAL was revealed. Interactions were detected for uridine phosphorylase 1 (UPP1) and guanine deaminase (GDA) with eight and six nucleoside prodrugs, respectively. The knowledge gained from this study can be important in the future search for drug lead candidates for UPP1 and GDA

System-wide analysis reveals a complex network of tumor-fibroblast interactions involved in tumorigenicity

Many fibroblast-secreted proteins promote tumorigenicity, and several factors secreted by cancer cells have in turn been proposed to induce these proteins. It is not clear whether there are single dominant pathways underlying these interactions or whether they involve multiple pathways acting in parallel. Here, we identified 42 fibroblast-secreted factors induced by breast cancer cells using comparative genomic analysis. To determine what fraction was active in promoting tumorigenicity, we chose five representative fibroblast-secreted factors for in vivo analysis. We found that the majority (three out of five) played equally major roles in promoting tumorigenicity, and intriguingly, each one had distinct effects on the tumor microenvironment. Specifically, fibroblast-secreted amphiregulin promoted breast cancer cell survival, whereas the chemokine CCL7 stimulated tumor cell proliferation while CCL2 promoted innate immune cell infiltration and angiogenesis. The other two factors tested had minor (CCL8) or minimally (STC1) significant effects on the ability of fibroblasts to promote tumor growth. The importance of parallel interactions between fibroblasts and cancer cells was tested by simultaneously targeting fibroblast-secreted amphiregulin and the CCL7 receptor on cancer cells, and this was significantly more efficacious than blocking either pathway alone. We further explored the concept of parallel interactions by testing the extent to which induction of critical fibroblast-secreted proteins could be achieved by single, previously identified, factors produced by breast cancer cells. We found that although single factors could induce a subset of genes, even combinations of factors failed to induce the full repertoire of functionally important fibroblast-secreted proteins. Together, these results delineate a complex network of tumor-fibroblast interactions that act in parallel to promote tumorigenicity and suggest that effective anti-stromal therapeutic strategies will need to be multi-targeted

Matrix Metalloproteinase 13 Is Induced in Fibroblasts in Polyomavirus Middle T Antigen-Driven Mammary Carcinoma without Influencing Tumor Progression

Matrix metalloproteinase (MMP) 13 (collagenase 3) is an extracellular matrix remodeling enzyme that is induced in myofibroblasts during the earliest invasive stages of human breast carcinoma, suggesting that it is involved in tumor progression. During progression of mammary carcinomas in the polyoma virus middle T oncogene mouse model (MMTV-PyMT), Mmp13 mRNA was strongly upregulated concurrently with the transition to invasive and metastatic carcinomas. As in human tumors, Mmp13 mRNA was found in myofibroblasts of invasive grade II and III carcinomas, but not in benign grade I and II mammary intraepithelial neoplasias. To determine if MMP13 plays a role in tumor progression, we crossed MMTV-PyMT mice with Mmp13 deficient mice. The absence of MMP13 did not influence tumor growth, vascularization, progression to more advanced tumor stages, or metastasis to the lungs, and the absence of MMP13 was not compensated for by expression of other MMPs or tissue inhibitor of metalloproteinases. However, an increased fraction of thin collagen fibrils was identified in MMTV-PyMT;Mmp13−/− compared to MMTV-PyMT;Mmp13+/+ tumors, showing that collagen metabolism was altered in the absence of MMP13. We conclude that the expression pattern of Mmp13 mRNA in myofibroblasts of invasive carcinomas in the MMTV-PyMT breast cancer model recapitulates the expression pattern observed in human breast cancer. Our results suggest that MMP13 is a marker of carcinoma-associated myofibroblasts of invasive carcinoma, even though it does not make a major contribution to tumor progression in the MMTV-PyMT breast cancer model

Isolation of mouse mammary carcinoma-derived macrophages and cancer cells for co-culture assays

We recently established an in vitro co-culture system in which monophosphoryl lipid A + interferon-γ (MPLA+IFNγ)-treated tumor-associated macrophages (TAMs) killed cancer cells. Here, we describe a step-by-step protocol for isolating TAMs and cancer cells from mouse primary mammary carcinomas, the setup of the co-culture system, and the image acquisition approach. The technical difficulties in the co-culture assay involve isolating pure TAMs and cancer cells from the same tumor and staining them with different dyes to track the macrophages’ tumoricidal activity. For complete details on the use and execution of this protocol, please refer to Sun et al. (2021).(1

Tissue doppler imaging predicts improved systolic performance and reversed left ventricular remodeling during long-term cardiac resynchronization therapy

AbstractObjectivesWe sought to evaluate the long-term impact of cardiac resynchronization therapy (CRT) on left ventricular (LV) performance and remodeling using three-dimensional echocardiography and tissue Doppler imaging (TDI).BackgroundThree-dimensional echocardiography and TDI allow rapid and accurate evaluation of LV volumes and performance.MethodsTwenty-five consecutive patients with severe heart failure and bundle branch block who underwent biventricular pacemaker implantation were included. Before and after implantation of the pacemaker, three-dimensional echocardiography and TDI were performed. These examinations were repeated at outpatient visits every six months.ResultsFive patients (20%) died during one-year follow-up. In the remaining 20 patients, significant reductions in LV end-diastolic volume and LV end-systolic volume of 9.6 ± 14% and 16.5 ± 15%, respectively (p < 0.01), could be demonstrated during long-term follow-up. Accordingly, LV ejection fraction increased by 21.7 ± 18% (p < 0.01). According to a newly developed TDI technique—tissue tracking—all regional myocardial segments improved their longitudinal systolic shortening (p < 0.01). The extent of the LV base displaying delayed longitudinal contraction, as detected by TDI before pacemaker implantation, predicted long-term efficacy of CRT. The QRS duration failed to predict resynchronization efficacy.ConclusionsCardiac resynchronization significantly improved LV function and reversed LV remodeling during long-term follow-up. Patients likely to benefit from CRT can be identified by TDI before implantation of a biventricular pacemaker

Visualizing stromal cell dynamics in different tumor microenvironments by spinning disk confocal microscopy

The tumor microenvironment consists of stromal cells and extracellular factors that evolve in parallel with carcinoma cells. To gain insights into the activities of stromal cell populations, we developed and applied multicolor imaging techniques to analyze the behavior of these cells within different tumor microenvironments in the same live mouse. We found that regulatory T-lymphocytes (Tregs) migrated in proximity to blood vessels. Dendriticlike cells, myeloid cells and carcinoma-associated fibroblasts all exhibited higher motility in the microenvironment at the tumor periphery than within the tumor mass. Since oxygen levels differ between tumor microenvironments, we tested if acute hypoxia could account for the differences in cell migration. Direct visualization revealed that Tregs ceased migration under acute systemic hypoxia, whereas myeloid cells continued migrating. In the same mouse and microenvironment, we experimentally subdivided the myeloid cell population and revealed that uptake of fluorescent dextran defined a low-motility subpopulation expressing markers of tumor-promoting, alternatively activated macrophages. In contrast, fluorescent anti-Gr1 antibodies marked myeloid cells patrolling inside tumor vessels and in the stroma. Our techniques allow real-time combinatorial analysis of cell populations based on spatial location, gene expression, behavior and cell surface molecules within intact tumors. The techniques are not limited to investigations in cancer, but could give new insights into cell behavior more broadly in development and disease