Differential Angiogenic Responses of Human Endothelial Colony-Forming Cells to Different Molecular Subtypes of Breast Cancer Cells

Objective@#Triple negative breast cancer (TNBC) is one subtype of breast cancer. It is characterized by lack of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2. Compared with non-TNBC, TNBC is more aggressive, of higher grade, and frequently metastatic with poor prognosis, which is correlated with upregulated microvascular density. Endothelial colony-forming cells (ECFCs) mediate neovascularization, which is the crucial contributor to cancer growth and metastasis. The present study aimed to determine whether angiogenic responses of ECFCs are regulated differently by TNBC compared with non-TNBC. @*Methods@#MDA-MB-231 and MCF7 cells were utilized for TNBC and non-TNBC, respectively.Bone-marrow-derived human ECFCs were treated with a conditioned medium (CM) of cancer cells to investigate the paracrine effect on angiogenesis. Also, ECFCs were co-cultured with cancer cells to evaluate the angiogenic effect of direct cell-to-cell interaction. Angiogenic responses of ECFCs were evaluated by proliferation, migration, and tube formation. Gene expression profiles of pro-angiogenic factors were also analyzed. @*Results@#Migration and tube formation of ECFCs were increased by treatment with CM of MDA-MB-231, which correlated with a higher gene expression profile of pro-angiogenic factors in MDA-MB-231 compared to MCF7. Interestingly, ECFCs co-cultured with MDAMB-231 showed further increase of tube formation, suggesting synergic mechanisms between the paracrine effect and direct interaction between the cells. @*Conclusion@#The angiogenic potential of ECFCs was enhanced by TNBC through both direct and indirect mechanisms. Therefore, the investigation of signaling pathways to regulate ECFC-mediated angiogenesis will be important to the discovery of anti-angiogenic therapies to treat TNBC patients.

Determination of Colistin Resistance by Simple Disk Diffusion Test Using Modified Mueller-Hinton Agar

BACKGROUND: Colistin has become a last-resort antibiotic for the management of multidrug-resistant gram-negative bacteria. The disk diffusion test is cheap and easy to perform but may be unreliable for colistin susceptibility testing due to poor diffusion of the large colistin molecule. An improved agar diffusion test would increase the reliability of colistin susceptibility testing. This study aimed to modify Muller-Hinton agar (MHA) to improve colistin diffusion in agar.METHODS: MHA was modified by reducing the agar concentration from 100% to 30% and supplementing with protamine. We tested 60 gram-negative clinical isolates of Pseudomonas aeruginosa (N=27) and Acinetobacter calcoaceticus-baumannii complex (N=33). Disk diffusion test results were interpreted based on minimum inhibitory concentrations determined by broth microdilution.RESULTS: The modified MHA yielded the best performance metrics, including 94.7% sensitivity, 100% specificity, and an area under the curve of 0.995 (95% confidence interval, 0.982–1.000), P<0.001, at a cut-off point of 13 mm.CONCLUSIONS: A reduction of the agar concentration from 100% to 30% and the addition of protamine improved colistin diffusion in agar and allowed routine colistin susceptibility testing in a clinical microbiology laboratory, but should be handled with caution.

Advanced tube formation assay using human endothelial colony forming cells for in vitro evaluation of angiogenesis

The tube formation assay is a widely used in vitro experiment model to evaluate angiogenic properties by measuring the formation of tubular structures from vascular endothelial cells (ECs). in vitro experimental results are crucial when considered the advisability of moving forward to in vivo studies. Thus, the additional attentions to the in vitro assay is necessary to improve the quality of the pre-clinical data, leading to better decision-making for successful drug discovery. In this study, we improved the tube formation assay system in three aspects. First, we used human endothelial colony forming cells (ECFCs), which are endothelial precursors that have a robust proliferative capacity and more defined angiogenic characteristics compared to mature ECs. Second, we utilized a real-time cell recorder to track the progression of tube formation for 48 hours. Third, to minimize analysis error due to the limited observation area, we used image-stitching software to increase the microscope field of view to a 2×2 stitched area from the 4× object lens. Our advanced tube formation assay system successfully demonstrated the time-dependent dynamic progression of tube formation in the presence and absence of VEGF and FGF-2. Vatalanib, VEGF inhibitor, was tested by our assay system. Of note, IC₅₀ values of vatalanib was different at each observation time point. Collectively, these results indicate that our advanced tube formation assay system replicates the dynamic progression of tube formation in response to angiogenic modulators. Therefore, this new system provides a sensitive and versatile assay model for evaluating pro- or anti-angiogenic drugs.

Clinicopathological values of NBS1 and DNA damage response genes in epithelial ovarian cancers

Epithelial ovarian cancers (EOCs) are highly lethal gynecological malignancies with a high recurrence rate. Therefore, developing prognostic markers for recurrence after chemotherapy is crucial for the treatment of ovarian cancers. As ovarian cancers frequently respond to DNA-damaging agents, we assessed the clinicopathological significance of key double-strand DNA break (DSB) repair genes, including BRCA1, BRCA2, BARD1, ATM, RAD51 and NBS1 in EOC cell lines and paraffin-embedded tissue sections from 140 EOC patients treated with cytoreductive surgery, followed by platinum-based chemotherapy. These samples were analyzed for the clinicopathological impact of DSB genes by western blot analysis, immunohistochemistry and quantitative real-time PCR. Of the DSB repair genes, BRCA1, ATM and NBS1, which are involved in the homologous recombination-mediated repair pathway, were related to aggressive parameters in EOC. When survival analysis was performed, NBS1 expression exhibited an association with EOC recurrence. Specifically, increased NBS1 expression was found in 107 out of 140 cases (76.0%) and correlated with advanced stage (P=0.001), high grade (P=0.001) and serous histology (P=0.008). The median recurrence-free survival in patients with positive and negative expression of NBS1 was 30 and 78 months, respectively (P=0.0068). In multivariate analysis, NBS1 was an independent prognostic factor for the recurrence of EOC. Together, these results suggest that NBS1 is a marker of poor prognosis for the recurrence of EOC and is associated with aggressive clinicopathological parameters.

N-acetyl-D-glucosamine kinase interacts with dynein light-chain roadblock type 1 at Golgi outposts in neuronal dendritic branch points

N-acetylglucosamine kinase (GlcNAc kinase or NAGK) is a ubiquitously expressed enzyme in mammalian cells. Recent studies have shown that NAGK has an essential structural, non-enzymatic role in the upregulation of dendritogenesis. In this study, we conducted yeast two-hybrid screening to search for NAGK-binding proteins and found a specific interaction between NAGK and dynein light-chain roadblock type 1 (DYNLRB1). Immunocytochemistry (ICC) on hippocampal neurons using antibodies against NAGK and DYNLRB1 or dynein heavy chain showed some colocalization, which was increased by treating the live cells with a crosslinker. A proximity ligation assay (PLA) of NAGK-dynein followed by tubulin ICC showed the localization of PLA signals on microtubule fibers at dendritic branch points. NAGK-dynein PLA combined with Golgi ICC showed the colocalization of PLA signals with somal Golgi facing the apical dendrite and with Golgi outposts in dendritic branch points and distensions. NAGK-Golgi PLA followed by tubulin or DYNLRB1 ICC showed that PLA signals colocalize with DYNLRB1 at dendritic branch points and at somal Golgi, indicating a tripartite interaction between NAGK, dynein and Golgi. Finally, the ectopic introduction of a small peptide derived from the C-terminal amino acids 74-96 of DYNLRB1 resulted in the stunting of hippocampal neuron dendrites in culture. Our data indicate that the NAGK-dynein-Golgi tripartite interaction at dendritic branch points functions to regulate dendritic growth and/or branching.

Glucose-Insulin-Potassium Solution Protects Ventricular Myocytes of Neonatal Rat in an In Vitro Coverslip Ischemia/Reperfusion Model

BACKGROUND AND OBJECTIVES: The benefit of high glucose-insulin-potassium (GIK) solution in clinical applications is controversial. We established a neonatal rat ventricular myocyte (NRVM) in vitro coverslip ischemia/reperfusion (I/R) model and investigated the effects of GIK solution on suppressing reactive oxygen species (ROS) and upregulating O-GlcNacylation, which protects cells from ischemic injury. MATERIALS AND METHODS: NRVMs were isolated from postnatal day 3-4 Sprague-Dawley rat pups and grown in Dulbecco's modified Eagle's medium containing high glucose (4.5 g/L), fetal bovine serum, and penicillin/streptomycin. The effects of the GIK solution on ROS production, apoptosis, and expression of O-GlcNAc and O-GlcNAc transferase (OGT) were investigated in the coverslip I/R model. RESULTS: Covering the 24-well culture plates for 3 hr with 12 mm diameter coverslips resulted in the appropriate ischemic shock. Glucose and insulin synergistically reduced ROS production, protected NRVM dose-dependently from apoptosis, and altered O-GlcNAc and OGT expression. CONCLUSION: The high GIK solution protected NRVM from I/R injury in vitro by reducing ROS and altering O-GlcNacylation.

Partial Hepatectomy in Acetylation-Deficient BubR1 Mice Corroborates that Chromosome Missegregation Initiates Tumorigenesis

BACKGROUND: Aneuploidy has been suggested as one of the major causes of cancer from the time of Boveri. In support of this notion, many studies have shown that cancer cells exhibit aneuploidy. However, there are evidences that do not support the aneuploidy hypothesis. We have previously reported that the spindle assembly checkpoint protein BubR1 is acetylated in mitosis and that the acetylation of BubR1 is crucial for checkpoint maintenance and chromosome-spindle attachment. Mice heterozygous for acetylation-deficient BubR1 (K243R/+) spontaneously develop cancer with chromosome instability. As K243R/+ mice develop hepatocellular carcinoma, we set out to test if chromosome mis-segregation was the cause of their liver cancer. METHODS: Primary hepatocytes in the regenerating liver after partial hepatectomy (PH) were analyzed and compared for various mitotic parameters. RESULTS: Primary hepatocytes isolated from K243R/+ mice after PH displayed a marked increase of chromosome misalignment, accompanied by an increase of micronuclei. In comparison, the number of nuclei per cell and the centrosome numbers were not different between wild-type and K243R/+ mice. Taken together, chromosome mis-segregation provokes tumorigenesis in mouse liver. CONCLUSION: Our results corroborate that PH provides a reliable tool for assessing mitotic infidelity and cancer in mice.

Implementation of a Next-Generation Electronic Nursing Records System Based on Detailed Clinical Models and Integration of Clinical Practice Guidelines / 대한의료정보학회지

OBJECTIVES: The purpose of this paper is to describe the components of a next-generation electronic nursing records system ensuring full semantic interoperability and integrating evidence into the nursing records system. METHODS: A next-generation electronic nursing records system based on detailed clinical models and clinical practice guidelines was developed at Seoul National University Bundang Hospital in 2013. This system has two components, a terminology server and a nursing documentation system. RESULTS: The terminology server manages nursing narratives generated from entity-attribute-value triplets of detailed clinical models using a natural language generation system. The nursing documentation system provides nurses with a set of nursing narratives arranged around the recommendations extracted from clinical practice guidelines. CONCLUSIONS: An electronic nursing records system based on detailed clinical models and clinical practice guidelines was successfully implemented in a hospital in Korea. The next-generation electronic nursing records system can support nursing practice and nursing documentation, which in turn will improve data quality.

Development of thymic lymphomas in mice disrupted of Brca2 allele in the thymus

Germ-line mutations in BRCA2 predispose to early-onset cancer. Homozygous mutant mouse, which has Brca2 truncated in exon 11 exhibit paradoxic occurrence of growth retardation and development of thymic lymphomas. However, due to its large embryonic lethality, cohort studies on the thymic lymphomas were not feasible. With the aid of Cre-loxP system, we demonstrate here that thymus-specific disruption of Brca2 allele without crossing it to p53-mutant background leads to the development of thymic lymphomas. Varying from 16 weeks to 66 weeks after birth, 25% of mice disrupted of Brca2 in the thymus died of thymic lymphomas, whereas previous report did not observe lymphomagenesis using similar Cre-loxP system. Future analysis of thymic lymphomas from these mice presented here will provide information on the cooperative mutations that are required for the BRCA2-associated pathogenesis of cancer.

Impaired phosphorylation and mis-localization of Bub1 and BubR1 are responsible for the defective mitotic checkpoint function in Brca2-mutant thymic lymphomas

Breast cancer susceptibility gene, BRCA2, is a tumor suppressor and individuals who inherit one defected copy of BRCA2 allele experience early onset breast cancer or ovarian cancer accompanied by the loss of the wild type allele. Mouse model for Brca2 mutation shows growth retardation and paradoxical occurrence of thymic lymphomas. Thymic lymphomas from Brca2-mutant mice harbor mutations in p53, Bub1, and BubR1, which function as mitotic checkpoint proteins. Therefore, interplay between Brca2 and mitotic checkpoint has been suggested in the maintenance of genetic fidelity, although it has not been assessed whether the unique mutations in Bub1 and BubR1 found in Brca2-mutant mice are responsible for the abolishment of mitotic checkpoint function. This report demonstrates that Bub1 and BubR1 mutant proteins from Brca2(-/-)thymic lymphomas have defects in the phosphorylation and kinetochore localization after spindle damage. Thus, the mutations of Bub1 and BubR1 found in Brca2- mutant mice indeed are responsible for the chromosome instability in Brca2-mutated tumors.