Regulation of matrix metalloproteinase-9 (MMP-9) in TNF-stimulated neutrophils: novel pathways for tertiary granule release

Matrix metalloproteinase-9 (MMP-9) is present in the tertiary granules of neutrophils and is rapidly released following stimulation. We examined the pathways that regulate tumor necrosis factor (TNF)-mediated MMP-9 release and found this to be dependent on the TNF receptor I. TNF rapidly activated extracellular signal-regulated kinase and p38 mitogen-activated protein kinases, but neither of these pathways was critical for MMP-9 release. Many neutrophil responses to TNF require beta2-integrin-dependent signaling and subsequent Src family kinase activation. In contrast, we found that MMP-9 release from tertiary granules was only partially affected by blocking beta2-integrin-mediated adhesion. Similarly, blocking Src family kinases with the inhibitor PP2 only attenuated TNF-induced MMP-9 release. Blocking beta2-integrin-mediated adhesion and Src family kinases did not result in additive inhibition of MMP-9 release. In contrast, inhibiting protein kinase C (PKC) with a pan-specific inhibitor blocked greater than 85% of MMP-9 release. Inhibitors against specific PKC isoforms suggested a role for PKC alpha and PKC delta in maximal MMP-9 release. These data suggest that MMP-9 release from tertiary granules uses beta2-integrin-independent signaling pathways. Furthermore, PKC isoforms play a critical role in regulating tertiary granule release.     

In Vivo Mutagenicity Testing of Arylboronic Acids and Esters

Arylboronic acids and esters (referred to collectively as arylboronic compounds) are commonly used intermediates in the synthesis of pharmaceuticals but pose a challenge for chemical syntheses because they are often positive for bacterial mutagenicity in vitro. As such, arylboronic compounds are then typically controlled to levels that are acceptable for mutagenic impurities, that is, the threshold of toxicological concern (TTC). This study used ICH M7 guidance to design and conduct a testing strategy to investigate the in vivo relevance of the in vitro positive findings of arylboronic compounds. Eight arylboronic compounds representing a variety of chemical scaffolds were tested in Sprague Dawley and/or Wistar rats in the in vivo Pig-a (peripheral blood reticulocytes and mature red blood cells) and/or comet assays (duodenum and/or liver). Five of the eight compounds were also tested in the micronucleus (peripheral blood) assay. The arylboronic compounds tested orally demonstrated high systemic exposure; thus the blood and bone marrow were adequately exposed to test article. One compound was administered intravenously due to formulation stability issues. This investigation showed that arylboronic compounds that were mutagenic in vitro were not found to be mutagenic in the corresponding in vivo assays. Therefore, arylboronic compounds similar to the scaffolds tested in this article may be considered non-mutagenic and managed in accordance with the ICH Q3A/Q3B guidelines. Environ. Mol. Mutagen. 2019. © 2019 Wiley Periodicals, Inc.     

Fine structure of progenitor cells in early ectopic human embryos

This study has documented the major types of lineage progenitor cells at the second level of cell differentiation after the establishment of the primary germ layers in ectopic human embryos in vivo. These correspond to stages 8 and 9 of embryogenesis (weeks 3–4) in the Carnegie collection. The aim of this study was to provide images of fine structure of tissue progenitor cells to compare them with current imaging of their equivalent stem cells identified using fluorescent stem cell markers. These include neural, mesenchymal, endodermal, ectodermal (epidermal) and haematopoietic progenitor cells, including those for amniotic, yolk sac and chorionic tissues that are used in current stem cell research. Neural induction by the notochord has been imaged. This study should give valuable clues to understand the pattern of cell differentiation of embryonic stem cells (ESC) in vitro, which are more or less mimicked in ESC colonies, embryoid bodies and neurospheres as documented in the literature.The fine structure of week-3 and week-4 human ectopic embryos is presented to demonstrate progenitor tissue cells that will eventually form the brain, spinal cord, skin, gut, heart, blood, muscle, bone and other tissues of the human body later on in development. These images should help stem cell researchers using fluorescent markers and other techniques to identify embryonic and adult stem cells in culture.     

Prevalence, mechanisms, and clinical significance of macroreentrant atrial tachycardia during and following left atrial ablation for atrial fibrillation.

OBJECTIVES: The purpose of this study was to determine the prevalence and clinical significance of macroreentrant atrial tachycardia (AT) after left atrial (LA) circumferential ablation for atrial fibrillation (AF). BACKGROUND: Linear ablation for AF may result in macroreentrant AT. METHODS: Three hundred forty-nine patients (age 54 +/- 11 years) underwent LA circumferential ablation for AF (paroxysmal in 227). Ablation lines were created around the left-sided and right-sided pulmonary veins, with additional ablation lines in the posterior LA and mitral isthmus. If macroreentrant AT was observed acutely in the electrophysiology laboratory, it was not ablated. If an organized AT occurred during follow-up, the initial strategy was rate control. If AT persisted for > 3 to 4 months, catheter ablation was performed. RESULTS: Seventy-one patients (20%) had spontaneous or induced macroreentrant AT (cycle length 244 +/- 31 ms) in the electrophysiology laboratory following LA circumferential ablation. During follow-up, 85 patients (24%) experienced spontaneous AT (cycle length 238 +/- 35 ms) at a mean of 44 +/- 62 days following LA circumferential ablation. Among the 71 patients with macroreentrant AT acutely following LA circumferential ablation, 39 (55%) developed AT during follow-up. Among the 85 patients with AT during follow-up, the tachycardia remitted without a repeat ablation procedure in 28 patients (33%), most commonly within 5 months. Twenty-eight of the 349 patients (8%) underwent a repeat ablation procedure for AT. The critical isthmus was localized to the mitral isthmus in 17 of 28 patients (61%). CONCLUSIONS: Macroreentrant AT is a common form of proarrhythmia after LA circumferential ablation for AF. Because it may resolve spontaneously, ablation of AT should be deferred for several months.     

Cell survival, cell death and cell cycle pathways are interconnected: implications for cancer therapy.

The partial cross-utilization of molecules and pathways involved in opposing processes like cell survival, proliferation and cell death, assures that mutations within one signaling cascade will also affect the other opposite process at least to some extent, thus contributing to homeostatic regulatory circuits. This review highlights some of the connections between opposite-acting pathways. Thus, we discuss the role of cyclins in the apoptotic process, and in the regulation of cell proliferation. CDKs and their inhibitors like the INK4-family (p16(Ink4a), p15(Ink4b), p18(Ink4c), p19(Ink4d)), and the Cip1/Waf1/Kip1-2-family (p21(Cip1/Waf1), p27(Kip1), p57(Kip2)) are shown both in the context of proliferation regulators and as contributors to the apoptotic machinery. Bcl2-family members (i.e. Bcl2, Bcl-X(L) Mcl-1(L); Bax, Bok/Mtd, Bak, and Bcl-X(S); Bad, Bid, Bim(EL), Bmf, Mcl-1(S)) are highlighted both for their apoptosis-regulating capacity and also for their effect on the cell cycle progression. The PI3-K/Akt cell survival pathway is shown as regulator of cell metabolism and cell survival, but examples are also provided where aberrant activity of the pathway may contribute to the induction of apoptosis. Myc/Mad/Max proteins are shown both as a powerful S-phase driving complex and as apoptosis-sensitizers. We also discuss multifunctional proteins like p53 and Rb (RBL1/p107, RBL2/p130) both in the context of G1-S transition and as apoptotic triggers. Finally, we reflect on novel therapeutic approaches that would involve redirecting over-active survival and proliferation pathways towards induction of apoptosis in cancer cells.     

Discrimination of a transformation phenotype in Syrian golden hamster embryo (SHE) cells using ATR-FTIR spectroscopy

Primary Syrian hamster embryo (SHE) cells might be used to assess morphological transformation following treatment with chemical carcinogens. We employed attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy to interrogate SHE colonies, as complex biomolecules absorb in the mid-infrared (IR; λ = 2–20 μm) giving vibrational spectra associated with structure and function. Early-passage SHE cells were cultured (pH 6.7) in the presence or absence of benzo[a]pyrene (B[a]P; 5.0 μg/ml). Unstained colonies were applied to an ATR crystal, and vibrational spectra were obtained in the ATR mode using a Bruker Vector 22 FTIR spectrometer with Helios ATR attachment. These were individually baseline-corrected and normalised. Spectra were then analysed using principal component analysis (PCA) plus linear discriminant analysis (LDA). PCA was used to reduce the dataset dimensions before LDA was employed to reveal clustering. This determined whether wavenumber–absorbance relationships expressed as single points (scores) in ‘hyperspace’ might on the basis of multivariate distance reveal biophysical differences associated with morphologies in vehicle control (non-transformed or transformed) or carcinogen-treated (non-transformed or transformed) cells. Retrospectively designated SHE colonies (following staining and microscopic analysis) clustered according to whether they were vehicle control (non-transformed), B[a]P-treated (non-transformed) or transformed (control and B[a]P-treated). Scores plots pointed to a B[a]P-treated phenotype and derived loadings plots highlighted distinguishing markers in control transformed vs. B[a]P-treated transformed; these were mostly associated with Amide I, Amide II and phosphate stretching (asymmetric and symmetric) vibrations. Combined application of ATR-FTIR spectroscopy and unsupervised (PCA)/supervised (LDA) may be a novel approach to scoring SHE colonies for morphological transformation.     

Evaluation of three fall-risk assessment tools in an acute care setting

AIM: This paper is a report of a study to evaluate the validity of three fall-risk assessment tools to identify patients at high risk for falls. BACKGROUND: Patient falls make up 38% of all adverse events occurring in hospital settings, and may result in physical injury and undesirable emotional and financial outcomes. No single fall-risk assessment tool has been conclusively validated. METHOD: The Morse Fall Scale, St Thomas Risk Assessment Tool in Falling Elderly Inpatients, and Hendrich II Fall Risk Model were validated in inter-rater reliability and validity studies in 2003. This included assessment of the probability of disagreement, kappa-values, sensitivity, specificity, positive predictive values and negative predictive values of the assessment tools with the associated 95% CI. FINDINGS: One hundred and forty-four patients were recruited for the inter-rater reliability study. The probabilities of disagreement were between 2.8% and 9.7%, and 95% CI for all tools ranged from 1.1% to 15.7%. The kappa-values were all higher than 0.80. In the validity study, 5489 patients were recruited to observe 60 falls. The Morse Fall Scale at a cutoff score of 25 and Heindrich II Fall Risk Model at a cutoff score of 5 had strong sensitivity values of 88% and 70%, respectively. However, in comparison with the Morse Fall Scale (specificity = 48.3%), only the Heindrich II Fall Risk Model had a more acceptable level of specificity (61.5%). CONCLUSION: The Heindrich II Fall Risk Model is potentially useful in identifying patients at high risk for falls in acute care facilities.