Cytoplasmic accumulation and plasma membrane association of anillin and Ect2 promote confined migration and invasion.

Cells migrating in confinement experience mechanical challenges whose consequences on cell migration machinery remain only partially understood. Here, we demonstrate that a pool of the cytokinesis regulatory protein anillin is retained during interphase in the cytoplasm of different cell types. Confinement induces recruitment of cytoplasmic anillin to plasma membrane at the poles of migrating cells, which is further enhanced upon nuclear envelope (NE) rupture(s). Rupture events also enable the cytoplasmic egress of predominantly nuclear RhoGEF Ect2. Anillin and Ect2 redistributions scale with microenvironmental stiffness and confinement, and are observed in confined cells in vitro and in invading tumor cells in vivo. Anillin, which binds actomyosin at the cell poles, and Ect2, which activates RhoA, cooperate additively to promote myosin II contractility, and promote efficient invasion and extravasation. Overall, our work provides a mechanistic understanding of how cytokinesis regulators mediate RhoA/ROCK/myosin II-dependent mechanoadaptation during confined migration and invasive cancer progression.

Cells in the polyaneuploid cancer cell (PACC) state have increased metastatic potential.

Although metastasis is the leading cause of cancer deaths, it is quite rare at the cellular level. Only a rare subset of cancer cells (~ 1 in 1.5 billion) can complete the entire metastatic cascade: invasion, intravasation, survival in the circulation, extravasation, and colonization (i.e. are metastasis competent). We propose that cells engaging a Polyaneuploid Cancer Cell (PACC) phenotype are metastasis competent. Cells in the PACC state are enlarged, endocycling (i.e. non-dividing) cells with increased genomic content that form in response to stress. Single-cell tracking using time lapse microscopy reveals that PACC state cells have increased motility. Additionally, cells in the PACC state exhibit increased capacity for environment-sensing and directional migration in chemotactic environments, predicting successful invasion. Magnetic Twisting Cytometry and Atomic Force Microscopy reveal that cells in the PACC state display hyper-elastic properties like increased peripheral deformability and maintained peri-nuclear cortical integrity that predict successful intravasation and extravasation. Furthermore, four orthogonal methods reveal that cells in the PACC state have increased expression of vimentin, a hyper-elastic biomolecule known to modulate biomechanical properties and induce mesenchymal-like motility. Taken together, these data indicate that cells in the PACC state have increased metastatic potential and are worthy of further in vivo analysis.

OBSCN restoration via OBSCN-AS1 long-noncoding RNA CRISPR-targeting suppresses metastasis in triple-negative breast cancer.

Mounting evidence implicates the giant, cytoskeletal protein obscurin (720 to 870 kDa), encoded by the OBSCN gene, in the predisposition and development of breast cancer. Accordingly, prior work has shown that the sole loss of OBSCN from normal breast epithelial cells increases survival and chemoresistance, induces cytoskeletal alterations, enhances cell migration and invasion, and promotes metastasis in the presence of oncogenic KRAS. Consistent with these observations, analysis of Kaplan-Meier Plotter datasets reveals that low OBSCN levels correlate with significantly reduced overall and relapse-free survival in breast cancer patients. Despite the compelling evidence implicating OBSCN loss in breast tumorigenesis and progression, its regulation remains elusive, limiting any efforts to restore its expression, a major challenge given its molecular complexity and gigantic size (~170 kb). Herein, we show that OBSCN-Antisense RNA 1 (OBSCN-AS1), a novel nuclear long-noncoding RNA (lncRNA) gene originating from the minus strand of OBSCN, and OBSCN display positively correlated expression and are downregulated in breast cancer biopsies. OBSCN-AS1 regulates OBSCN expression through chromatin remodeling involving H3 lysine 4 trimethylation enrichment, associated with open chromatin conformation, and RNA polymerase II recruitment. CRISPR-activation of OBSCN-AS1 in triple-negative breast cancer cells effectively and specifically restores OBSCN expression and markedly suppresses cell migration, invasion, and dissemination from three-dimensional spheroids in vitro and metastasis in vivo. Collectively, these results reveal the previously unknown regulation of OBSCN by an antisense lncRNA and the metastasis suppressor function of the OBSCN-AS1/OBSCN gene pair, which may be used as prognostic biomarkers and/or therapeutic targets for metastatic breast cancer.

Monoclonal antibody Y01 prevents tauopathy progression induced by lysine 280-acetylated tau in cell and mouse models.

The spatiotemporal pattern of the spread of pathologically modified tau through brain regions in Alzheimer's disease (AD) can be explained by prion-like cell-to-cell seeding and propagation of misfolded tau aggregates. Hence, to develop targeted therapeutic antibodies, it is important to identify the seeding- and propagation-competent tau species. The hexapeptide 275VQIINK280 of tau is a critical region for tau aggregation, and K280 is acetylated in various tauopathies, including AD. However, the mechanism that links tau acetylated on lysine 280 (tau-acK280) to subsequent progression to neurodegenerative disease remains unclear. Here, we demonstrate that tau-acK280 is critical for tau propagation processes including secretion, aggregation, and seeding. We developed an antibody, Y01, that specifically targets tau-acK280 and solved the crystal structure of Y01 in complex with an acK280 peptide. The structure confirmed that Y01 directly recognizes acK280 and the surrounding residues. Strikingly, upon interaction with acetylated tau aggregates, Y01 prevented tauopathy progression and increased neuronal viability in neuron cultures and in tau-Tg mice through antibody-mediated neutralization and phagocytosis, respectively. Based on our observations that tau-acK280 is a core species involved in seeding and propagation activities, the Y01 antibody that specifically recognizes acK280 represents a promising therapeutic candidate for AD and other neurodegenerative diseases associated with tauopathy.

Derivation of site-specific derived concentration guideline levels at Korea Research Reactor 1 and 2 sites using probabilistic analysis.

The purpose of this study is to apply a probabilistic method to derive the derived concentration guideline levels for decommissioning of Korea Research Reactor 1 and 2. A total of seven parameters were found to be the sensitive parameters of the target nuclides. The DCGLs of Co-60 and H-3 were 0.063 Bq/g and 85.470 Bq/g, respectively. The concentrations of the gamma ray-emitting nuclides in the actual reactor sites were 7.7-215 times lower than the derived DCGLs for gamma ray-emitting nuclides.

Cytokinesis machinery promotes cell dissociation from collectively migrating strands in confinement.

Cells tune adherens junction dynamics to regulate epithelial integrity in diverse (patho)physiological processes, including cancer metastasis. We hypothesized that the spatially confining architecture of peritumor stroma promotes metastatic cell dissemination by remodeling cell-cell adhesive interactions. By combining microfluidics with live-cell imaging, FLIM/FRET biosensors, and optogenetic tools, we show that confinement induces leader cell dissociation from cohesive ensembles. Cell dissociation is triggered by myosin IIA (MIIA) dismantling of E-cadherin cell-cell junctions, as recapitulated by a mathematical model. Elevated MIIA contractility is controlled by RhoA/ROCK activation, which requires distinct guanine nucleotide exchange factors (GEFs). Confinement activates RhoA via nucleocytoplasmic shuttling of the cytokinesis-regulatory proteins RacGAP1 and Ect2 and increased microtubule dynamics, which results in the release of active GEF-H1. Thus, confining microenvironments are sufficient to induce cell dissemination from primary tumors by remodeling E-cadherin cell junctions via the interplay of microtubules, nuclear trafficking, and RhoA/ROCK/MIIA pathway and not by down-regulating E-cadherin expression.

Extracellular fluid viscosity enhances cell migration and cancer dissemination.

Cells respond to physical stimuli, such as stiffness1, fluid shear stress2 and hydraulic pressure3,4. Extracellular fluid viscosity is a key physical cue that varies under physiological and pathological conditions, such as cancer5. However, its influence on cancer biology and the mechanism by which cells sense and respond to changes in viscosity are unknown. Here we demonstrate that elevated viscosity counterintuitively increases the motility of various cell types on two-dimensional surfaces and in confinement, and increases cell dissemination from three-dimensional tumour spheroids. Increased mechanical loading imposed by elevated viscosity induces an actin-related protein 2/3 (ARP2/3)-complex-dependent dense actin network, which enhances Na+/H+ exchanger 1 (NHE1) polarization through its actin-binding partner ezrin. NHE1 promotes cell swelling and increased membrane tension, which, in turn, activates transient receptor potential cation vanilloid 4 (TRPV4) and mediates calcium influx, leading to increased RHOA-dependent cell contractility. The coordinated action of actin remodelling/dynamics, NHE1-mediated swelling and RHOA-based contractility facilitates enhanced motility at elevated viscosities. Breast cancer cells pre-exposed to elevated viscosity acquire TRPV4-dependent mechanical memory through transcriptional control of the Hippo pathway, leading to increased migration in zebrafish, extravasation in chick embryos and lung colonization in mice. Cumulatively, extracellular viscosity is a physical cue that regulates both short- and long-term cellular processes with pathophysiological relevance to cancer biology.

Polarized NHE1 and SWELL1 regulate migration direction, efficiency and metastasis.

Cell migration regulates diverse (patho)physiological processes, including cancer metastasis. According to the Osmotic Engine Model, polarization of NHE1 at the leading edge of confined cells facilitates water uptake, cell protrusion and motility. The physiological relevance of the Osmotic Engine Model and the identity of molecules mediating cell rear shrinkage remain elusive. Here, we demonstrate that NHE1 and SWELL1 preferentially polarize at the cell leading and trailing edges, respectively, mediate cell volume regulation, cell dissemination from spheroids and confined migration. SWELL1 polarization confers migration direction and efficiency, as predicted mathematically and determined experimentally via optogenetic spatiotemporal regulation. Optogenetic RhoA activation at the cell front triggers SWELL1 re-distribution and migration direction reversal in SWELL1-expressing, but not SWELL1-knockdown, cells. Efficient cell reversal also requires Cdc42, which controls NHE1 repolarization. Dual NHE1/SWELL1 knockdown inhibits breast cancer cell extravasation and metastasis in vivo, thereby illustrating the physiological significance of the Osmotic Engine Model.

Giant obscurin regulates migration and metastasis via RhoA-dependent cytoskeletal remodeling in pancreatic cancer.

Obscurins, encoded by the OBSCN gene, are giant cytoskeletal proteins with structural and regulatory roles. Large scale omics analyses reveal that OBSCN is highly mutated across different types of cancer, exhibiting a 5-8% mutation frequency in pancreatic cancer. Yet, the functional role of OBSCN in pancreatic cancer progression and metastasis has to be delineated. We herein show that giant obscurins are highly expressed in normal pancreatic tissues, but their levels are markedly reduced in pancreatic ductal adenocarcinomas. Silencing of giant obscurins in non-tumorigenic Human Pancreatic Ductal Epithelial (HPDE) cells and obscurin-expressing Panc5.04 pancreatic cancer cells induces an elongated, spindle-like morphology and faster cell migration via cytoskeletal remodeling. Specifically, depletion of giant obscurins downregulates RhoA activity, which in turn results in reduced focal adhesion density, increased microtubule growth rate and faster actin dynamics. Although OBSCN knockdown is not sufficient to induce de novo tumorigenesis, it potentiates tumor growth in a subcutaneous implantation model and exacerbates metastasis in a hemispleen murine model of pancreatic cancer metastasis, thereby shortening survival. Collectively, these findings reveal a critical role of giant obscurins as tumor suppressors in normal pancreatic epithelium whose loss of function induces RhoA-dependent cytoskeletal remodeling, and promotes cell migration, tumor growth and metastasis.

Cancer cells display increased migration and deformability in pace with metastatic progression.

In this study, we explored the relation between metastatic states vs the capacity of confined migration, amoeboid transition, and cellular stiffness. We compared across an isogenic panel of human breast cancer cells derived from MDA-MB-231 cells. It was observed that cells after lung metastasis have the fastest migration and lowest stiffness, with a significantly higher capacity to transition into an amoeboid mode. Our findings illustrate that metastasis is a selective process favoring motile and softer cells. Moreover, the observation that circulating tumor cells resemble the parental cell line, but not lung-metastatic cells, suggests that cells with higher deformability and motility are likely selected during extravasation and colonization.

TLQP-21 mediated activation of microglial BV2 cells promotes clearance of extracellular fibril amyloid-ß.

ß-Amyloid (Aß) plaque in the brains of patients with Alzheimer's disease (AD) is mainly caused by impaired clearance of Aß by glial cells, including microglia and astrocytes. Because microglia play an important protective role in the central nervous system, many efforts have been made to identify agents that effectively improve microglial Aß phagocytosis. This study found that TLQP-21, which is cleaved from VGF (VGF nerve growth factor inducible) precursor protein, enhanced Aß phagocytosis and degradation by microglial BV2 cells. TLQP-21 also improved microglial phagocytic activity and promoted fibrillar amyloid-ß (fAß) uptake by microglial BV2 cells via a C3AR1-dependent mechanism. Moreover, TLQP-21 stimulated Aß degradation by enhancing lysosome activity, thereby enhancing fAß clearance. These results suggest that treatment with TLQP-21 may be a novel therapeutic strategy to efficiently enhance microglial Aß clearance in AD.

A feasibility study for the continuous measurement of pupillary response using the pupillography during CPR in out-of-hospital cardiac arrest patients.

PURPOSE: We investigated the change in pupil size and pupil light reflex (PLR) using a pupillography capable of continuous measurement both during CPR and immediately following the return-of-spontaneous circulation (ROSC) in out-of-hospital cardiac arrest (OHCA) comatose patients in an emergency department. METHODS: Pupil size and PLR were continuously measured both during CPR and immediately following ROSC until intensive care unit (ICU) admission. The changes in pupil sizes during CPR were categorized into three groups (no change - N, decreased - D, and increased - I groups). RESULTS: Pupillography was applied for 118 and 60 patients during CPR and immediately following ROSC, respectively. Only two patients had a PLR during CPR. The number of patients included each group were 58 (N-group), 21 (D-group) and 39 (I-group). In the D-group, the proportion of witnessed arrest was higher than in the N-group and I-group (81% vs. 55% and 49%, respectively; p = 0.049). There were statistically significant shorter prehospital time in the D-group than the N-group and I-group (13 vs. 23 and 24 min, respectively; p = 0.012). PLR was observed immediately following ROSC in 14 patients. PLR was maintained in seven of these patients until admission to intensive care unit. Six of the seven patients who remained with PLR until ICU admission had survival to hospital discharge, and three of them had good neurological recovery. CONCLUSION: Our study demonstrated that measurement of the continuous pupillary response can be feasible. Patients with the presence of PLR following ROSC had better outcomes.

Clinical Effects of Activated Charcoal Unavailability on Treatment Outcomes for Oral Drug Poisoned Patients.

BACKGROUND: Activated charcoal is the most frequently and widely used oral decontaminating agent in emergency departments (EDs). However, there is some debate about its clinical benefits and risks. In Korea, activated charcoal with sorbitol was unavailable as of the mid-2015, and our hospital had been unable to use it from September 2015. This study examined the differences of clinical features and outcomes of patients during the periods charcoal was and was not available. METHODS: We retrospectively reviewed the electronic medical records of patients who had visited an urban tertiary academic ED for oral drug poisoning between January 2013 and January 2017. RESULTS: For the charcoal-available period, 413 patients were identified and for the charcoal-unavailable period, 221. Activated charcoal was used in the treatment of 141 patients (34%) during the available period. The mortality rates during the available and unavailable periods were 1.9 and 0.9%, respectively (p = 0.507). There was also no interperiod difference in the development of aspiration pneumonia (9.9 versus 9.5%, p = 0.864), the endotracheal intubation rate (8.4 versus 7.2%, p = 0.586), and vasopressor use (5.3 versus 5.0%, p = 0.85). Intensive care unit (ICU) admission was higher in the unavailable period (5.8 versus 13.6%, p = 0.001). ICU days were lower in the unavailable period (10 [4.5-19] versus 4 [3-9], p = 0.01). Hospital admission (43.3 versus 29.9%, p = 0.001) was lower in the unavailable period. CONCLUSIONS: In this single center study, there appeared to be no difference in mortality, intubation rates, or vasopressor use between the charcoal-available and charcoal-unavailable periods.

Initial blood pH during cardiopulmonary resuscitation in out-of-hospital cardiac arrest patients: a multicenter observational registry-based study.

BACKGROUND: When an out-of-hospital cardiac arrest (OHCA) patient receives cardiopulmonary resuscitation (CPR) in the emergency department (ED), blood laboratory test results can be obtained by using point-of-care testing during CPR. In the present study, the relationship between blood laboratory test results during CPR and outcomes of OHCA patients was investigated. METHODS: This study was a multicenter retrospective analysis of prospective registered data that included 2716 OHCA patients. Data from the EDs of three university hospitals in different areas were collected from January 2009 to December 2014. Univariate and multivariable analyses were conducted to elucidate the factors associated with survival to discharge and neurological outcomes. A final analysis was conducted by including patients who had no prehospital return of spontaneous circulation and those who underwent rapid blood laboratory examination during CPR. RESULTS: Overall, 2229 OHCA patients were included in the final analysis. Among them, the rate of survival to discharge and a good Cerebral Performance Categories Scale score were 14% and 4.4%, respectively. The pH level was independently related to survival to hospital discharge (adjusted OR 6.287, 95% CI 2.601-15.197; p < 0.001) and good neurological recovery (adjusted OR 15.395, 95% CI 3.439-68.911; p < 0.001). None of the neurologically intact patients had low pH levels (< 6.8) or excessive potassium levels (> 8.5 mEq/L) during CPR. CONCLUSIONS: Among the blood laboratory test results during CPR of OHCA patients, pH and potassium levels were observed as independent factors associated with survival to hospital discharge, and pH level was considered as an independent factor related to neurological recovery.

Prehospital Supraglottic Airway Was Associated With Good Neurologic Outcome in Cardiac Arrest Victims Especially Those Who Received Prolonged Cardiopulmonary Resuscitation.

OBJECTIVES: We performed this study to investigate the association of prehospital supraglottic airway (SGA) on neurologic outcome in cardiac arrest victims with adjustment of postresuscitation variables as well as prehospital and resuscitation variables. METHODS: This study was a retrospective study based on a multicenter prospective cohort registry from December 2013 to April 2016. According to the 28-day cerebral performance categories (CPCs) scale, patients were divided into the good-outcome group (CPC 1-2) and the poor-outcome group (CPC 3-5). We compared the two groups with respect to demographic variables, prehospital and in-hospital resuscitation variables, and postresuscitation variables. RESULTS: A total of 869 cardiac arrest victims who received in-progress cardiopulmonary resuscitation (CPR) were delivered to the emergency department of three hospitals, and 310 patients were admitted to the intensive care unit. The use of a prehospital SGA was independently associated with 28-day good neurologic outcome (odds ratio [OR] = 7.88; 95% confidence interval [CI] = 1.33-46.53; p = 0.023] when postresuscitation variables were adjusted, although there were no significant association with the acquisition of sustained return of spontaneous circulation (OR = 0.992; 95% CI = 0.591-1.666; p = 0.976). Furthermore, a prehospital SGA was significantly associated with good neurologic outcome, especially in patients who received prolonged CPR (low flow time > 15 minutes; OR = 3.41; 95% CI = 1.23-9.45; p = 0.018) rather than in patients with nonprolonged CPR (OR = 4.50; 95% CI = 0.75-27.13; p = 0.101). CONCLUSIONS: When postresuscitation variables were adjusted, the prehospital SGA was independently associated with 28-day good neurologic outcome in cardiac arrest victims.

Incidence and clinical features of intracranial hemorrhage causing out-of-hospital cardiac arrest: a multicenter retrospective study.

OBJECTIVE: The general incidence of intracranial hemorrhage (ICH) as a cause of out-of-hospital cardiac arrest (OHCA) remains unclear, although the incidence of subarachnoid hemorrhage has been determined to be 4% to 18%. The main objectives of our study were to describe the incidence of ICH in OHCA and the different laboratory findings between ICH and non-ICH groups. METHODS: A retrospective cohort study using the prospective OHCA registry was conducted at three university hospitals in Korea. All cases of OHCA that occurred over a period of 6 years, from January 2009 to December 2014, were examined. Pre-hospital and in-hospital variables and laboratory data taken during CPR were examined in order to compare the ICH and non-ICH groups. RESULTS: A total of 2716 patients with OHCA were registered in the database. Among the 804 patients included in the final analysis, ICH was the cause of cardiac arrest in 92 patients (11.4%). Of those with ICH, 79 (86%) patients also had subarachnoid hemorrhage. No patient had a good neurological outcome in the ICH group. There were statistically significant differences in gender, age, pre-hospital return of spontaneous circulation, survival to hospital discharge, good neurologic outcomes, serum sodium, potassium, glucose, Pco2, and Po2 during CPR between the ICH and non-ICH groups. In multivariate analysis, gender, age, potassium, glucose and Po2 levels differed significantly between the two groups. CONCLUSIONS: OHCA patients with confirmed ICH were identified in about 11% of cases after return of spontaneous circulation. Gender, age, higher glucose, and lower potassium and Po2 levels during CPR were associated with ICH.

A simple model to predict bacteremia in women with acute pyelonephritis.

OBJECTIVES: To construct a simple model to predict bacteremia in women with uncomplicated acute pyelonephritis (APN) for the judicious use of blood cultures. METHODS: A prospective database including 735 women with uncomplicated APN at an academic urban emergency department was analyzed retrospectively. Independent risk factors were determined using multivariate logistic regression in two-thirds of patients. Cutoff values representing 10% and 30% of risk were selected for the stratification. This model was internally and externally validated using a remaining one-thirds of patients and 169 independent patients, respectively. RESULTS: Independent risk factors were as follows: age ≥65 years (odds ratio [OR]=5.18, 4 points), vomiting (OR=2.40, 2 points), heart rate >110 beats/min (OR=2.35, 2 points), segmented neutrophils >90% (OR=3.17, 3 points), and urine WBC ≥50/HPF (OR=4.27, 4 points). Patients were stratified as low (points <4), intermediate (points, 4-6), or high risk (7≤ points). The areas under receiver operating characteristics curves were 0.707 and 0.792 in internal and external validation cohorts, respectively. The model stratified internal and external validation cohort into low (8.5% and 5.7%), intermediate (16.5% and 14.8%), and high risk of bacteremia (42.0% and 56.4%). CONCLUSIONS: This model provides a useful tool to predict the risk of bacteremia, which can be helpful to decide whether to perform blood cultures and whether to admit the patient for the intravenous antibiotics in women with uncomplicated APN.

Effects of the polarizability and packing density of transparent oxide films on water vapor permeation.

The tin oxide and silicon oxide films have been deposited on polycarbonate substrates as gas barrier films, using a thermal evaporation and ion beam assisted deposition process. The oxide films deposited by ion beam assisted deposition show a much lower water vapor transmission rate than those by thermal evaporation. The tin oxide films show a similar water vapor transmission rate to the silicon oxide films in thermal evaporation but a lower water vapor transmission rate in IBAD. These results are related to the fact that the permeation of water vapor with a large dipole moment is affected by the chemistry of oxides and the packing density of the oxide films. The permeation mechanism of water vapor through the oxide films is discussed in terms of the chemical interaction with water vapor and the microstructure of the oxide films. The chemical interaction of water vapor with oxide films has been investigated by the refractive index from ellipsometry and the OH group peak from X-ray photoelectron spectroscopy, and the microstructure of the composite oxide films was characterized using atomic force microscopy and a transmission electron microscope. The activation energy for water vapor permeation through the oxide films has also been measured in relation to the permeation mechanism of water vapor. The diffusivity of water vapor for the tin oxide films has been calculated from the time lag plot, and its implications are discussed.