PKA mediates modality-specific modulation of the mechanically gated ion channel PIEZO2.

PKA is a downstream effector of many inflammatory mediators that induce pain hypersensitivity by increasing the mechanosensitivity of nociceptive sensory afferent. Here, we examine the molecular mechanism underlying PKA-dependent modulation of the mechanically activated ion channel PIEZO2, which confers mechanosensitivity to many nociceptors. Using phosphorylation site prediction algorithms, we identified multiple putative and highly conserved PKA phosphorylation sites located on intracellular intrinsically disordered regions of PIEZO2. Site-directed mutagenesis and patch-clamp recordings showed that substitution of one or multiple putative PKA sites within a single intracellular domain does not alter PKA-induced PIEZO2 sensitization, whereas mutation of a combination of nine putative sites located on four different intracellular regions completely abolishes PKA-dependent PIEZO2 modulation, though it remains unclear whether all or just some of these nine sites are required. By demonstrating that PIEZO1 is not modulated by PKA, our data also reveal a previously unrecognized functional difference between PIEZO1 and PIEZO2. Moreover, by demonstrating that PKA only modulates PIEZO2 currents evoked by focal mechanical indentation of the cell, but not currents evoked by pressure-induced membrane stretch, we provide evidence suggesting that PIEZO2 is a polymodal mechanosensor that engages different protein domains for detecting different types of mechanical stimuli.

Rhizochalinin Exhibits Anticancer Activity and Synergizes with EGFR Inhibitors in Glioblastoma In Vitro Models.

Rhizochalinin (Rhiz) is a recently discovered cytotoxic sphingolipid synthesized from the marine natural compound rhizochalin. Previously, Rhiz demonstrated high in vitro and in vivo efficacy in various cancer models. Here, we report Rhiz to be highly active in human glioblastoma cell lines as well as in patient-derived glioma-stem like neurosphere models. Rhiz counteracted glioblastoma cell proliferation by inducing apoptosis, G2/M-phase cell cycle arrest, and inhibition of autophagy. Proteomic profiling followed by bioinformatic analysis suggested suppression of the Akt pathway as one of the major biological effects of Rhiz. Suppression of Akt as well as IGF-1R and MEK1/2 kinase was confirmed in Rhiz-treated GBM cells. In addition, Rhiz pretreatment resulted in a more pronounced inhibitory effect of γ-irradiation on the growth of patient-derived glioma-spheres, an effect to which the Akt inhibition may also contribute decisively. In contrast, EGFR upregulation, observed in all GBM neurospheres under Rhiz treatment, was postulated to be a possible sign of incipient resistance. In line with this, combinational therapy with EGFR-targeted tyrosine kinase inhibitors synergistically increased the efficacy of Rhiz resulting in dramatic inhibition of GBM cell viability as well as a significant reduction of neurosphere size in the case of combination with lapatinib. Preliminary in vitro data generated using a parallel artificial membrane permeability (PAMPA) assay suggested that Rhiz cannot cross the blood brain barrier and therefore alternative drug delivery methods should be used in the further in vivo studies. In conclusion, Rhiz is a promising new candidate for the treatment of human glioblastoma, which should be further developed in combination with EGFR inhibitors.

The effect of the enhanced recovery programme on long-term survival following liver resection for colorectal liver metastases.

BACKGROUND: Enhanced recovery programmes are associated with improved short-term outcomes following liver surgery. The impact of enhanced recovery programmes on medium- and long-term outcomes is incompletely understood. This study aimed to assess the impact of an enhanced recovery programme on long-term survival in patients undergoing surgery for colorectal liver metastases. METHODS: At a tertiary hepatobiliary centre, we analysed short-, medium- and long-term outcomes in consecutive patients undergoing liver resection for colorectal liver metastases. A five-year retrospective review was carried out comparing the enhanced recovery programme to standard care. RESULTS: A total of 172 patients were included in the analysis: 87 on standard care and 85 on an enhanced recovery programme. Open surgery was performed in 122 patients: 74 (85.1%) and 48 (56.5%) patients in the standard care and enhanced recovery programme, respectively (p < 0.001). There was a significant reduction in the median (IQR) length of hospital stay in the enhanced recovery programme compared with standard care (7 (5) days vs. 8 (3) days, p = 0.0009). There was no significant difference in survival between standard care and the Enhanced Recovery Programme at one (p = 0.818), three (p = 0.203), and five years (p = 0.247). CONCLUSION: An enhanced recovery programme was associated with a reduced length of hospital stay. There was no effect on the one-, three- and five-year survival.

Implementation of a Point-of-Care Radiologist-Technologist Communication Tool in a Quality Assurance Program.

OBJECTIVE: We implemented an Image Quality Reporting and Tracking Solution (IQuaRTS), directly linked from the PACS, to improve communication between radiologists and technologists. MATERIALS AND METHODS: IQuaRTS launched in May 2015. We compared MRI issues filed in the period before IQuaRTS implementation (May-September 2014) using a manual system with MRI issues filed in the IQuaRTS period (May-September 2015). The unpaired t test was used for analysis. For assessment of overall results in the IQuaRTS period alone, all issues filed across all modalities were included. Summary statistics and charts were generated using Excel and Tableau. RESULTS: For MRI issues, the number of issues filed during the IQuaRTS period was 498 (2.5% of overall MRI examination volume) compared with 78 issues filed during the period before IQuaRTS implementation (0.4% of total examination volume) (p = 0.0001), representing a 625% relative increase. Tickets that documented excellent work were 8%. Other issues included images not pushed to PACS (20%), film library issues (19%), and documentation or labeling (8%). Of the issues filed, 55% were MRI-related and 25% were CT-related. The issues were stratified across six sites within our institution. Staff requiring additional training could be readily identified, and 80% of the issues were resolved within 72 hours. CONCLUSION: IQuaRTS is a cost-effective online issue reporting tool that enables robust data collection and analytics to be incorporated into quality improvement programs. One limitation of the system is that it must be implemented in an environment where staff are receptive to quality improvement.

Linking metabolism and histone acetylation dynamics by integrated metabolic flux analysis of Acetyl-CoA and histone acetylation sites.

OBJECTIVES: Histone acetylation is an important epigenetic modification that regulates various biological processes and cell homeostasis. Acetyl-CoA, a hub molecule of metabolism, is the substrate for histone acetylation, thus linking metabolism with epigenetic regulation. However, still relatively little is known about the dynamics of histone acetylation and its dependence on metabolic processes, due to the lack of integrated methods that can capture site-specific histone acetylation and deacetylation reactions together with the dynamics of acetyl-CoA synthesis. METHODS: In this study, we present a novel proteo-metabo-flux approach that combines mass spectrometry-based metabolic flux analysis of acetyl-CoA and histone acetylation with computational modelling. We developed a mathematical model to describe metabolic label incorporation into acetyl-CoA and histone acetylation based on experimentally measured relative abundances. RESULTS: We demonstrate that our approach is able to determine acetyl-CoA synthesis dynamics and site-specific histone acetylation and deacetylation reaction rate constants, and that consideration of the metabolically labelled acetyl-CoA fraction is essential for accurate determination of histone acetylation dynamics. Furthermore, we show that without correction, changes in metabolic fluxes would be misinterpreted as changes in histone acetylation dynamics, whereas our proteo-metabo-flux approach allows to distinguish between the two processes. CONCLUSIONS: Our proteo-metabo-flux approach expands the repertoire of metabolic flux analysis and cross-omics and represents a valuable approach to study the regulatory interplay between metabolism and epigenetic regulation by histone acetylation.

The Histogenetic Origin of Malignant Cells Predicts Their Susceptibility towards Synthetic Lethality Utilizing the TK.007 System.

BACKGROUND: Remarkable differences exist in the outcome of systemic cancer therapies. Lymphomas and leukemias generally respond well to systemic chemotherapies, while solid cancers often fail. We engineered different human cancer cells lines to uniformly express a modified herpes simplex virus thymidine kinase TK.007 as a suicide gene when ganciclovir (GCV) is applied, thus in theory achieving a similar response in all cell lines. METHODS: Fifteen different cell lines were engineered to express the TK.007 gene. XTT-cell proliferation assays were performed and the IC50-values were calculated. Functional kinome profiling, mRNA sequencing, and bottom-up proteomics analysis with Ingenuity pathway analysis were performed. RESULTS: GCV potency varied among cell lines, with lymphoma and leukemia cells showing higher susceptibility than solid cancer cells. Functional kinome profiling implies a contribution of the SRC family kinases and decreased overall kinase activity. mRNA sequencing highlighted alterations in the MAPK pathways and bottom-up proteomics showed differences in apoptotic and epithelial junction signaling proteins. CONCLUSIONS: The histogenetic origin of cells influenced the susceptibility of human malignant cells towards cytotoxic agents with leukemias and lymphomas being more sensitive than solid cancer cells.

Multiomic profiling of medulloblastoma reveals subtype-specific targetable alterations at the proteome and N-glycan level.

Medulloblastomas (MBs) are malignant pediatric brain tumors that are molecularly and clinically heterogenous. The application of omics technologies-mainly studying nucleic acids-has significantly improved MB classification and stratification, but treatment options are still unsatisfactory. The proteome and their N-glycans hold the potential to discover clinically relevant phenotypes and targetable pathways. We compile a harmonized proteome dataset of 167 MBs and integrate findings with DNA methylome, transcriptome and N-glycome data. We show six proteome MB subtypes, that can be assigned to two main molecular programs: transcription/translation (pSHHt, pWNT and pG3myc), and synapses/immunological processes (pSHHs, pG3 and pG4). Multiomic analysis reveals different conservation levels of proteome features across MB subtypes at the DNA methylome level. Aggressive pGroup3myc MBs and favorable pWNT MBs are most similar in cluster hierarchies concerning overall proteome patterns but show different protein abundances of the vincristine resistance-associated multiprotein complex TriC/CCT and of N-glycan turnover-associated factors. The N-glycome reflects proteome subtypes and complex-bisecting N-glycans characterize pGroup3myc tumors. Our results shed light on targetable alterations in MB and set a foundation for potential immunotherapies targeting glycan structures.

Outcomes of first emergency admissions for alcohol-related liver disease in England over a 10-year period: retrospective observational cohort study using linked electronic databases.

OBJECTIVES: To examine time trends in patient characteristics, care processes and case fatality of first emergency admission for alcohol-related liver disease (ARLD) in England. DESIGN: National population-based, retrospective observational cohort study. SETTING: Clinical Practice Research Datalink population of England, 2008/2009 to 2017/2018. First emergency admissions were identified using the Liverpool ARLD algorithm. We applied survival analyses and binary logistic regression to study prognostic trends. OUTCOME MEASURES: Patient characteristics; 'recent' General Practitioner (GP) consultations and hospital admissions (preceding year); higher level care; deaths in-hospital (including certified cause) and within 365 days. Covariates were age, sex, deprivation status, coding pattern, ARLD stage, non-liver comorbidity, coding for ascites and varices. RESULTS: 17 575 first admissions (mean age: 53 years; 33% women; 32% from most deprived quintile). Almost half had codes suggesting advanced liver disease. In year before admission, only 47% of GP consulters had alcohol-related problems recorded; alcohol-specific diagnostic codes were absent in 24% of recent admission records. Overall, case fatality rate was 15% in-hospital and 34% at 1 year. Case-mix-adjusted odds of in-hospital death reduced by 6% per year (adjusted OR (aOR): 0.94; 95% CI: 0.93 to 0.96) and 4% per year at 365 days (aOR: 0.96; 95% CI: 0.95 to 0.97). Exploratory analyses suggested the possibility of regional inequalities in outcome. CONCLUSIONS: Despite improving prognosis of first admissions, we found missed opportunities for earlier recognition and intervention in primary and secondary care. In 2017/2018, one in seven were still dying during index admission, rising to one-third within a year. Nationwide efforts are needed to promote earlier detection and intervention, and to minimise avoidable mortality after first emergency presentation. Regional variation requires further investigation.

Effects of Human RelA Transgene on Murine Macrophage Inflammatory Responses.

The NFκB transcription factors are major regulators of innate immune responses, and NFκB signal pathway dysregulation is linked to inflammatory disease. Here, we utilised bone marrow-derived macrophages from the p65-DsRedxp/IκBα-eGFP transgenic strain to study the functional implication of xenogeneic (human) RelA(p65) protein introduced into the mouse genome. Confocal imaging showed that human RelA is expressed in the cells and can translocate to the nucleus following activation of Toll-like receptor 4. RNA sequencing of lipid A-stimulated macrophages, revealed that human RelA impacts on murine gene transcription, affecting both non-NFκB and NFκB target genes, including immediate-early and late response genes, e.g., Fos and Cxcl10. Validation experiments on NFκB targets revealed markedly reduced mRNA levels, but similar kinetic profiles in transgenic cells compared to wild-type. Enrichment pathway analysis of differentially expressed genes revealed interferon and cytokine signaling were affected. These immune response pathways were also affected in macrophages treated with tumor necrosis factor. Data suggests that the presence of xenogeneic RelA protein likely has inhibitory activity, altering specific transcriptional profiles of key molecules involved in immune responses. It is therefore essential that this information be taken into consideration when designing and interpreting future experiments using this transgenic strain.

Metabolic synchronization by traveling waves in yeast cell layers.

The coordination of cellular behavior is a prerequisite of functionality of tissues and organs. Generally, this coordination occurs by signal transduction, neuronal control, or exchange of messenger molecules. The extent to which metabolic processes are involved in intercellular communication is less understood. Here, we address this question in layers of resting yeast cells and report for the first time the observation of intercellular glycolytic waves. We use a combined experimental and theoretical approach and explain the radial velocity of the waves to arise from the substrate gradient due to local substrate addition. Our results show that metabolic processes introduce an additional level of local intercellular coordination.

Inward rotating spiral waves in glycolysis.

We report on the first observation of inward rotating spiral waves (antispirals) in a biochemical reaction-diffusion system. Experiments are performed with extracts from yeast cells in an open spatial reactor. By increasing the protein concentration of the extract we observe a transition from outward to inward propagating waves of glycolytic activity. Numerical simulations with an allosteric model for the phosphofructokinase can reproduce these inward propagating waves over a wide range of parameters if the octameric structure of yeast phosphofructokinase is taken into account.

Spatial control of the energy metabolism of yeast cells through electrolytic generation of oxygen.

The metabolic dynamics of yeast cells is controlled by electric pulses delivered through a spatially extended yeast cell/Au electrode interface. Concomitant with voltage pulses, oxygen is generated electrolytically at the electrode surface and delivered to the cells. The generation of oxygen was investigated in dependence of the applied voltage, width of the voltage pulses and temperature of the electrolytic solution. The local oxygen pulses at the electrodes lead to a transient activation of the aerobic energy metabolism of the yeast cells causing a perturbation in their energy balance. The effect of these local perturbations on the temporal dynamics of glycolysis in yeast cells is quantified in dependence of the energy state of cells.

Spatial desynchronization of glycolytic waves as revealed by Karhunen-Loeve analysis.

The dynamics of glycolytic waves in a yeast extract have been investigated in an open spatial reactor. At low protein contents in the extract, we find a transition from inwardly moving target patterns at the beginning of the experiment to outwardly moving spiral- or circular-shaped waves at later stages. These two phases are separated by a transition phase of more complex spatiotemporal dynamics. We have analyzed the pattern dynamics in these three intervals at different spatial scales by means of a Karhunen-Loeve (KL) decomposition. During the initial phase of the experiment, the observed patterns are sufficiently described by the two dominant KL modes independently of the spatial scale. However, during the last stage of the experiment, at least 6 KL modes are needed to account for the observed patterns at spatial scales larger than 3 mm, while for smaller scales, 2 KL modes are still sufficient. This indicates that in the course of the experiment, the local glycolytic oscillators become desynchronized at spatial scales larger than 3 mm. Possible reasons for the desynchronization of the glycolytic waves are discussed.

Analysis of the oscillatory kinetics of glycolytic intermediates in a yeast extract by FT-IR spectroscopy.

In the present work we demonstrate that FT-IR spectroscopy is a powerful tool for the time resolved and noninvasive measurement of multi-substrate/product interactions in complex metabolic networks as exemplified by the oscillating glycolysis in yeast extract. We found that many of the glycolytic intermediates can be identified with FT-IR spectroscopy. For this, we have constructed a spectral library of most of the glycolytic intermediates and obtained the kinetics of single components in spectra from glycolysing yeast extract by the use of mathematical fitting procedures. The results are in good agreement with the known phase relationships of oscillatory glycolysis. They provide the basis for future application of this method to investigate the energy metabolism of living cells.

Glycolytic oscillations and waves in an open spatial reactor: Impact of feedback regulation of phosphofructokinase.

An open spatial reactor has been designed for the investigation of spatio-temporal dynamics of glycolysis. The reactor consists of a diffusive layer made of gel-fixed yeast extract which is in contact with a continuously stirred reservoir to supply this layer with substrates. The coupling between reaction and diffusion in the gel layer enables the formation of spatio-temporal patterns. Temporal oscillations of glycolysis are simply induced by feeding the yeast extract with sugar. Under properly chosen conditions, these oscillations sustain for more than 12 h. A necessary prerequisite for the generation of oscillations is that the ATP concentration in the feeding solution must be high enough to allow for negative feedback of phosphofructokinase. Otherwise, the interplay between ATP-consuming and ATP-producing reactions leads to an unfavorable low ATP/AMP ratio. The generation of travelling NADH-waves is observed in the diffusive layer, when feeding the yeast extract with substrates. Break-up of circular-shaped waves is repeatedly observed, resulting in the formation of rotating NADH-spirals.

Spatiotemporal dynamics of glycolytic waves provides new insights into the interactions between immobilized yeast cells and gels.

The immobilization of cells or enzymes is a promising tool for the development of biosensors, yet the interactions between the fixative materials and the cells are not fully understood, especially with respect to their impact on both cell metabolism and cell-to-cell signaling. We show that the spatiotemporal dynamics of waves of metabolic synchronization of yeast cells provides a new criterion to distinguish the effect of different gels on the cellular metabolism, which otherwise could not be detected. Cells from the yeast Saccharomyces carlsbergensis were immobilized into agarose gel, silica gel (TMOS), or a mixture of TMOS and alginate. We compared these immobilized cells with respect to their ability to generate temporal, intracellular oscillations in glycolysis as well as propagating, extracellular synchronization waves. While the temporal dynamics, as measured by the period and the number of oscillatory cycles, was similar for all three immobilized cell populations, significant differences have been observed with respect to the shape of the waves, wave propagation direction and velocity in the three gel matrices used.

Excitation-induced dynamics of external pH pattern in Chara corallina cells and its dependence on external calcium concentration.

The influence of cell excitation and external calcium level on the dynamics of light-induced pH bands along the length of Chara corallina cells is studied in the present paper. Generation of an action potential (AP) transiently quenched these pH patterns, which was more pronounced at 0.05-0.1 mM Ca2+ than at higher concentrations of Ca2+ (0.6-2 mM) in the medium. After transient smoothing of the pH bands, some alkaline peaks reemerged at slightly shifted positions in media with low Ca2+ concentrations, while at high Ca2+ concentrations, the alkaline spots reappeared exactly at their initial positions. This Ca2+ dependency has been revealed by both digital imaging and pH microelectrodes. The stabilizing effect of external Ca2+ on the locations of recovering alkaline peaks is supposedly due to formation of a physically heterogeneous environment around the cell owing to precipitation of CaCO3 in the alkaline zones at high Ca2+ during illumination. The elevation of local pH by dissolving CaCO3 facilitates the reappearance of alkaline spots at their initial locations after temporal suppression caused by cell excitation. At low Ca2+ concentrations, when the solubility product of CaCO3 is not attained, the alkaline peaks are not stabilized by CaCO3 dissolution and may appear at random locations.

Control of glycolytic oscillations by temperature.

External control of oscillatory glycolysis in yeast extract has been performed by application of either homogeneous temperature oscillations or stationary, spatial temperature gradients. Entrainment of the glycolytic oscillations by the 1/2- and 1/3-harmonic, as well as the fundamental input frequency, could be observed. From the phase response curve to a single temperature pulse, a distinct sensitivity of NADH-oxidizing processes, compared with NAD-reducing processes, is visible. Determination of glycolytic intermediates shows that the feedback-regulated phosphofructokinase as well as the glyceraldehyde-3-phosphate dehydrogenase are the most temperature-sensitive steps of glycolysis. We also find strong concentration changes in ATP and AMP at varying temperatures and, accordingly, in the energy charge. Construction of a feedback loop for spatial control of temperature by means of a Peltier element allowed us to apply a temperature gradient to the yeast extract. With this setup it is possible to initiate traveling waves and to control the wave velocity.

Extracellular potassium alters frequency and profile of retinal spreading depression waves.

The phenomenon of spreading depression (SD) was observed in chicken retina by means of optical registration via a microscope and a CCD camera applying modern methods of image processing for optimized evaluation of the wave profiles. The propagation dynamics of SD waves was investigated as a function of extracellular potassium. Two main findings were obtained. Firstly, the frequency of spontaneous wave generation increased with the increase of K+ concentration. Secondly, there was an effect of potassium on the wave profile. In particular, the recovery zone of SD waves was shortened at increased K+. This effect was not only due to the dispersion relation of waves in excitable media as shown by the result of the mechanically induced wave trains. Applying the basic principles of chemical excitability for the interpretation of the data led us to the conclusion that these potassium effects are due to perturbations of an autocatalytic reaction to be further explored.