Zone Of Cell Death Research Articles

Increased susceptibility to ischemia causes exacerbated response to microinjuries in the cirrhotic liver.

Fractional laser ablation is a technique developed in dermatology to induce remodeling of skin scars by creating a dense pattern of microinjuries. Despite remarkable clinical results, this technique has yet to be tested for scars in other tissues. As a first step toward determining the suitability of this technique, we aimed to (1) characterize the response to microinjuries in the healthy and cirrhotic liver, and (2) determine the underlying cause for any differences in response. Healthy and cirrhotic rats were treated with a fractional laser then euthanized from 0 h up to 14 days after treatment. Differential expression was assessed using RNAseq with a difference-in-differences model. Spatial maps of tissue oxygenation were acquired with hyperspectral imaging and disruptions in blood supply were assessed with tomato lectin perfusion. Healthy rats showed little damage beyond the initial microinjury and healed completely by 7 days without scarring. In cirrhotic rats, hepatocytes surrounding microinjury sites died 4-6 h after ablation, resulting in enlarged and heterogeneous zones of cell death. Hepatocytes near blood vessels were spared, particularly near the highly vascularized septa. Gene sets related to ischemia and angiogenesis were enriched at 4 h. Laser-treated regions had reduced oxygen saturation and broadly disrupted perfusion of nodule microvasculature, which matched the zones of cell death. Our results demonstrate that the cirrhotic liver has an exacerbated response to microinjuries and increased susceptibility to ischemia from microvascular damage, likely related to the vascular derangements that occur during cirrhosis development. Modifications to the fractional laser tool, such as using a femtosecond laser or reducing the spot size, may be able to prevent large disruptions of perfusion and enable further development of a laser-induced microinjury treatment for cirrhosis.

Response of Articular Cartilage to Hyperosmolar Stress: Report of an Ex Vivo Injury Model

Background: Physiological 0.9% saline is commonly used as an irrigation fluid in modern arthroscopy. There is a growing body of evidence that a hyperosmolar saline solution has chondroprotective effects, especially if iatrogenic injury occurs. Purpose: To (1) corroborate the superiority of a hyperosmolar saline solution regarding chondrocyte survival after mechanical injury and (2) observe the modulatory response of articular cartilage to osmotic stress and injury. Study Design: Controlled laboratory study. Methods: Osteochondral explants were isolated from bovine stifle joints and exposed to either 0.9% saline (308 mOsm) or hyperosmolar saline (600 mOsm) and then damaged with a sharp dermatome blade to attain a confined full-thickness cartilage injury site, incubated in the same fluids for another 3 hours, and transferred to chondropermissive medium for further culture for 1 week. Chondrocyte survival was assessed by confocal imaging, while the cellular response was evaluated over 1 week by relative gene expression for apoptotic and inflammatory markers and mediator release into the medium. Results: The full-thickness cartilage cut resulted in a confined zone of cell death that mainly affected superficial zone chondrocytes. Injured samples that were exposed to hyperosmolar saline showed less expansion of cell death in both the axial (P < .007) and the coronal (P < .004) plane. There was no progression of cell death during the following week of culture. Histological assessment revealed an intact cartilage matrix and normal chondrocyte morphology. Inflammatory and proapoptotic genes were upregulated on the first days postexposure with a notable downregulation toward day 7. Mediator release into the medium was concentrated on day 3. Conclusion: This in vitro cartilage injury model provides further evidence for the chondroprotective effect of a hyperosmolar saline irrigation fluid, as well as novel data on the capability of articular cartilage to quickly regain joint homeostasis after osmotic stress and injury. Clinical Relevance: Raising the osmolarity of an irrigating solution may be a simple and safe strategy to protect articular cartilage during arthroscopic surgery.

Evolution Pattern in Bruised Tissue of 'Red Delicious' Apple.

The study of apple damage mechanisms is key to improving post-harvest apple treatment techniques, and the evolution pattern of damaged tissue is fundamental to the study of apple damage mechanisms. In the study, 'Red Delicious' apples were used to explore the relationship between damage and time. A cell death zone was found in the pulp of the damaged tissue after the apple had been bruised. The tissue damage was centered in the cell death zone and developed laterally, with the width of the damage increasing with injury time. The extent of tissue damage in the core and pericarpal directions varied. About 60% of the damaged tissue developed in the core direction and 40% in the pericarpal direction, and the damage ratios in both directions remained consistent throughout the injury. The depth of damage and the rate of damage were influenced by the impact force size and the difference in the size of the damaged part of the apple, but the damage development pattern was independent of the impact force size and the difference in the damaged part. The maximum damage rate was reached at about 30 min, and the depth of damage was stabilized at about 72 min. By studying the evolution pattern of the damaged tissue of the bruised 'Red Delicious' apple, it provides the research idea and theoretical basis for enhancing the prediction accuracy and robustness of early stage damage in apples.

OPTIMIZING WOUNDED ARTICULAR CARTILAGE FOR IMPROVED CARTILAGE INTEGRATION: EVALUATING CHONDROPROTECTIVE MEASURES FOR MINIMIZING IN SITU CHONDROCYTE DEATH

AbstractObjectiveThe preparation of host degenerate cartilage for repair typically requires cutting and/or scraping to remove the damaged tissue. This can lead to mechanical injury and cartilage cell (chondrocytes) death, potentially limiting the integration of repair material. This study evaluated cell death at the site of cutting injury and determined whether raising the osmotic pressure (hyper-osmolarity) prior to injury could be chondroprotective.MethodsEx vivo human femoral head cartilage was obtained from 13 patients (5 males and 8 females: 71.8 years old) with Ethical Permission and Patient consent. Cartilage wells were created using 3 or 5mm biopsy punches. Cell death at the wounded edge of the host cartilage and the edge of the extracted explants were assessed by quantifying the percentage of cell death (PCD) and measuring the width of the cell death zone at identified regions of interest (ROI) using the confocal laser scanning microscopy and image analysis software. To assess the chondroprotective effect of hyper-osmolarity, cartilage specimens were incubated in 340 or 600mOsm media, five minutes prior to injury to allow the chondrocytes to respond to the altered osmolarity. Wounded cartilage explants and cartilage wells were then cultured for a further 150 minutes following injury.ResultsIn 340mOsm media, the PCD around the 3mm cartilage wells was significantly less compared to the corresponding explants (20.05±10.24% vs 35.25±4.86%; P=0.0003). When using the 5mm biopsy punch, the PCD at the wound edges was significantly lower when compared to the 3mm cartilage wells (13.33±7.80% vs 20.05±10.24%; P=0.0121) at the same osmolarity. The width of the cell death zone for the well edges for both 3 and 5mm punches was significantly narrower when compared to their corresponding harvested cartilage explants in 340mOsm media (P&lt;0.0001; P=0.0218, respectively). Exposing cartilage to raised osmolarity (600mOsm) prior to injury significantly reduced the PCD for cartilage wells produced by the 3mm biopsy punches (from 20.05±10.24% to 12.24±6.00%; P=0.0025). In addition, the zone of cell death was marginally reduced at the edges of the 5mm cartilage wells (19.25±15.78mm to 12.72±9.09mm; P=0.0499).ConclusionsThe choice of biopsy punch and the osmolarity of the incubation medium prior to cartilage injury markedly affected the extent of chondrocyte death both at the edges of the cartilage wells and the explants. The smaller biopsy punch caused more chondrocyte death in the native cartilage wells compared to the larger punch, but this could be compensated for by the chondroprotective effect of raising the osmotic pressure. In general, there was less cell death at the wounded edges of the cartilage wells, compared to the explants. These results suggest that there is scope for further optimising the cutting implements used to create the cartilage wells and protecting chondrocytes by hyper-osmolarity in order to minimize cell death at cut edges and potentially enhance integration between cartilage repair material and host cartilage.Declaration of Interest(b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project.

Dynamic Prediction Model for Initial Apple Damage.

Prediction models of damage severity are crucial for the damage expression of fruit. In light of issues such as the mismatch of existing models in actual damage scenarios and the failure of static models to meet research needs, this article proposes a dynamic prediction model for damage severity throughout the entire process of apple damage and studies the relationship between the initial bruise form and impact energy distribution of apple damage. From the experiments, it was found that after impact a "cell death zone" appeared in the internal pulp of the damaged part of Red Delicious apples. The reason for the appearance of the cell death zone was that the impact force propagated in the direction of the fruit kernel in the form of stress waves; the continuous action of which continuously compressed the pulp's cell tissue. When the energy absorbed via elastic deformation reached the limit value, intercellular disadhesion of parenchyma cells at the location of the stress wave peak occurred to form cell rupture. The increase in intercellular space for the parenchyma cells near the rupture site caused a large amount of necrocytosis and, ultimately, formed the cell death zone. The depth of the cell death zone was closely related to the impact energy. The correlation coefficient r between the depth of the cell death zone and the distribution of impact energy was slightly lower at the impact height of 50 mm. As the impact height increased, the correlation coefficient r increased, approaching of value of 1. When the impact height was lower (50 mm), the correlation coefficient r had a large distribution range (from 0.421 to 0.983). As the impact height increased, the distribution range significantly decreased. The width of the cell death zone had a poor correlation with the pressure distribution on the impact surface of the apples that was not related to the impact height. In this article, the corresponding relationship between the form and impact energy distribution of the internal damaged tissues in the initial damage of Red Delicious apples was analyzed. This analysis aimed to provide a research concept and theoretical basis for more reliable research on the morphological changes in the damaged tissues of apples in the future, further improving the prediction accuracy of damage severity.

Ccn2a-FGFR1-SHH signaling is necessary for intervertebral disc homeostasis and regeneration in adult zebrafish.

Intervertebral disc (IVD) degeneration is the primary cause of back pain in humans. However, the cellular and molecular pathogenesis of IVD degeneration is poorly understood. This study shows that zebrafish IVDs possess distinct and non-overlapping zones of cell proliferation and cell death. We find that, in zebrafish, cellular communication network factor 2a (ccn2a) is expressed in notochord and IVDs. Although IVD development appears normal in ccn2a mutants, the adult mutant IVDs exhibit decreased cell proliferation and increased cell death leading to IVD degeneration. Moreover, Ccn2a overexpression promotes regeneration through accelerating cell proliferation and suppressing cell death in wild-type aged IVDs. Mechanistically, Ccn2a maintains IVD homeostasis and promotes IVD regeneration by enhancing outer annulus fibrosus cell proliferation and suppressing nucleus pulposus cell death through augmenting FGFR1-SHH signaling. These findings reveal that Ccn2a plays a central role in IVD homeostasis and regeneration, which could be exploited for therapeutic intervention in degenerated human discs.

Chloroplast redox state changes mark cell-to-cell signaling in the hypersensitive response.

Hypersensitive response (HR)-conferred resistance is associated with induction of programmed cell death and pathogen spread restriction in its proximity. The exact role of chloroplastic reactive oxygen species and its link with salicylic acid (SA) signaling in HR remain unexplained. To unravel this, we performed a detailed spatiotemporal analysis of chloroplast redox response in palisade mesophyll and upper epidermis to potato virus Y (PVY) infection in a resistant potato genotype and its transgenic counterpart with impaired SA accumulation and compromised resistance. Besides the cells close to the cell death zone, we detected individual cells with oxidized chloroplasts further from the cell death zone. These are rare in SA-deficient plants, suggesting their role in signaling for resistance. We confirmed that chloroplast redox changes play important roles in signaling for resistance, as blocking chloroplast redox changes affected spatial responses at the transcriptional level. Through spatiotemporal study of stromule induction after PVY infection, we show that stromules are induced by cell death and also as a response to PVY multiplication at the front of infection. Overall induction of stromules is attenuated in SA-deficient plants.

Therapeutic Bronchoscopy for Lung Nodules: Where Are We Now?

Lobar resection has been the established standard of care for peripheral early-stage non-small cell lung cancer (NSCLC). Over the last few years, surgical lung sparing approach (sublobar resection [SLR]) has been compared with lobar resection in T1N0 NSCLC. Three nonsurgical options are available in those patients who have a prohibitive surgical risk, and those who refuse surgery: stereotactic body radiotherapy (SBRT), percutaneous ablation, and bronchoscopic ablation. Local ablation involves placement of a probe into a tumor, and subsequent application of either heat or cold energy, pulsing electrical fields, or placement of radioactive source under an image guidance to create a zone of cell death that encompasses the targeted lesion and an ablation margin. Despite being in their infancy, the bronchoscopic ablative techniques are undergoing rapid research, as they extrapolate a significant knowledge-base from the percutaneous techniques that have been in the radiologist's armamentarium since 2000. Here, we discuss selected endoscopic and percutaneous thermal and non-thermal therapies with the focus on their efficacy and safety.

Analysis of Virus Spread Around the Cell Death Zone at Spatiotemporal Resolution Using Confocal Microscopy.

The role of programmed cell death (PCD) in hypersensitive response (HR)-conferred resistance depends on the type of host-pathogen interaction and therefore has to be studied for each individual pathosystem. Here we present and explain the protocol for studying the role of PCD in HR-conferred resistance in potato plants in the interaction with the viral pathogen. As an experimental system, we use genotype Rywal, where the virus spread is restricted and HR PCD develops 3days post potato virus Y (PVY) inoculation. As a control of virus multiplication and spread, we include its transgenic counterpart impaired in salicylic acid (SA) accumulation (NahG-Rywal), in which the HR-PCD occurs but the spread of the virus is not restricted. To follow the occurrence of virus-infected cells and/or virus multiplication outside the cell death zone, we use GFP-tagged PVY (PVY-N605(123)-GFP) which can be monitored by confocal microscopy. Any other plant-pathogen system which results in PCD development could be studied using a modified version of this protocol.

Histologically Confirmed Recellularization is a Key Factor that Affects Meniscal Healing in Immature and Mature Meniscal Tears.

Healing outcomes of meniscal repair are better in younger than in older. However, exact mechanisms underlying superior healing potential in younger remain unclear from a histological perspective. This study included 24 immature rabbits and 24 mature rabbits. Tears were created in the anterior horn of medial meniscus of right knee in each rabbit. Animals were sacrificed at 1, 3, 6, and 12 weeks postoperatively. We performed macroscopic and histological evaluations of post-meniscal repair specimens. Cells were counted within a region of interest to confirm cellularization at tear site in immature menisci. The width of cell death zone was measured to determine the region of cell death in mature menisci. Apoptosis was evaluated by TUNEL assay. Vascularization was assessed by CD31 immunofluorescence. The glycosaminoglycans and the types 1 and 2 collagen content was evaluated by calculating average optical density of corresponding histological specimens. Cartilage degeneration was also evaluated. Healing outcomes following untreated meniscal tears were superior in immature group. Recellularization with meniscus-like cell morphology was observed at tear edge in immature menisci. Superior recellularization was observed at meniscal sites close to joint capsule than at sites distant from the capsule. Recellularization did not occur at tear site in mature group; however, we observed gradual enlargement of cell death zone. Apoptosis was presented at 1, 3, 6, 12 weeks in immature and mature menisci after untreated meniscal tears. Vascularization was investigated along the tear edges in immature menisci. Glycosaminoglycans and type 2 collagen deposition were negatively affected in immature menisci. We observed glycosaminoglycan degradation in mature menisci and cartilage degeneration, specifically in immature cartilage of the femoral condyle. In conclusion, compared with mature rabbits, immature rabbits showed more robust healing response after untreated meniscal tears. Vascularization contributed to the recellularization after meniscal tears in immature menisci. Meniscal injury fundamentally alters extracellular matrix deposition.

Does pulsed field ablation regress over time? A quantitative temporal analysis of pulmonary vein isolation

The tissue specificity of pulsed field ablation (PFA) makes it an attractive energy source for pulmonary vein (PV) isolation (PVI). However, beyond each PFA lesion's zone of irreversible electroporation and cell death, there may be a surrounding zone of reversible electroporation and cell injury that could potentially normalize with time. The purpose of this study was to assess whether the level of electrical PVI that is observed acutely after PFA regresses over time. In a clinical trial, patients with paroxysmal atrial fibrillation underwent PVI using a biphasic PFA waveform delivered through a dedicated, variably deployable multielectrode basket/flower catheter. Detailed voltage maps were created using a multispline diagnostic catheter immediately after PFA and again ∼3 months later in a prospective, protocol-specified reassessment procedure. We analyzed 20 patients who underwent PFA with durable PVI and available maps from both time points. To compare the ablated zones, the left- and right-sided PV antral isolation areas and nonablated posterior wall area were quantified and the distances between left and right PV low-voltage edges were measured. A comparison of voltage maps immediately after PFA and at a median of 84 days (interquartile range 69-90 days) later revealed that there was no significant difference in either the left- and right-sided PV antral isolation areas or nonablated posterior wall area. The distances between low-voltage edges on the posterior wall were also not significantly different between the 2 time points. This study demonstrates that the level of PV antral isolation after PFA with a multielectrode PFA catheter persists without regression.

Precision transcriptomics of viral foci reveals the spatial regulation of immune-signaling genes and identifies RBOHD as an important player in the incompatible interaction between potato virus Y and potato.

SUMMARYWhereas the activation of resistance (R) proteins has been intensively studied, the downstream signaling mechanisms leading to the restriction of the pathogen remain mostly unknown. We studied the immunity network response conditioned by the potato Ny‐1 gene against potato virus Y. We analyzed the processes in the cell death zone and surrounding tissue on the biochemical and gene expression levels in order to reveal the spatiotemporal regulation of the immune response. We show that the transcriptional response in the cell death zone and surrounding tissue is dependent on salicylic acid (SA). For some genes the spatiotemporal regulation is completely lost in the SA‐deficient line, whereas other genes show a different response, indicating multiple connections between hormonal signaling modules. The induction of NADPH oxidase RBOHD expression occurs specifically on the lesion border during the resistance response. In plants with silenced RBOHD, the functionality of the resistance response is perturbed and the spread of the virus is not arrested at the site of infection. RBOHD is required for the spatial accumulation of SA, and conversely RBOHD is under the transcriptional regulation of SA. Using spatially resolved RNA‐seq, we also identified spatial regulation of an UDP‐glucosyltransferase, another component in feedback activation of SA biosynthesis, thus deciphering a novel aspect of resistance signaling.

A Collagen Fiber Membrane Aided Chondroprotective-Based Strategy for Enhancing Integration of Regenerative Neo-Cartilage.

The lack of neo-cartilage integration with host tissues is a great challenge for the clinical translation of new technologies for the repair of articular cartilage (AC) defect. Recently, we developed a promising double-layered collagen-based system for targeted delivery of fibroblast growth factor 2 (FGF2) to the subchondral bone for AC repair. The system effectively promoted the regeneration of both cartilage and subchondral bone. However, neo-cartilage integration was unsatisfactory, which might be due to the presence of a zone of cell death (ZCD) in the cartilage induced by injury. Here, we hypothesized that maintaining cell viability in the region surrounding the defect and decreasing the size of ZCD by using chondroprotective agents such as insulin-like growth factor-1 (IGF-1), might be an effective strategy to improve neo-cartilage integration. A targeted delivery system for IGF-1 to cartilage based on the FGF2 delivery system was formulated to weaken the impact on the effects of FGF2. The two growth factors were incorporated into the different layers of the membrane without interdiffusion. Due to the different densities of collagen fibers in the different layers, the in vitro and in vivo assays demonstrated that both proteins were released via unidirectional diffusion without mixing or lateral diffusion. Particularly, the released IGF-1 increased the viability of chondrocytes, decreased the ZCD size, and enhanced the integration of regenerative neo-cartilage with host tissues, without any undesirable effects on the FGF2mediated regeneration of cartilage and subchondral bone. Taken together, our findings demonstrate that the collagen fiber membrane-aided chondroprotective-based strategy is an effective way to improve neo-cartilage integration.

04:03 PM Abstract No. 370 Real-time MRI-observed electrolytic ablation zones strongly correlate with regions of histologic liver tissue death in vitro

Electrolytic ablation (EA) causes cell death by creating an inhospitably alkaline or acidic microenvironmental pH (1). We hypothesized that MRI may enable the monitoring of the ablation zone created by electrolytic ablation (EA) in vitro in real time. First, a series of phantoms containing Corgel™ (tyramine-substituted hyaluronate) (Lifecore Biomedical) were created and titrated to pHs ranging from 2 to 12. T1 and T2 mapping studies using the TSE and SE sequences, respectively, were performed in a 1.5 T scanner (Siemens). Second, agarose gels containing encapsulated human hepatocellular carcinoma (HCC) Huh-7 cells were treated with 10 V EA for 120, 240, 360, 480, and 600 sec. The LIVE/DEAD® Viability/Cytotoxicity Kit (ThermoFisher Scientific) was used to assess cell death. This experiment was repeated under MRI guidance, and a correlation between the MRI ablation zone and the histologic ablation zone was performed. T2 mapping studies of the Corgel™ phantoms revealed that acidic and basic pH consistently elongated the T2 relaxation time of the phantoms, suggesting that pH change may be visible on conventional T2-weighted imaging without contrast. In the in vitro ablation experiment, increasing ablation duration produced larger zones of cellular death, with a peak reached at 360 seconds. Furthermore, when EA was observed on T2-weighted MRI, zones of hypointensity closely approximated the region of cell death as confirmed by the fluorescent live / dead reporter. Correlational analysis revealed r = 0.985, R2 = 0.916, and p < 0.0001, suggesting that real-time T2-weighted MRI may be an accurate predictor of cell death induced by EA. T2-weighted MRI reliably detects alkaline and acidic pHs as relative signal hypointensity. This T2 prolongation enables the real-time visualization of the ablation zone created by EA, which has a strong correlation to the zone of histologic cell death in vitro.

Influence of mitochondrial DNA loss on the development of complex structured colonies of SK1 strains of yeast Saccharomyces cerevisiae

Aim. Determine the effect of mitochondrial DNA loss on the formation of complex yeast colonies of Saccharomyces cerevisiae. Methods. Development of giant colonies of the parent strain SK1 (rho+) and the "petit" strain SK1p, which lost mtDNA (rho0 mutation), was observed for 40 days,. To find out zones of cell death in colonies, both strains were cultured on solid YPD media containing dyes that accumulate in dead cells. The survival of the cells within colony was estimated by the ability to create microcolonies. Results. The loss of mitochondrial DNA in SK1p cells led to a decrease in colony area and to simplification of colony morphology on the YPD medium. When SK1p colonies were cultivated on media with addition of dyes, bright spot was formed in the center due to the dyes accumulation in dead cells. At the same time, parent strain developed a uniform insignificant coloration. Conclusions. rho0 mutation in SK1p yeast strain Saccharomyces cerevisiae led to a significant simplification of complex colony structure that formed on the nutrient medium YPD. The mitochondrial DNA loss in strain SK1p results in an accelerated cell death in the center of colony on YPD.Keywords: Saccharomyces cerevisiae, rho0, colonies, cell survival.

Abstract 195: Electrochemical treatment produces pH changes in the tumor microenvironment that are toxic to cancer cells

Abstract Background: Locoregional therapy is playing an increasingly central role in the management of hepatocellular carcinoma (HCC), where ablation is distinguished among these techniques by its capacity to effect cure. Electrochemical treatment (EChT), a technique that facilitates necrosis via the application of direct current at low voltages shows promise; however, widespread use has been limited as studies of the underlying biology and the mechanism of action have been limited. Methods: A novel assay for the study of EChT mechanism was designed, wherein HCC cells (2.5e5 cells/mL) were embedded in low-melting temperature agarose (1.5%). EChT was performed on these assays between nitinol cathodes (d = 0.5 mm) and platinum anodes (d = 0.5 mm) in a variety of geometries. Buffering capacity of the encapsulation assay was varied via the addition of HEPES buffer at final concentrations of 10mM, 50mM, or 200mM. Either during or immediately following EChT, the following measurements were recorded: cell viability, pH, ROS burden, transmembrane potential, and temperature. Results: Following EChT, acidic pH was appreciated surrounding the anodes and basic pH surrounding the cathodes in regions where cell death was observed by fluorescence microscopy. Upon increasing the buffering capacity of the assay, the total area of cell death decreased (p &amp;lt; 10-6). There was not sufficient heat generation nor transmembrane potential to account for the observed cell death. A pH-threshold was identified, where a pH &amp;gt; 10.8 or &amp;lt; 4.4 was found to cause cell necrosis in the three different buffer conditions studied. By varying the number of electrodes, their spacing, and length of ablation, it was possible to shape the zone of cell death observed to an arbitrary geometry. Conclusions: The mechanism of EChT-induced cancer cell death is through the spread of acidic and basic species generated by hydrolysis of water upon electron transfer at the electrodes. The diffusion of these species leads to cell death in a predictable, pH dependent manner. These results suggest that an electrochemical ablation device could treat complex HCC tumor geometries with appropriate electrode placement and charge delivery. Citation Format: Nicholas Perkons, Elliot Stein, Chike Nwaezeapu, Joseph Wildenberg, Daniel Ackerman, Gregory Nadolski, Stephen Hunt, Terence Gade. Electrochemical treatment produces pH changes in the tumor microenvironment that are toxic to cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 195.

Cell Death Is Not Sufficient for the Restriction of Potato Virus Y Spread in Hypersensitive Response-Conferred Resistance in Potato

Hypersensitive response (HR)-conferred resistance to viral infection restricts the virus spread and is accompanied by the induction of cell death, manifested as the formation of necrotic lesions. While it is known that salicylic acid is the key component in the orchestration of the events restricting viral spread in HR, the exact function of the cell death in resistance is still unknown. We show that potato virus Y (PVY) can be detected outside the cell death zone in Ny-1-mediated HR in potato plants (cv. Rywal), observed as individual infected cells or small clusters of infected cells outside the cell death zone. By exploiting the features of temperature dependent Ny-1-mediated resistance, we confirmed that the cells at the border of the cell death zone are alive and harbor viable PVY that is able to reinitiate infection. To get additional insights into this phenomenon we further studied the dynamics of both cell death zone expansion and occurrence of viral infected cell islands outside it. We compared the response of Rywal plants to their transgenic counterparts, impaired in SA accumulation (NahG-Rywal), where the lesions occur but the spread of the virus is not restricted. We show that the virus is detected outside the cell death zone in all lesion developmental stages of HR lesions. We also measured the dynamics of lesions expansion in both genotypes. We show that while rapid lesion expansion is observed in SA-depleted plants, virus spread is even faster. On the other hand the majority of analyzed lesions slowly expand also in HR-conferred resistance opening the possibility that the infected cells are eventually engulfed by cell death zone. Taken altogether, we suggest that the HR cell death is separated from the resistance mechanisms which lead to PVY restriction in Ny-1 genetic background. We propose that HR should be regarded as a process where the dynamics of events is crucial for effectiveness of viral arrest albeit the exact mechanism conferring this resistance remains unknown.

Reduction Of Metastasis With Plasma-Treated Liquid In Mice: The Role Of Myleoid Cells In The Tumor Microenvironment

Plasma-treated liquids have been proposed to be effective against tumor growth in vitro and in vivo. Recently, we have shown antitumor effects of DMEM exposed to the plasma of the kINPen MED in a murine, orthotopic pancreatic cancer model of diffuse peritoneal tumor metastasis [1]. Mice in the treatment group showed less metastasis formation, slower metastasis growth, increased apoptosis in tumor nodes, and better overall survival. At the same time, treatment was safe and did not affect other organs. In this study, we followed up on cellular and molecular events in the tumor microenvironment of peritoneal cancer lesions of treated compared to control mice. Special emphasize was given to myeloid cells. Tumor nodes were sectioned onto microscopy slides and stained with fluorescently labeled antibodies targeting subsets of myeloid cells. Congruent to the zone of apoptotic cell death, we identified a strong and significant influx of murine macrophages. As apoptosis was only on the tumor side exposed to the peritoneal cavity, macrophages likely influxed from this site. We next sought to identify their subtype with regard to the polarization paradigm. Almost all macrophages stained positive for CD206, a marker associated with M2 macrophages. Macrophages positive for inducible nitric oxide synthase (iNOS), a prominent marker for M1 macrophages, were nearly absent. Staining was confirmed with mouse tissues containing the respective population. This finding suggested that tumor growth was halted despite presence of M2 macrophages, which are thought to support tumor growth in general when becoming tumor-associated macrophages. Next, we stained for granulocytes influx. While we detected fewer granulocytes in tissues, we found indices for extracellular trap formation within tumor nodes, which are here linked to improved anticancer responses. Finally, we co-cultured murine PDA6606 pancreatic cancer cells with murine RAW-macrophages in vitro. Cancer cells strongly polarized macrophages to M2 but plasma treatment halted this to some extent. Plasma also increased migratory activity of macrophages but only when co-cultured with pancreatic cancer cells. Together with a rather inflammatory cytokine profile and cell surface marker expression with properties of M2 as well as M1, we propose a mixed response of murine RAW-macrophages in the tumor microenvironment upon plasma treatment. Download : Download high-res image (365KB) Download : Download full-size image Fig. 1: Tumor nodes were collected after 21 days with daily injection of NTP or medium (control), respectively and stained for F4/80 (A). The number of macrophages was significantly enhanced in treated tumors (B).

Factors Affecting Lethal Isotherms During Cryoablation Procedures.

Background:Creating appropriately-sized, lethal isotherms during cryoablation of renal tumors is critical in order to achieve sufficiently-sized zones of cell death. To ensure adequate cell death in target treatment locations, surgeons must carefully select the type, size, location, and number of probes to be used, as well as various probe operating parameters.Objective:The current study investigates the effects of probe type, operating pressure, and clinical method on the resulting sizes of isotherms in an in vitro gelatin model.Method:Using a total of four cryoprobes from two manufacturers, freeze procedures were conducted in gelatin in order to compare resulting sizes of constant temperature zones (isotherms). The effects of certain procedural parameters which are clinically adjustable were studied.Results:Test results show that the sizes of 0 °C,-20 °C and -40 °C isotherms created by similarly-sized probes from two different manufacturers were significantly different for nearly all comparisons made, and that size differences resulting from changing the operating pressure were not as prevalent. Furthermore, isotherm sizes created using a multiple freeze procedure (a ten minute freeze, followed by a five minute passive thaw, followed by another ten minute freeze) did not result in statistically-significant differences when compared to those created using a single freeze procedure in all cases.Conclusion:These results indicate that selection of the probe manufacturer and probe size may be more important for dictating the size of kill zones during cryoablation than procedural adjustments to operating pressures or freeze times.

Quantitative proteomics reveals protein profiles underlying major transitions in aspen wood development.

BackgroundWood development is of outstanding interest both to basic research and industry due to the associated cellulose and lignin biomass production. Efforts to elucidate wood formation (which is essential for numerous aspects of both pure and applied plant science) have been made using transcriptomic analyses and/or low-resolution sampling. However, transcriptomic data do not correlate perfectly with levels of expressed proteins due to effects of post-translational modifications and variations in turnover rates. In addition, high-resolution analysis is needed to characterize key transitions. In order to identify protein profiles across the developmental region of wood formation, an in-depth and tissue specific sampling was performed.ResultsWe examined protein profiles, using an ultra-performance liquid chromatography/quadrupole time of flight mass spectrometry system, in high-resolution tangential sections spanning all wood development zones in Populus tremula from undifferentiated cambium to mature phloem and xylem, including cell expansion and cell death zones. In total, we analyzed 482 sections, 20–160 μm thick, from four 47-year-old trees growing wild in Sweden. We obtained high quality expression profiles for 3,082 proteins exhibiting consistency across the replicates, considering that the trees were growing in an uncontrolled environment. A combination of Principal Component Analysis (PCA), Orthogonal Projections to Latent Structures (OPLS) modeling and an enhanced stepwise linear modeling approach identified several major transitions in global protein expression profiles, pinpointing (for example) locations of the cambial division leading to phloem and xylem cells, and secondary cell wall formation zones. We also identified key proteins and associated pathways underlying these developmental landmarks. For example, many of the lignocellulosic related proteins were upregulated in the expansion to the early developmental xylem zone, and for laccases with a rapid decrease in early xylem zones. We observed upregulation of two forms of xylem cysteine protease (Potri.002G005700.1 and Potri.005G256000.2; Pt-XCP2.1) in early xylem and their downregulation in late maturing xylem. Our data also show that Pt-KOR1.3 (Potri.003G151700.2) exhibits an expression pattern that supports the hypothesis put forward in previous studies that this is a key xyloglucanase involved in cellulose biosynthesis in primary cell walls and reduction of cellulose crystallinity in secondary walls.ConclusionOur novel multivariate approach highlights important processes and provides confirmatory insights into the molecular foundations of wood development.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2458-z) contains supplementary material, which is available to authorized users.