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PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e11395
Author(s):  
Tai-Hsin Tsai ◽  
Chih-Hui Chang ◽  
Szu-Huai Lin ◽  
Yu-Feng Su ◽  
Yi-Cheng Tsai ◽  
...  

Objectives There is much evidence suggesting that inflammation contributes majorly to subarachnoid hemorrhage (SAH)-induced cerebral vasospasm and brain injury. miRNAs have been found to modulate inflammation in several neurological disorders. This study investigated the effect of miR-195-5p on SAH-induced vasospasm and early brain injury in experimental rats. Methods Ninety-six Sprague-Dawley male rats were randomly and evenly divided into a control group (no SAH, sham surgery), a SAH only group, a SAH + NC-mimic group, and a SAH + miR-195-5p group. SAH was induced using a single injection of blood into the cisterna magna. Suspensions containing NC-mimic and miR-195-5p were intravenously injected into rat tail 30 mins after SAH was induced. We determined degree of vasospasm by averaging areas of cross-sections the basilar artery 24h after SAH. We measured basilar artery endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κ B), phosphorylated NF-κ B (p-NF-κ B), inhibitor of NF-κ B (Iκ Bα) and phosphorylated-Iκ Bα (p-Iκ Bα). Cell death assay was used to quantify the DNA fragmentation, an indicator of apoptotic cell death, in the cortex, hippocampus, and dentate gyrus. Tumor necrosis factor alpha (TNF-α) levels were measured using sample protein obtained from the cerebral cortex, hippocampus and dentate gyrus. Results Prior to fixation by perfusion, there were no significant physiological differences among the control and treatment groups. SAH successfully induced vasospasm and early brain injury. MiR-195-5p attenuated vasospasam-induced changes in morphology, reversed SAH-induced elevation of iNOS, p-NF-κ B, NF-κ B, and p-Iκ Bα and reversed SAH-induced suppression of eNOS in the basilar artery. Cell death assay revealed that MiR-195-5p significantly decreased SAH-induced DNA fragmentation (apoptosis) and restored TNF-α level in the dentate gyrus. Conclusion In conclusion, MiRNA-195-5p attenuated SAH-induced vasospasm by up-regulating eNOS, down-regulating iNOS and inhibiting the NF-κ B signaling pathway. It also protected neurons by decreasing SAH-induced apoptosis-related cytokine TNF-α expression in the dentate gyrus. Further study is needed to elucidate the detail mechanism underlying miR-195-5p effect on SAH-induced vasospasm and cerebral injury. We believe that MiR-195-5p can potentially be used to manage SAH-induced cerebral vasospasm and brain injury.


Cancers ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 653
Author(s):  
Sharath Bhagavatula ◽  
Devon Thompson ◽  
Sebastian W. Ahn ◽  
Kunj Upadhyaya ◽  
Alex Lammers ◽  
...  

By observing the activity of anti-cancer agents directly in tumors, there is potential to greatly expand our understanding of drug response and develop more personalized cancer treatments. Implantable microdevices (IMD) have been recently developed to deliver microdoses of chemotherapeutic agents locally into confined regions of live tumors; the tissue can be subsequently removed and analyzed to evaluate drug response. This method has the potential to rapidly screen multiple drugs, but requires surgical tissue removal and only evaluates drug response at a single timepoint when the tissue is excised. Here, we describe a “lab-in-a-tumor” implantable microdevice (LIT-IMD) platform to image cell-death drug response within a live tumor, without requiring surgical resection or tissue processing. The LIT-IMD is inserted into a live tumor and delivers multiple drug microdoses into spatially discrete locations. In parallel, it locally delivers microdose levels of a fluorescent cell-death assay, which diffuses into drug-exposed tissues and accumulates at sites of cell death. An integrated miniaturized fluorescence imaging probe images each region to evaluate drug-induced cell death. We demonstrate ability to evaluate multi-drug response over 8 h using murine tumor models and show correlation with gold-standard conventional fluorescence microscopy and histopathology. This is the first demonstration of a fully integrated platform for evaluating multiple chemotherapy responses in situ. This approach could enable a more complete understanding of drug activity in live tumors, and could expand the utility of drug-response measurements to a wide range of settings where surgery is not feasible.


2021 ◽  

Background: MicroRNAs have been recently declared to be contributed to the various aspects of osteosarcoma cells, including growth and survival, apoptosis, invasion, and chemoresistance. Objectives: The present study aimed to investigate the potentiating effects of miR-129 on the chemosensitivity of Saose-2 osteosarcoma cells to methotrexate (MTX) and underlying mechanisms. Methods: Saose-2 cells were transfected with miR-129 mimics using Lipofectamine. The cytotoxic effects of miR-129 and MTX on Saose-2 cells were measured using MTT assay. Scratch wound healing assay was used to evaluate cell migration. The apoptosis rate of cancer cells was also measured using ELISA Cell Death Assay and flow cytometry. The mRNA expression levels of target genes were measured using quantitative RT-PCR. Results: miR-129 mimic transfection significantly increased the expression levels of this miRNA in Saose-2 cells (P<0.05). The combination of MTX with miR-129 transfection led to enhanced cytotoxic effects of MTX in lower concentrations. In addition, miR-129 significantly increased MTX-induced apoptosis levels and decreased invasion behavior in Saose-2 cells. The mRNA expression levels of c-Myc, K-Ras, CXCR4, MMP9, and ADAMTS, as main genes involved in chemoresistance and invasion, were downregulated in miR-129 transfected cells. Conclusion: The obtained results revealed the importance of miR-129 in the sensitivity of osteosarcoma cells to MTX and its underlying mechanisms. Therefore, miR-129 might be an appropriate candidate for reversing MTX resistance in osteosarcoma cells.


Pharmaceutics ◽  
2020 ◽  
Vol 12 (6) ◽  
pp. 497
Author(s):  
Farhatun Najat Maluin ◽  
Mohd Zobir Hussein ◽  
Nor Azah Yusof ◽  
Abu Seman Idris ◽  
Leona Daniela Jeffery Daim ◽  
...  

Health risks which result from exposure to pesticides have sparked awareness among researchers, triggering the idea of developing nanoencapsulation pesticides with the aim to enhance cytoprotection as well as genoprotection of the pesticides. In addition, nanocapsules of pesticides have slow release capability, high bioavailability, and site-specific delivery, which has attracted great interest from researchers. Hence, the objective of this work is to synthesize a nanoformulation of a fungicide of different sizes, namely, chitosan-hexaconazole nanoparticles (18 nm), chitosan-dazomet nanoparticles (7 nm), and chitosan-hexaconazole-dazomet nanoparticles (5 nm), which were then subjected to toxicological evaluations, including cytotoxicity, genotoxicity, cell death assay, and dermal irritation assays. Incubation of chitosan-based nanofungicides with V79-4 hamster lung cell did not reveal cytotoxicity or genotoxicity, potentially suggesting that encapsulation with chitosan reduces direct toxicity of the toxic fungicides. Meanwhile, pure fungicide revealed its high cytotoxic effect on V79-4 hamster lung cells. In addition, dermal exposure assessment on rabbits revealed that chitosan-hexaconazole nanoparticles are classified under corrosive subcategory 1C, while chitosan-dazomet nanoparticles are classified under corrosive subcategory 1B. Moreover, both chitosan-hexaconazole nanoparticles and chitosan-dazomet nanoparticles are classified as causing mild irritation.


Cells ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 703 ◽  
Author(s):  
Robin Demuynck ◽  
Iuliia Efimova ◽  
Abraham Lin ◽  
Heidi Declercq ◽  
Dmitri V. Krysko

The failure of drug efficacy in clinical trials remains a big issue in cancer research. This is largely due to the limitations of two-dimensional (2D) cell cultures, the most used tool in drug screening. Nowadays, three-dimensional (3D) cultures, including spheroids, are acknowledged to be a better model of the in vivo environment, but detailed cell death assays for 3D cultures (including those for ferroptosis) are scarce. In this work, we show that a new cell death analysis method, named 3D Cell Death Assay (3DELTA), can efficiently determine different cell death types including ferroptosis and quantitatively assess cell death in tumour spheroids. Our method uses Sytox dyes as a cell death marker and Triton X-100, which efficiently permeabilizes all cells in spheroids, was used to establish 100% cell death. After optimization of Sytox concentration, Triton X-100 concentration and timing, we showed that the 3DELTA method was able to detect signals from all cells without the need to disaggregate spheroids. Moreover, in this work we demonstrated that 2D experiments cannot be extrapolated to 3D cultures as 3D cultures are less sensitive to cell death induction. In conclusion, 3DELTA is a more cost-effective way to identify and measure cell death type in 3D cultures, including spheroids.


2019 ◽  
Vol 8 (2) ◽  
pp. 101-106
Author(s):  
Amin Mohammadi ◽  
Ali Mostafaie ◽  
Ahmad Bagheri ◽  
Sarah Kiani ◽  
Maryam Chalabi

Background: Breast cancer is the most common cause of cancer-related death in women worldwide. Therefore, there is an urget need to identify and develop therapeutic strategies against this deadly disease. This study is the first to investigate the effects of Hemolymph Serum of Potamon persicum Crab (HSPPC) on MCF-7 and MDA-231 breast cancer cell lines. Materials and Methods: LDH and MTT assays were performed on MCF-7 and MDA-231 breast cancer cell lines as well as human umbilical vein endothelial cells (HUVEC) to determine the cytotoxic and antiproliferative activity of the HSPPC at different concentrations. Further, the apoptosis inducing action of the hemolymph serum was determined by TUNEL (terminal deoxynucleotidyl transferasemediated dUTP nick end labeling) and cell death assay. Results: The IC50 values of HSPPC for MCF-7 and MDA-231 cell lines were 960±0.369 and 850±1.422 μg/mL, respectively. The growth of both MCF-7 and MDA-231 cell lines were significantly (P<0.001) inhibited by HSPPC as compared with untreated controls at 48 hours. The results showed that HSPPC had no cytotoxic effects but significantly inhibited cell growth in a dose and time dependent manner. In addition, DNA fragmentation analysis (TUNEL) and cell death assay indicated induction of apoptosis by HSPPC in MCF-7 and MDA-231 cell lines. Conclusion: The results suggest that HSPPC contains bioactive compound(s) with potentials for the treatment of breast cancer.


Plant Methods ◽  
2019 ◽  
Vol 15 (1) ◽  
Author(s):  
Isabel M. L. Saur ◽  
Saskia Bauer ◽  
Xunli Lu ◽  
Paul Schulze-Lefert

Abstract Background Plant disease resistance to host-adapted pathogens is often mediated by host nucleotide-binding and leucine-rich repeat (NLR) receptors that detect matching pathogen avirulence effectors (AVR) inside plant cells. AVR-triggered NLR activation is typically associated with a rapid host cell death at sites of attempted infection and this response constitutes a widely used surrogate for NLR activation. However, it is challenging to assess this cell death in cereal hosts. Results Here we quantify cell death upon NLR-mediated recognition of fungal pathogen AVRs in mesophyll leaf protoplasts of barley and wheat. We provide measurements for the recognition of the fungal AVRs AvrSr50 and AVRa1 by their respective cereal NLRs Sr50 and Mla1 upon overexpression of the AVR and NLR pairs in mesophyll protoplast of both, wheat and barley. Conclusions Our data demonstrate that the here described approach can be effectively used to detect and quantify death of wheat and barley cells induced by overexpression of NLR and AVR effectors or AVR effector candidate genes from diverse fungal pathogens within 24 h.


2018 ◽  
Vol 56 ◽  
pp. 48-54
Author(s):  
Daniel Camara Teixeira ◽  
Elizabeth L. Cordonier ◽  
Subhashinee S.K. Wijeratne ◽  
Patricia Huebbe ◽  
Augusta Jamin ◽  
...  

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3790-3790
Author(s):  
Line Groth-Pedersen ◽  
Yen-Hsi Chen ◽  
Marianne Faber ◽  
Birgitte Klug Albertsen ◽  
Peder Skov Wehner ◽  
...  

Abstract The survival rate of childhood acute lymphoblastic leukemia (ALL) is now close to 90 %. However, 15-20 % of patients still experience relapse, which is caused by persistence of minimal residual disease (MRD) in the bone marrow during chemotherapy. Thus, a method for identification of drugs that are capable of eliminating this cell population is necessary, but not currently available. Indeed, current and past ALL in vitro sensitivity assays demand high cell numbers (100,000 ALL cells per well in a 96-well plate), which is beyond what can routinely be harvested from the MRD population in the BM at the end of induction therapy. Accordingly, we developed a cell death assay that is far more sensitive and needs only 1,000 cells per well in a 96-well plate. It is based on stainings with annexinV-V450, a marker for early apoptosis, and Live/Dead Yellow, a marker for loss of cell membrane integrity, which can be quantified by flow cytometry. In order to compare patient samples, the viable fraction of treated cells were normalized to the viable fraction of untreated cells and classified as the survival index (SI). Assay time was set to 24 hours. When developing the assay we included the drugs that are currently used in the NOPHO ALL2008 induction therapy, namely vincristine, doxorubicin, dexamethasone and prednisolone. We determined one empirically derived cut-off concentration per drug and per cell density that produced a large scatter of SIs. Thereby only one drug concentration per cell density was needed for in vitro sensitivity testing (Vincristine: 0.09 and 9.23 mg/ml; doxorubicin: 0.5 and 2 mg/ml; prednisolone: 50 and 200 mg/ml; dexamethasone 2 and 100 mg/ml). The cell death assay showed only minor day-to-day variation (e.g. 0.09 mg/ml vincristine: N=15; rS= 0.92, p < 0.001) (Figure 1). In order to simulate a situation where FACS would be used for isolation of ALL subpopulations or MRD prior to in vitro sensitivity testing, ALL cells were mixed with peripheral blood mononuclear cells (MNCs) from healthy individuals to a final leukemic cell concentration of 10-2 and thereafter Fluorescence-activated cell sorted (FACSed) directly into a 96-well plate for exposure to chemotherapeutic drugs. The SI obtained was highly correlated to that of ALL cells that were not sorted, but directly and manually pipetted into the 96-well plate (e.g. 0.09 mg/ml vincristine: N=9; rS= 0.90, p = 0.001) (Figure 2). Next, we tested whether the in vitro sensitivity results obtained using 1,000 cells per well would resemble results obtained using 100,000 cells per well (i.e. the current standard). We exposed 1,000 and 100,000 cells per well in a 96-well plate from 36 ALL patients and three healthy individuals to the drugs listed above. For all drugs the SI for 1,000 and 100,000 cells per well were significantly correlated (vincristine: rs=0.65, p < 0.001; doxorubicin: rs= 0.48, p = 0.003; dexamethasone: rs= 0.55, p < 0.001; prednisolone: rs=0.35, p=0.04). Thus, we have developed a sensitive, yet robust in vitro sensitivity testing platform that includes FACS prior to cell death analysis. It demands only 3,000 ALL cells per drug to be tested in triplicates, and 3,000 cells that serve as untreated controls. Since a standard follow-up BM sample at day 28 after diagnosis contains at least 20 million MNCs in total, it enables in vitro sensitivity profiling of patients with MRD value > 0.1 %, potentially allowing future tailored chemotherapy according to the chemosensitivity profile of residual leukemia. Disclosures No relevant conflicts of interest to declare.


2014 ◽  
Vol 2014 (6) ◽  
pp. pdb.prot080267-pdb.prot080267 ◽  
Author(s):  
K. J. Falkenberg ◽  
D. N. Saunders ◽  
K. J. Simpson

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