scholarly journals CRISPR/Cas9 assisted gene targeting efficiently inhibits bovine herpesvirus-1 replication

2021 ◽  
Author(s):  
Pallavi Deol ◽  
Sonalika Mahajan ◽  
Sukdeb Nandi ◽  
Vishal Chander ◽  
Ashwini R. Chaple ◽  
...  
Keyword(s):  
Virus Replication ◽  
Gene Editing ◽  
Essential Gene ◽  
Complete Inhibition ◽  
Specific Gene ◽  
Specific Method ◽  
Induced Apoptosis ◽  
The Impact ◽  
Constant Expression

Abstract Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/ CRISPR associated protein (cas) are now being accepted as a highly specific method of gene editing. Among many other applications, CRISPR/cas has an immense potential to be used as antivirals. In this study, we successfully demonstrated CRISPR/Cas9 mediated inhibition of Bovine Herpes virus -1 (BHV-1) replication. BHV-1 causes economically important diseases in bovines with establishment of latency. Six essential genes and one non-essential gene of BHV-1 were targeted to assess the impact on virus replication. Inhibition of UL52, circ, and UL27 genes showed promising results, whereas the other three genes US6, UL18, and UL34 resulted in lower level of inhibition. Non-specific gene editing in host and virus was in-silico evaluated and was demonstrated by inhibition of virus induced apoptosis. Successful editing of one viral non-essential gene without any alterations in virus replication demonstrated the potential of CRISPR/Cas9 in replicating viral genome. Complete abrogation of virus replication was observed transiently (~24 hours post-transfection/hpt) when transfected with short lived in-vitro transcribed sgRNAs. Whereas, under constant expression of sgRNAs through plasmid, complete inhibition of virus replication was observed till ~72 hours post-infection. Complete inhibition of replication was also observed with in-vitro transcribed sgRNA when booster dose of sgRNA was trasnfected at 24hpt. It has been speculated that constant expression with plasmid based delivery may result in off-target activity which can be ruled out with short lived in-vitro transcribed sgRNA.

VDAC1 Mediated Anticancer Activity of Gallic Acid in Human Lung Adenocarcinoma A549 Cells

2018 ◽  
Vol 18 (2) ◽  
pp. 255-262 ◽  
Author(s):  
Aikebaier Maimaiti ◽  
Amier Aili ◽  
Hureshitanmu Kuerban ◽  
Xuejun Li
Keyword(s):  
Western Blot ◽  
Cell Viability ◽  
Gallic Acid ◽  
Molecular Mechanisms ◽  
A549 Cells ◽  
Dependent Manner ◽  
Lung Carcinoma Cells ◽  
Induced Apoptosis ◽  
The Impact

Aims: Gallic acid (GA) is generally distributed in a variety of plants and foods, and possesses cell growth-inhibiting activities in cancer cell lines. In the present study, the impact of GA on cell viability, apoptosis induction and possible molecular mechanisms in cultured A549 lung carcinoma cells was investigated. Methods: In vitro experiments showed that treating A549 cells with various concentrations of GA inhibited cell viability and induced apoptosis in a dose-dependent manner. In order to understand the mechanism by which GA inhibits cell viability, comparative proteomic analysis was applied. The changed proteins were identified by Western blot and siRNA methods. Results: Two-dimensional electrophoresis revealed changes that occurred to the cells when treated with or without GA. Four up-regulated protein spots were clearly identified as malate dehydrogenase (MDH), voltagedependent, anion-selective channel protein 1(VDAC1), calreticulin (CRT) and brain acid soluble protein 1(BASP1). VDAC1 in A549 cells was reconfirmed by western blot. Transfection with VDAC1 siRNA significantly increased cell viability after the treatment of GA. Further investigation showed that GA down regulated PI3K/Akt signaling pathways. These data strongly suggest that up-regulation of VDAC1 by GA may play an important role in GA-induced, inhibitory effects on A549 cell viability.

The Impact of Two Embryo Culture Media, SOF and Commercial BO, on Pre- and Post-Implantation Development of Cloned Sannen Goat Embryos

2020 ◽  
Author(s):  
Mehdi Hajian ◽  
Farnoosh Jafarpour ◽  
Sayed Morteza Aghamiri ◽  
Shiva Rouhollahi Varnosfaderani ◽  
Mohsen Rahimi ◽  
...  
Keyword(s):  
Embryo Culture ◽  
Culture Media ◽  
Specific Gene ◽  
Limited Information ◽  
Major Drawback ◽  
Animal Cloning ◽  
The Impact ◽  
Post Implantation

Abstract Background: The ingredients of embryo culture media developed by different companies are disclosed. Thus, it is impossible to determine which ingredients might be responsible for differences in pre-and post-implantation embryo development. To address this gap, we performed an experiment to compare two embryo culture media, namely, SOF and commercial BO, on pre- and post-implantation development of cloned Sannen goat embryos. Cumulus oocyte complexes derived from slaughterhouse ovaries were used for in vitro embryo production . In vitro development of IVF, parthenogenetic and SCNT embryos were assessed in both BO and SOF media. The expression of 16 genes, including AKT , OCT4 , SOX2 , BMPR1 , FGFR4 , CDC25 , CDX2 , GCN5 , PCAF , FOXD3 , SMAD5 , FZD , LIFR1 , CTNNB , ERK1 , and IFNT , belonging to 7 important pathways, i.e. pluripotency, FGF, TGFβ, cell cycle and proliferation, histone transferase, trophectoderm, and WNT, were examined in the goat SCNT and IVF blastocysts from both BO and SOF media. Results: The blastocyst rate in BO medium was significantly higher than that of the SOF medium in SCNT embryos ( P < 0.05). All of the genes examined showed increased expression levels in SCNT embryos compared to IVF embryos. In the IVF group, OCT4 , BMPR1 , and GCN5 showed significantly higher expression in the SOF medium compared to the BO medium. In this group, AKT , FGFR4 , SOX2 showed significantly lower expression in the SOF medium compared to the BO medium. In the SCNT group, FGFR4 , GCN5 , FZD , CTNNB , BMPR1 , and FGFR4 showed significantly higher expression in SOF medium compared to BO medium. In vivo development did not differ significantly between the two groups. Conclusions: Based on these results, we concluded that the limited information available on the allocations of ICM and TE cells in SCNT embryos and embryo-specific gene expression may be the major drawback IVC medium and an impediment to successful animal cloning.

Azacytidine Suppressors Autocrine IL-6 Secretion and Demonstrates In Vitro and In Vivo Activity Against Multiple Myeloma.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1566-1566
Author(s):  
Tiffany Khong ◽  
Janelle Sharkey ◽  
Andrew Spencer
Keyword(s):  
Multiple Myeloma ◽  
Murine Model ◽  
Dna Methyltransferase ◽  
Myeloma Cell ◽  
Apoptotic Pathway ◽  
P16 Gene ◽  
Induced Apoptosis ◽  
The Impact

Abstract Azacytidine (AZA), a DNA methyltransferase inhibitor, has been shown to inhibit cell growth and induce apoptosis in some cancer cells. We determined the impact of AZA on a panel of human myeloma cell lines (HMCL); KMS 12PE, KMS 18, LP-1, NCI-H929, OPM-2, RPMI-8226 and U266 and in an in vivo murine model of multiple myeloma (5T33 model). Dose responsiveness to AZA was determined via MTS assays with a range of AZA doses (1–10mM) for 72 hours. FACS and cell cycle analysis were used to evaluate the profile of the cells after exposure to AZA for 72 hours. MTS assays demonstrated a dose and time dependent AZA-induced inhibition of HMCL viability with effective concentrations of AZA ranging from 1–10 mM. This was associated with accumulation of cells in the Go/G1 phase with decreasing number of cells in the S and G2/M phases. Western Blot analysis using antibodies against caspases 3,8,10, PARP, phospho-ERK, ERK, Stat3 and phospho -Stat3 were performed to help characterize the mechanism(s) of cell killing. Cleavage of caspases 3,8,10 and PARP within 24 hours of AZA treatment confirmed early AZA-induced HMCL apoptosis. phospho-ERK which was absent in untreated U266 appeared after 48 hours exposure to 5mM AZA. Similarly inhibitors of caspases 3,8 and 9 were used to determine which apoptotic pathway was being preferentially activated by AZA. Inhibitors of both caspase 3 and 9 effectively abrogated AZA-induced apoptosis in U266 and NCI-H929. In contrast caspase 8 inhibitor was less effective which is consistent with AZA acting via the mitochondrial apoptotic pathway. Reactivation of p16 gene by AZA-induced hypomethylation was assessed with methylation specific PCR. MSP-PCR of the p16 gene indicated a loss of methylation and up-regulated transcription after 48 hours treatment with 5 mM AZA. The level of IL-6 in conditioned media from U266 cells treated with AZA was determined by ELISA assay and demonstrated a rapid fall in autocrine IL-6 production. RT-PCR demonstrated rapid AZA-induced cessation of IL-6 transcription temporarily associated with the disappearance of upstream phospho -Stat3. Addition of exogenous IL-6 did not rescue U266 from AZA-induced apoptosis. AZA was also administered to a 5T33 murine model of multiple myeloma at increasing concentrations (1, 3, 10 mg/kg). At 10 mg/kg the median survival of vehicle versus AZA treated mice was 28 days versus 30+ days (p=0.003). These findings justify further evaluation of AZA as a potential therapeutic agent for multiple myeloma.

scholarly journals Biphasic Modulation of Apoptotic Pathways in Cryptosporidium parvum-Infected Human Intestinal Epithelial Cells

2008 ◽  
Vol 77 (2) ◽  
pp. 837-849 ◽  
Author(s):  
Jin Liu ◽  
Mingqi Deng ◽  
Cheryl A. Lancto ◽  
Mitchell S. Abrahamsen ◽  
Mark S. Rutherford ◽  
...  
Keyword(s):  
Cryptosporidium Parvum ◽  
Expression Patterns ◽  
Nuclear Condensation ◽  
Successful Infection ◽  
Cell Gene Expression ◽  
Infected Cells ◽  
Apoptotic Pathways ◽  
Induced Apoptosis ◽  
The Impact

ABSTRACT The impact of Cryptosporidium parvum infection on host cell gene expression was investigated by microarray analysis with an in vitro model using human ileocecal HCT-8 adenocarcinoma cells. We found changes in 333 (2.6%) transcripts at at least two of the five (6, 12, 24, 48, and 72 h) postinfection time points. Fifty-one of the regulated genes were associated with apoptosis and were grouped into five clusters based on their expression patterns. Early in infection (6 and 12 h), genes with antiapoptotic roles were upregulated and genes with apoptotic roles were downregulated. Later in infection (24, 48, and 72 h), proapoptotic genes were induced and antiapoptotic genes were downregulated, suggesting a biphasic regulation of apoptosis: antiapoptotic state early and moderately proapoptotic state late in infection. This transcriptional profile matched the actual occurrence of apoptosis in the infected cultures. Apoptosis was first detected at 12 h postinfection and increased to a plateau at 24 h, when 20% of infected cells showed nuclear condensation. In contrast, experimental silencing of Bcl-2 induced apoptosis in 50% of infected cells at 12 h postinfection. This resulted in a decrease in the infection rate and a reduction in the accumulation of meront-containing cells. To test the significance of the moderately proapoptotic state late in the infection, we inhibited apoptosis using pancaspase inhibitor Z-VAD-FMK. This treatment also affected the progression of C. parvum infection, as reinfection, normally seen late (24 h to 48 h), did not occur and accumulation of mature meronts was impaired. Control of host apoptosis is complex and crucial to the life of C. parvum. Apoptosis control has at least two components, early inhibition and late moderate promotion. For a successful infection, both aspects appear to be required.

scholarly journals 4-[1-ethyl-1-methylhexy]-phenol Induces Apoptosis and Interrupts Ca2+ Homeostasis via ROS Pathway in Sertoli TM4 cells

2021 ◽  
Author(s):  
Xiaozhen Liu ◽  
Fuxiang Li ◽  
Zhaoliang Zhu ◽  
Gaoyi Peng ◽  
Danfei Huang ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Atpase Activity ◽  
Chain Structure ◽  
Side Chain ◽  
Ros Generation ◽  
Intracellular Ca ◽  
Ros Scavenger ◽  
Induced Apoptosis ◽  
The Impact

Abstract Biological effect of an individual nonylphenol (NP) isomer extremely relies upon the side chain structure. This research was designed to evaluate the impact of NP isomer, 4-[1-ethyl-1-methylhexy]-phenol (NP65), on Sertoli cells in vitro. Sertoli TM4 cells were exposed to various concentration (0, 0.1, 1, 10, or 20 μM) of NP65 for 24 h, and the outcomes indicated that treatment of NP65 induced reactive oxygen species (ROS) generation, oxidative stress as well as apoptosis for Sertoli TM4 cells. In addition, it was found that NP65 exposure affected homeostasis of Ca2+ in Sertoli TM4 cells by increasing cytoplasm [Ca2+]i, inhibiting Ca2+-ATPase activity and decreasing cAMP concentration. Pretreatment with ROS scavenger, N-acetylcysteine (NAC), attenuated NP65-induced oxidative stress as well as apoptosis for TM4 cells. Furthermore, NAC blocked NP65-induced disorders of Ca2+ homeostasis by attenuating the growth of intracellular [Ca2+]i and the inhibition of Ca2+-ATPase and cAMP activities. Thus, we have demonstrated that NP65 induced apoptosis as well as acted as a potent inhibitor of Ca2+-ATPase activity and resulted in disorder of Ca2+ homeostasis in Sertoli TM4 cells, ROS participated in the process. Our results supported the view that oxidative stress acted an essential role within the development of apoptosis and Ca2+ overload in TM4 cells as a consequence of NP65 stimulation.

Evaluation of Mechanisms of Resistance to Arsenic Trioxide In Patients with Acute Promyelocytic Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2746-2746
Author(s):  
Ezhilarasi Chendamarai ◽  
Ansu Abu Alex ◽  
Saravanan Ganesh ◽  
Kavitha M Lakshmi ◽  
Sachin Jadhav ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Arsenic Trioxide ◽  
Acute Promyelocytic Leukemia ◽  
Promyelocytic Leukemia ◽  
Newly Diagnosed ◽  
Mechanisms Of Resistance ◽  
Induced Apoptosis ◽  
The Impact

Abstract Abstract 2746 Introduction: Newly diagnosed and relapsed acute promyelocytic leukemia (APL) patients respond to therapy with arsenic trioxide (ATO) based regimens. Significantly more patients with relapsed APL have disease recurrence after ATO based therapy than newly diagnosed cases. We undertook a series of experiments to evaluate the potential mechanisms to explain this increased recurrence rate in patients with relapsed APL. Patients and methods: From April 2007 to March 2009 bone marrow samples from newly diagnosed and relapsed cases admitted at our center were utilized for these studies. If required the bone marrow blasts and promyelocyte component was enriched to above 90% using a lineage depletion cocktail and VarioMACS (Miltenyi Biotec, Germany). For in-vitro intracellular ATO concentration measurement, 2 × 107 cells were washed and suspended in RPMI media with 0.5 μM concentration of ATO and incubated for 24 hours. Cells were then washed, made into a pellet and solubilized with HNO3 and H2O2 and ATO content measured using an atomic absorption spectrophotometer. An in-vitro sensitivity assay of malignant cells as previously reported was standardized using an MTT assay system. The impact of co-culture of mesenchymal stromal cells (MSC) and malignant promyelocytes on ATO induced apoptosis was studied with 7AAD and Annexin staining using a flowcytometer. A gene expression array using 44k human microarray chip analysis (Agilent technologies) was done on 8 newly diagnosed and 8 relapsed cases. Results: Sixty five patients were included in this study. Of these 47 (72%) were newly diagnosed and 18 (28%) were relapsed cases. On immunophenotyping, CD34 was positive (>20%) in 3.6% of newly diagnosed and 50% of relapsed cases (P=0.001). The mean MFI of the relapsed cases for expression of CD38, VLA-5 and CD13 was significantly lower in the relapsed group. The ability of both newly diagnosed and relapsed primary APL cells to concentrate ATO intracellular was not significantly different (15.2±9 nG/107 cell Vs. 16.3±9.7 nG/107 cell). Similarly the in-vitro IC-50 assay was not significantly different between the two groups (5.5±3.8 Vs. 4.7±4.5 μM). Neither of these assays correlate with clinical parameters such as relapse, event free (EFS) or overall survival (OS). Evaluation of the impact of MSC on ATO induced apoptosis demonstrates a protective effect in newly diagnosed and relapsed cases (Fig 1). This effect was mediated partly by the MSC conditioning media and could not be overcome by addition of VLA-4 or VLA-5 blocking antibodies (data not shown). Gene expression studies comparing the two cohorts revealed 1744 genes that were differentially expressed (>2 fold) between samples at diagnosis and at relapse. Quantitative Real-time RT-PCR using SYBR- Green detection system was done to confirm the gene expression results obtained from microarray analysis. Using ΔΔCT method the fold difference was calculated for five selected genes which validated the microarray data. Conclusion: Relapsed patients have significant immuno-phenotypic differences from newly diagnosed cases. Mechanisms of resistance to ATO are probably multi-factorial but are unlikely to be related to intra-cellular ATO concentration. In-vitro IC-50 does not appear to predict clinical outcomes. Stromal interaction protects malignant promyelocytes from the apoptotic action of ATO which appears more pronounced in relapsed than in newly diagnosed cases. Further evaluation of parameters that enhance such stromal interaction and protect malignant promyelocytes from the apoptotic effect of ATO along with evaluation of dysregulated genes and pathways are required. Disclosure: No relevant conflicts of interest to declare.

scholarly journals CRISPR/Cas9 in Cancer Immunotherapy: Animal Models and Human Clinical Trials

Genes ◽  
2020 ◽  
Vol 11 (8) ◽  
pp. 921 ◽  
Author(s):  
Khalil Khalaf ◽  
Krzysztof Janowicz ◽  
Marta Dyszkiewicz-Konwińska ◽  
Greg Hutchings ◽  
Claudia Dompe ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Animal Models ◽  
Risk Evaluation ◽  
Gene Editing ◽  
Immunological Response ◽  
Specific Gene ◽  
Success Rates ◽  
Treatment Resistant ◽  
In Vitro Systems

Even though chemotherapy and immunotherapy emerged to limit continual and unregulated proliferation of cancer cells, currently available therapeutic agents are associated with high toxicity levels and low success rates. Additionally, ongoing multi-targeted therapies are limited only for few carcinogenesis pathways, due to continually emerging and evolving mutations of proto-oncogenes and tumor-suppressive genes. CRISPR/Cas9, as a specific gene-editing tool, is used to correct causative mutations with minimal toxicity, but is also employed as an adjuvant to immunotherapy to achieve a more robust immunological response. Some of the most critical limitations of the CRISPR/Cas9 technology include off-target mutations, resulting in nonspecific restrictions of DNA upstream of the Protospacer Adjacent Motifs (PAM), ethical agreements, and the lack of a scientific consensus aiming at risk evaluation. Currently, CRISPR/Cas9 is tested on animal models to enhance genome editing specificity and induce a stronger anti-tumor response. Moreover, ongoing clinical trials use the CRISPR/Cas9 system in immune cells to modify genomes in a target-specific manner. Recently, error-free in vitro systems have been engineered to overcome limitations of this gene-editing system. The aim of the article is to present the knowledge concerning the use of CRISPR Cas9 technique in targeting treatment-resistant cancers. Additionally, the use of CRISPR/Cas9 is aided as an emerging supplementation of immunotherapy, currently used in experimental oncology. Demonstrating further, applications and advances of the CRISPR/Cas9 technique are presented in animal models and human clinical trials. Concluding, an overview of the limitations of the gene-editing tool is proffered.

scholarly journals Introduction of Virulence Markers in PB2 of Pandemic Swine-Origin Influenza Virus Does Not Result in Enhanced Virulence or Transmission

2010 ◽  
Vol 84 (8) ◽  
pp. 3752-3758 ◽  
Author(s):  
Sander Herfst ◽  
Salin Chutinimitkul ◽  
Jianqiang Ye ◽  
Emmie de Wit ◽  
Vincent J. Munster ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Virus Replication ◽  
Reverse Genetics ◽  
The Past ◽  
Virulence Markers ◽  
Genetic Signatures ◽  
Respiratory Droplets ◽  
The Impact

ABSTRACT In the first 6 months of the H1N1 swine-origin influenza virus (S-OIV) pandemic, the vast majority of infections were relatively mild. It has been postulated that mutations in the viral genome could result in more virulent viruses, leading to a more severe pandemic. Mutations E627K and D701N in the PB2 protein have previously been identified as determinants of avian and pandemic influenza virus virulence in mammals. These mutations were absent in S-OIVs detected early in the 2009 pandemic. Here, using reverse genetics, mutations E627K, D701N, and E677G were introduced into the prototype S-OIV A/Netherlands/602/2009, and their effects on virus replication, virulence, and transmission were investigated. Mutations E627K and D701N caused increased reporter gene expression driven by the S-OIV polymerase complex. None of the three mutations affected virus replication in vitro. The mutations had no major impact on virus replication in the respiratory tracts of mice and ferrets or on pathogenesis. All three mutant viruses were transmitted via aerosols or respiratory droplets in ferrets. Thus, the impact of key known virulence markers in PB2 in the context of current S-OIVs was surprisingly small. This study does not exclude the possibility of emergence of S-OIVs with other virulence-associated mutations in the future. We conclude that surveillance studies aimed at detecting S-OIVs with increased virulence or transmission should not rely solely on virulence markers identified in the past but should include detailed characterization of virus phenotypes, guided by genetic signatures of viruses detected in severe cases of disease in humans.

scholarly journals CircFNDC3B inhibits cell growth in abdominal aortic aneurysm by targeting the miR-1270/PDCD10 axis

2021 ◽  
Author(s):  
Baoping Deng ◽  
Yue Wei ◽  
Jinfeng Zhang ◽  
Na Zeng ◽  
Yulan He ◽  
...  
Keyword(s):  
Abdominal Aortic Aneurysm ◽  
Aortic Aneurysm ◽  
Luciferase Reporter ◽  
Vsmc Proliferation ◽  
Normal Tissues ◽  
Qrt Pcr ◽  
Abdominal Aortic ◽  
Induced Apoptosis ◽  
The Impact

Abstract BackgroudIn certain cancers, circRNA fibronectin type III domain containing 3B (circFNDC3B) may serve as a specific target for the treatment. However, the role and underlying regulatory mechanisms of circFNDC3B in abdominal aortic aneurysm (AAA) remain unknown.MaterialsCircFNDC3B expression in AAA and normal tissues were assessed by qRT-PCR. The biological functions of circFNDC3B were evaluated by MTT, flow cytometry and Caspase-3 activity assays. Furthermore, the molecular mechanism of circFNDC3B was demonstrated by RNA immunoprecipitation (RIP), dual-luciferase reporter assay, western blotting, qRT-PCR and rescue experiments. ResultsWe found that the expression of circFNDC3B was significantly upregulated in AAA clinical specimens. Functionally, overexpression of circFNDC3B inhibited vascular smooth muscle cell (VSMC) proliferation and induced apoptosis in vitro, yet, knockdown of circFNDC3B had the opposite effects. Mechanistically, circFNDC3B upregulated the expression of programmed cell death 10 (PDCD10) by acting as a molecular sponge for miR-1270. Notably, forced expression of PDCD10 countervailed the impact of circFNDC3B knockdown on AAA biological processes.ConclusionsOur data indicated that circFNDC3B promoted the progression of AAA by targeting the miR-1270/PDCD10 pathway, and may be a potential therapeutic target in the treatment of AAA.

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