RNA interference (RNAi) is an effective and important tool used to study gene function. For large-scale screens, RNAi is used to systematically down-regulate genes of interest and analyze their roles in a biological process. However, RNAi... more
RNA interference (RNAi) is an effective and important tool used to study gene function. For large-scale screens, RNAi is used to systematically down-regulate genes of interest and analyze their roles in a biological process. However, RNAi is associated with off-target effects (OTEs), including microRNA (miRNA)-like OTEs. The contribution of reagent-specific OTEs to RNAi screen data sets can be significant. In addition, the post-screen validation process is time and labor intensive. Thus, the availability of robust approaches to identify candidate off-targeted transcripts would be beneficial. Significant efforts have been made to eliminate false positive results attributable to sequence-specific OTEs associated with RNAi. These approaches have included improved algorithms for RNAi reagent design, incorporation of chemical modifications into siRNAs, and the use of various bioinformatics strategies to identify possible OTEs in screen results. Genome-wide Enrichment of Seed Sequence mat...
Protein kinase CK2, a protein serine/threonine kinase, plays a global role in activities related to cell growth, cell death, and cell survival. CK2 has a large number of potential substrates localized in diverse locations in the cell... more
Protein kinase CK2, a protein serine/threonine kinase, plays a global role in activities related to cell growth, cell death, and cell survival. CK2 has a large number of potential substrates localized in diverse locations in the cell including, for example, NF-κB as an important downstream target of the kinase. In addition to its involvement in cell growth and proliferation it is also a potent suppressor of apoptosis, raising its key importance in cancer cell phenotype. CK2 interacts with diverse pathways which illustrates the breadth of its impact on the cellular machinery of both cell growth and cell death giving it the status of a “master regulator” in the cell. With respect to cancer, CK2 has been found to be dysregulated in all cancers examined demonstrating increased protein expression levels and nuclear localization in cancer cells compared with their normal counterparts. We originally proposed CK2 as a potentially important target for cancer therapy. Given the ubiquitous and essential for cell survival nature of the kinase, an important consideration would be to target it specifically in cancer cells while sparing normal cells. Towards that end, our design of a tenascin based sub-50 nm (i.e., less than 50 nm size) nanocapsule in which an anti-CK2 therapeutic agent can be packaged is highly promising because this formulation can specifically deliver the cargo intracellularly to the cancer cells in vivo. Thus, appropriate strategies to target CK2 especially by molecular approaches may lead to a highly feasible and effective approach to eradication of a given cancer.
Aim: Rabies virus infections are a global threat to human and animal health, yet no progressive curative therapy has been developed. In this study, the nucleoprotein gene of rabies virus which is responsible for viral infection was used... more
Aim: Rabies virus infections are a global threat to human and animal health, yet no progressive curative therapy has been developed. In this study, the nucleoprotein gene of rabies virus which is responsible for viral infection was used as a target to design our desired siRNA. Methods: The conserved regions were analyzed by doing alignment of sequences from different strains. Subsequently, different computational tools were used for designing and validation of siRNA molecules. Results: We identified four probable siRNA molecules from twelve different strains of rabies virus which may silence the nucleoprotein gene and inhibit the multiplication of the virus. Conclusion: Our study may help to take an effective therapeutic approach against rabies virus and lead to better control of rabies in humans.
The liver is the second largest organ in the human body and is responsible for several functions that directly contribute to homeostasis. Hepatocytes are the main parenchymal liver cells that regulate multiple biochemical and metabolic... more
The liver is the second largest organ in the human body and is responsible for several functions that directly contribute to homeostasis. Hepatocytes are the main parenchymal liver cells that regulate multiple biochemical and metabolic functions and the synthesis of substances important to the body. Mesenchymal stem cells (MSCs) are a group of stem cells derived from the mesoderm, which can be obtained from various tissues. Under certain conditions , MSCs can differentiate into several cell types, including hepatocytes. Post-transcriptional regulations of liver development signalling and hepatocyte differentiation have been demonstrated. At the post-transcriptional level, microRNAs have emerged as precursors for determining cell fate during differentiation. MicroRNAs (miRNAs) are small non-coding RNAs involved in the post-transcriptional regulation of gene expression. They can determine the stem cell fate by repressing the translation of target mRNAs. In this review, we outline signalling pathways involved in stem cell differentiation to hepatocytes and its interplay with liver development. He-patic differentiation models in two-dimensional and three-dimensional cultures used to analyse signalling mechanisms will be described. We also highlight the possible miRNAs involved in this process and the transdifferentiation signalling mechanisms present in hepatocytes.
Polymer hydrogels have been widely explored as therapeutic delivery matrices because of their ability to present sustained, localized and controlled release of bioactive factors. Bioactive factor delivery from injectable biopolymer... more
Polymer hydrogels have been widely explored as therapeutic delivery matrices because of their ability to present sustained, localized and controlled release of bioactive factors. Bioactive factor delivery from injectable biopolymer hydrogels provides a versatile approach to treat a wide variety of diseases, to direct cell function and to enhance tissue regeneration. The innovative development and modification of both natural-(e.g., alginate (ALG), chitosan, hyaluronic acid (HA), gelatin, heparin (HEP), etc.) and synthetic-(e.g., polyesters, polyethyleneimine (PEI), etc.) based polymers has resulted in a variety of approaches to design drug delivery hydrogel systems from which loaded therapeutics are released. This review presents the state-of-the-art in a wide range of hydrogels that are formed though self-assembly of polymers and peptides, chemical crosslinking, ionic crosslinking and biomolecule recognition. Hydrogel design for bioactive factor delivery is the focus of the first s...
The SARS-CoV-2 or COVID-19 pandemic has reached a new height with an unprecedented infection rate and mortality post-world war II history. However, there is no particular designed drug for COVID-19 up to this point. Thus, there exist... more
The SARS-CoV-2 or COVID-19 pandemic has reached a new height with an unprecedented infection rate and mortality post-world war II history. However, there is no particular designed drug for COVID-19 up to this point. Thus, there exist three strategies for COVID-19 drug design; drug repurposing option, herbal medicine development, and transcriptomics-based drug lead. As the most underutilized option, transcriptomics-based drug lead could be leveraged to deal with SARS-CoV-2 infection. One of the main methods to block the SARS-CoV-2 infection is to inhibit the RNA polymerase enzyme that is responsible to the viral replication. In this regard, the objective of the strategy is to design the anti-sense siRNA drug and lead to inhibit the mRNA of the RNA Polymerase Enzyme (RdRp) gene that encodes the viral RNA Polymerase of the SARS-CoV-2. The Computer-Aided Drug Design (CADD)-based method was leveraged with sequence retrieval of 24 RdRp gene sequences, multiple sequence alignment, phylogenetic tree reconstruction, 2D/3D RNA structure predictions, and RNA-RNA docking. Both the RNAalifold conserved structure from the RdRp genes and the RNAfold structure of the siRNA for blocking the conserved structure are negative or less than 0 kcal/mol. The predicted RNA docking occurred with the best RMSD score of 22.53 Å, which is beyond the accepted threshold of 10-20 Å. Based on the findings, the 2D/3D structures of both the siRNA and mRNA could be elucidated, and the docking between them is feasible. However, this finding should be elucidated in the wet laboratory setting for the final lead validation.
Nanotechnology, which deals with features as small as a 1 billionth of a meter, began to enter into mainstream physical sciences and engineering some 20 years ago. Recent applications of nanoscience include the use of nanoscale materials... more
Nanotechnology, which deals with features as small as a 1 billionth of a meter, began to enter into mainstream physical sciences and engineering some 20 years ago. Recent applications of nanoscience include the use of nanoscale materials in electronics, catalysis, and biomedical research. Among these applications, strong interest has been shown to biological processes such as blood coagulation control and multimodal bioimaging, which has brought about a new and exciting research field called nanobiotechnology. Biotechnology, which itself also dates back ∼30 years, involves the manipulation of macroscopic biological systems such as cells and mice in order to understand why and how molecular level mechanisms affect specific biological functions, e.g., the role of APP (amyloid precursor protein) in Alzheimer’s disease (AD). This review aims (1) to introduce key concepts and materials from nanotechnology to a non-physical sciences community; (2) to introduce several state-of-the-art examples of current nanotechnology that were either constructed for use in biological systems or that can, in time, be utilized for biomedical research; (3) to provide recent excerpts in nanotoxicology and multifunctional nanoparticle systems (MFNPSs); and (4) to propose areas in neuroscience that may benefit from research at the interface of neurobiologically important systems and nanostructured materials.
Національний університет біоресурсів і природокористування України НДІ фітомедицини, біотехнологій та екології Факультет захисту рослин, біотехнологій та екології Агенство з трансферу технологій, освіти і науки daRostim Німецький... more
Національний університет біоресурсів і природокористування України НДІ фітомедицини, біотехнологій та екології Факультет захисту рослин, біотехнологій та екології Агенство з трансферу технологій, освіти і науки daRostim Німецький приватний інститут прикладної біотехнології daRostim БІОЛОГІЧНО АКТИВНІ ПРЕПАРАТИ В РОСЛИННИЦТВІ НАУКОВЕ ОБҐРУНТУВАННЯ-РЕКОМЕНДАЦІЇ-ПРАКТИЧНІ РЕЗУЛЬТАТИ Матеріали XV Міжнародної науково-практичної конференції Київ, 25-29 червня 2019 року
The frontline of plant defense against non-viral pathogens such as bacteria, fungi and oomycetes is provided by transmembrane pattern recognition receptors that detect conserved pathogen-associated molecular patterns (PAMPs), leading to... more
The frontline of plant defense against non-viral pathogens such as bacteria, fungi and oomycetes is provided by transmembrane pattern recognition receptors that detect conserved pathogen-associated molecular patterns (PAMPs), leading to pattern-triggered immunity (PTI). To counteract this innate defense, pathogens deploy effector proteins with a primary function to suppress PTI. In specific cases, plants have evolved intracellular resistance (R) proteins detecting isolate-specific pathogen effectors, leading to effector-triggered immunity (ETI), an amplified version of PTI, often associated with hypersensitive response (HR) and programmed cell death (PCD). In the case of plant viruses, no conserved PAMP was identified so far and the primary plant defense is thought to be based mainly on RNA silencing, an evolutionary conserved, sequence-specific mechanism that regulates gene expression and chromatin states and represses invasive nucleic acids such as transposons. Endogenous silencin...
Human immunodeficiency virus type 1 (HIV-1) following integration hijacks host cell machineries where chromatinization of the viral genome regulates its latency, transcription, and replication. The cooperation among ATP-dependent... more
Human immunodeficiency virus type 1 (HIV-1) following integration hijacks host cell machineries where chromatinization of the viral genome regulates its latency, transcription, and replication. The cooperation among ATP-dependent chromatin remodeling factors, posttranslational modifying enzymes, and histone chaperones is well established during transcriptional activation in eukaryotes. However, the role of histone chaperones in transcription of the HIV promoter is poorly understood. Previous studies from our group have established the role of the human histone chaperone nucleophosmin (NPM1) in the acetylation-dependent chromatin transcription. NPM1 is known to interact with HIV-Tat. Here, we report that infection by HIV-1 induces the acetylation of histone chaperone NPM1. Acetylation of NPM1 was found to be critical for nuclear localization of Tat as well as Tat-mediated transcription alluding to the critical role for the host factor towards viral pathogenesis. Furthermore, knockdown experiments mediated by small interfering RNA identified the critical role played by the chaperone NPM1 in transcriptional activation of the integrated provirus. These results shed further insights into the possible role of histone chaperone NPM1 acetylation in viral gene transcription, which could be a potential therapeutic target.► In this study, we report that HIV-1 infection induces the acetylation of NPM1. ► Acetylation of NPM1 is critical for Tat-mediated transcription. ► It is also required for the nuclear localization of Tat. ► Acetylated NPM1 is enriched on the HIV-1 promoter in a Tat-dependent manner.
In this article, we identify and characterise the miRNA machinery components Drosha, Dicer-1 and Argonaute-1 of the desert locust. By means of phylogenetic analyses, we reveal important insights in the evolutionary context of these... more
In this article, we identify and characterise the miRNA machinery components Drosha, Dicer-1 and Argonaute-1 of the desert locust. By means of phylogenetic analyses, we reveal important insights in the evolutionary context of these components. Our data illustrate that insect Argonaute-1 proteins form a monophyletic group with ALG-1 and ALG-2 of Caenorhabditis elegans and with the four (non-Piwi) Argonaute proteins present in humans. On the other hand, humans apparently lack clear homologues of the insect Argonaute-2 proteins. In addition, we demonstrate that drosha, dicer-1 and argonaute-1 display wide transcript tissue-distribution in adult desert locusts, and that during locust phase transition and feeding of starved locusts the expression levels of the miRNA pathway are regulated at the transcript level.
Cotton leaf curl virus disease (CLCuD) is caused by a suite of whitefly-transmitted begomovirus species and strains, resulting in extensive losses annually in India and Pakistan. RNA-interference (RNAi) is a proven technology used for... more
Cotton leaf curl virus disease (CLCuD) is caused by a suite of whitefly-transmitted begomovirus species and strains, resulting in extensive losses annually in India and Pakistan. RNA-interference (RNAi) is a proven technology used for knockdown of gene expression in higher organisms and viruses. In this study, a small interfering RNA (siRNA) construct was designed to target the AC1 gene of Cotton leaf curl Kokhran virus-Burewala (CLCuKoV-Bu) and the βC1 gene and satellite conserved region of the Cotton leaf curl Multan betasatellite (CLCuMB). The AC1 gene and CLCuMB coding and non-coding regions function in replication initiation and suppression of the plant host defense pathway, respectively. The construct, Vβ, was transformed into cotton plants using the Agrobacterium-mediated embryo shoot apex cut method. Results from fluorescence in situ hybridization and karyotyping assays indicated that six of the 11 T₁ plants harbored a single copy of the Vβ transgene. Transgenic cotton plant...
Cellular uptake of therapeutic oligonucleotides and subsequent intracellular trafficking to their target sites represents the major technical hurdle for the biological effectiveness of these potential drugs. Accordingly, laboratories... more
Cellular uptake of therapeutic oligonucleotides and subsequent intracellular trafficking to their target sites represents the major technical hurdle for the biological effectiveness of these potential drugs. Accordingly, laboratories worldwide focus on the development of suitable delivery systems. Among the different available non-viral systems like cationic polymers, cationic liposomes and polymeric nanoparticles, cell-penetrating peptides (CPPs) represent an attractive concept to bypass the problem of poor membrane permeability of these charged macromolecules. While uptake per se in most cases does not represent the main obstacle of nucleic acid delivery in vitro, it becomes increasingly apparent that intracellular trafficking is the bottleneck. As a consequence, in order to optimize a given delivery system, a side-by-side analysis of nucleic acid cargo internalized and the corresponding biological effect is required to determine the overall efficacy. In this review, we will concentrate on peptide-mediated delivery of siRNAs and steric block oligonucleotides and discuss different methods for quantitative assessment of the amount of cargo taken up and how to correlate those numbers with biological effects by applying easy to handle reporter systems. To illustrate current limitations of non-viral nucleic acid delivery systems, we present own data as an example and discuss options of how to enhance trafficking of molecules entrapped in cellular compartments.
microRNAs (miRNAs) are tiny ribo-regulatory molecules involved in various essential pathways for persistence of cellular life, such as development, environmental adaptation, and stress response. In recent years, miRNAs have become a major... more
microRNAs (miRNAs) are tiny ribo-regulatory molecules involved in various essential pathways for persistence of cellular life, such as development, environmental adaptation, and stress response. In recent years, miRNAs have become a major focus in molecular biology because of their functional and diagnostic importance. This interest in miRNA research has resulted in the development of many specific software and pipelines for the identification of miRNAs and their specific targets, which is the key for the elucidation of miRNA-modulated gene expression. While the well-recognized importance of miRNAs in clinical research pushed the emergence of many useful computational identification approaches in animals, available software and pipelines are fewer for plants. Additionally, existing approaches suffers from mis-identification and annotation of plant miRNAs since the miRNA mining process for plants is highly prone to false-positives, particularly in cereals which have a highly repetitive genome. Our group developed a homology-based in silico miRNA identification approach for plants, which utilizes two Perl scripts " SUmirFind " and " SUmirFold " and since then, this method helped identify many miRNAs particularly from crop species such as Triticum or Aegliops. Herein, we describe a comprehensive updated guideline by the implementation of two new scripts, " SUmirPredictor " and " SUmirLocator, " and refinements to our previous method in order to identify genuine miRNAs with increased sensitivity in consideration of miRNA identification problems in plants. Recent updates enable our method to provide more reliable and precise results in an automated fashion in addition to solutions for elimination of most false-positive predictions, miRNA naming and miRNA mis-annotation. It also provides a comprehensive view to genome/transcriptome-wide location of miRNA precursors as well as their association with transposable elements. The " SUmirPredictor " and " SUmirLocator " scripts are freely available together with a reference high-confidence plant miRNA list.
The non-nucleoside analog gemcitabine has been the standard of care for treating pancreatic cancer. The drug shows good potency in pancreatic cancer cells in vitro but, due to poor bioavailability, requires administration in large doses... more
The non-nucleoside analog gemcitabine has been the standard of care for treating pancreatic cancer. The drug shows good potency in pancreatic cancer cells in vitro but, due to poor bioavailability, requires administration in large doses by infusion and this systemic exposure results in significant toxicity for the patient. Genes have been identified that, when silenced by siRNA, synergize with gemcitabine treatment and offer a means of reducing the gemcitabine dosage required for efficacy. However, benefiting from the synergism between the two agents requires that the gemcitabine and siRNA penetrate the same cells. To ensure co-delivery, we incorporated gemcitabine covalently within siRNAs against targets synergistic with gemcitabine (CHK1 or RAD17). We demonstrated that specific bases within an siRNA can be replaced with gemcitabine to increase efficacy. The result is a single drug molecule that simultaneously co-delivers gemcitabine and a synergistic siRNA. The siRNA–gemcitabine constructs demonstrate a 5–30-fold improvement in potency compared with gemcitabine alone. Co-delivering a CHK1 siRNA–gemcitabine construct together with a WEE1 siRNA resulted in a 10-fold improvement in IC50 compared with gemcitabine alone. These constructs demonstrate efficacy across a wide array of pancreatic tumor cells and may represent a novel therapeutic approach for treating pancreatic cancer.
Although infections of vertebrate hosts by arthropod-borne viruses may lead to pathogenic outcomes, infections of vector mosquitoes result in persistent infections, where the virus replicates in the host without causing apparent... more
Although infections of vertebrate hosts by arthropod-borne viruses may lead to pathogenic outcomes, infections of vector mosquitoes result in persistent infections, where the virus replicates in the host without causing apparent pathological effects. It is unclear how persistent infections are established and maintained in mosquitoes. Several reports revealed the presence of flavivirus-like DNA sequences in the mosquito genome, and recent studies have shown that DNA forms of RNA viruses restrict virus re-plication in Drosophila, suggesting that DNA forms may have a role in developing persistent infections. Here, we sought to investigate whether arboviruses generate DNA forms following infection in mosquitoes. Our results with West Nile, Dengue, and La Crosse viruses demonstrate that DNA forms of the viral RNA genome are generated in mosquito cells; however, not the entire viral genome, but patches of viral RNA in DNA forms can be detected 24 h post infection.
For more than a decade, RNA interference (RNAi) has brought about an entirely new approach to functional genomics screening. Enabling high-throughput loss-of-function (LOF) screens against the human genome, identifying new drug targets,... more
For more than a decade, RNA interference (RNAi) has brought about an entirely new approach to functional genomics screening. Enabling high-throughput loss-of-function (LOF) screens against the human genome, identifying new drug targets, and significantly advancing experimental biology, RNAi is a fast, flexible technology that is compatible with existing high-throughput systems and processes; however, the recent advent of clustered regularly interspaced palindromic repeats (CRISPR)-Cas, a powerful new precise genome-editing (PGE) technology, has opened up vast possibilities for functional genomics. CRISPR-Cas is novel in its simplicity: one piece of easily engineered guide RNA (gRNA) is used to target a gene sequence, and Cas9 expression is required in the cells. The targeted double-strand break introduced by the gRNA-Cas9 complex is highly effective at removing gene expression compared to RNAi. Together with the reduced cost and complexity of CRISPR-Cas, there is the realistic opportunity to use PGE to screen for phenotypic effects in a total gene knockout background. This review summarizes the exciting development of CRISPR-Cas as a high-throughput screening tool, comparing its future potential to that of well-established RNAi screening techniques, and highlighting future challenges and opportunities within these disciplines. We conclude that the two technologies actually complement rather than compete with each other, enabling greater understanding of the genome in relation to drug discovery.
To cite this article: Peyman Eivazy, Fatemeh Atyabi, Farhad Jadidi-Niaragh, Leili Aghebati Maleki, Abolfazl Miahipour, Jalal Abdolalizadeh & Mehdi Yousefi (2016): The impact of the codelivery of drug-siRNA by trimethyl chitosan... more
To cite this article: Peyman Eivazy, Fatemeh Atyabi, Farhad Jadidi-Niaragh, Leili Aghebati Maleki, Abolfazl Miahipour, Jalal Abdolalizadeh & Mehdi Yousefi (2016): The impact of the codelivery of drug-siRNA by trimethyl chitosan nanoparticles on the efficacy of chemotherapy for metastatic breast cancer cell line (MDA-MB-231), Artificial Cells, Nanomedicine, and Biotechnology,
Nonspecific effects triggered by small interfering RNAs (siRNAs) complicate the use of RNA interference (RNAi) to specifically downregulate gene expression
