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Triosephosphate isomerase deficiency (TPI Df) is a rare, aggressive genetic disease that typically affects young children and currently has no established treatment. TPI Df is characterized by hemolytic anemia, progressive neuromuscular degeneration, and a markedly reduced lifespan. The disease has predominately been studied using invertebrate and in vitro models, which lack key aspects of the human disease. While other groups have generated mammalian Tpi1 mutant strains, specifically with the mouse mus musculus, these do not recapitulate key characteristic phenotypes of the human disease. Reported here is the generation of a novel murine model of TPI Df. CRISPR-Cas9 was utilized to engineer the most common human disease-causing mutation, Tpi1 E105D , and Tpi1 null mice were also isolated as a frame-shifting deletion. Tpi1 E105D/null mice experience a markedly shortened lifespan, postural abnormalities consistent with extensive neuromuscular dysfunction, hemolytic anemia, pathological changes in spleen, and decreased body weight. There is a â¼95% reduction in TPI protein levels in Tpi1 E105D/null animals compared to wild-type littermates, consistent with decreased TPI protein stability, a known cause of TPI Df. This work illustrates the capability of Tpi1 E105D/null mice to serve as a mammalian model of human TPI Df. This work will allow for advancement in the study of TPI Df within a model with physiology similar to humans. The development of the model reported here will enable mechanistic studies of disease pathogenesis and, importantly, efficacy testing in a mammalian system for emerging TPI Df treatments.
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Epigenomics has become a significant research interest at a time when rapid environmental changes are occurring. Epigenetic mechanisms mainly result from systems like DNA methylation, histone modification, and RNA interference. Epigenetic mechanisms are gaining importance in classical genetics, developmental biology, molecular biology, cancer biology, epidemiology, and evolution. Epigenetic mechanisms play important role in the action and interaction of plant genes during development, and also have an impact on classical plant breeding programs, inclusive of novel variation, single plant heritability, hybrid vigor, plant-environment interactions, stress tolerance, and performance stability. The epigenetics and epigenomics may be significant for crop adaptability and pliability to ambient alterations, directing to the creation of stout climate-resilient elegant crop cultivars. In this review, we have summarized recent progress made in understanding the epigenetic mechanisms in plant responses to biotic and abiotic stresses and have also tried to provide the ways for the efficient utilization of epigenomic mechanisms in developing climate-resilient crop cultivars, especially in chickpea, and other legume crops.
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Background: The roots of Panax ginseng contain two types of tetracyclic triterpenoid saponins, namely, protopanaxadiol (PPD)-type saponins and protopanaxatiol (PPT)-type saponins. In P. ginseng, the protopanaxadiol 6-hydroxylase (PPT synthase) enzyme catalyses protopanaxatriol (PPT) production from protopanaxadiol (PPD). In this study, we constructed homozygous mutant lines of ginseng by CRISPR/Cas9-mediated mutagenesis of the PPT synthase gene and obtained the mutant ginseng root lines having complete depletion of the PPT-type ginsenosides. Methods: Two sgRNAs (single guide RNAs) were designed for target mutations in the exon sequences of the two PPT synthase genes (both PPTa and PPTg sequences) with the CRISPR/Cas9 system. Transgenic ginseng roots were generated through Agrobacterium-mediated transformation. The mutant lines were screened by ginsenoside analysis and DNA sequencing. Result: Ginsenoside analysis revealed the complete depletion of PPT-type ginsenosides in three putative mutant lines (Cr4, Cr7, and Cr14). The reduction of PPT-type ginsenosides in mutant lines led to increased accumulation of PPD-type ginsenosides. The gene editing in the selected mutant lines was confirmed by targeted deep sequencing. Conclusion: We have established the genome editing protocol by CRISPR/Cas9 system in P. ginseng and demonstrated the mutated roots producing only PPD-type ginsenosides by depleting PPT-type ginsenosides. Because the pharmacological activity of PPD-group ginsenosides is significantly different from that of PPT-group ginsenosides, the new type of ginseng mutant producing only PPD-group ginsenosides may have new pharmacological characteristics compared to wild-type ginseng. This is the first report to generate target-induced mutations for the modification of saponin biosynthesis in Panax species using CRISPR-Cas9 system.
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RNA interference (RNAi) is a highly conserved pathway for the post-transcriptional regulation of gene expression. It has become a crucial tool in life science research, with promising potential for pest-management applications. To induce an RNAi response, long double-stranded RNA (dsRNA) sequences specific to the target gene must be delivered to the cells. This dsRNA substrate is then processed to small RNA (sRNA) fragments that direct the silencing response. A major obstacle to applying this technique is the need to produce sufficiently large amounts of dsRNA in a very cost-effective manner. To overcome this issue, much attention has been given to the development and optimization of biological production systems. One such system is the E. coli HT115 strain transformed with the L4440 vector. While its effectiveness at inducing knockdowns in animals through feeding of the bacteria has been demonstrated, there is only limited knowledge on the applicability of bacteria-derived dsRNA for in vitro experiments. In this paper, we describe and compare methods for the economical (43.2 /mg) and large-scale (mg range) production of high-quality dsRNA from the HT115 bacterial system. We transformed the bacteria with constructs targeting the Helicoverpa-specific gene Dicer2 and, as a non-endogenous control, the Green Fluorescent Protein gene (GFP). First, we compared the total RNA extraction yields of four cell-lysis treatments: heating, lysozyme digestion, sonication, and a control protocol. Second, we assessed the quality and purity of these extracted dsRNAs. Third, we compared methods for the further purification of dsRNAs from crude RNA extracts. Finally, we demonstrated the efficiency of the produced dsRNAs at inducing knockdowns in a lepidopteran cell line. The insights and results from this paper will empower researchers to conduct otherwise prohibitively expensive knockdown studies, and greatly reduce the production times of routinely or large-scale utilized dsRNA substrates.
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Objective: The acute cases of pneumonia (COVID-19) were first reported from China in December 2019, and the pathogen was identified as SARS-CoV-2. Currently, many vaccines have been developed against this virus by using multiple genes, applying different platforms, and used for the vaccinations of the human population. Spike protein genes play an important role in host cell attachment and viral entry and have been extensively used for the development of vaccine and antiviral therapeutics. Short interfering RNA is also known as silencing RNA and contribute a significant role to regulate the expression of a specific gene. By using this technology, virus inhibition has been demonstrated against many viral diseases. Methods: In this work, we have reported the Insilico prediction, designing, and experimental validation of siRNAs antiviral potency against SARS-CoV-2-S-RBD. The siDirect 2.0 was selected for siRNAs prediction, and secondary structure was predicted by RNAfold while the HNADOCK was used for molecular docking analysis and specific binding of siRNAs to the selected target. We have used and evaluated four siRNAs for cellular toxicity and determination of antiviral efficiency based on the Ct value of q-real-time PCR in Vero E6 cells. Results: Based on the experimental evaluation and analysis of results from generated data, we observed that there is no cytotoxicity for any tested siRNAs in Vero E6 cells. Total four siRNA were filtered out from twenty-one siRNAs following the strict selection and scoring criteria. The better antiviral efficiency was observed in 3rd siRNAs based on the Ct value of q-real-time PCR. The results that emerged from this study encouraged us to validate the efficiency of these siRNAs in multiple cells by using alone and in a combination of two or more siRNAs to inhibit the SARS-CoV-2 proliferation. Conclusion: The Insilico prediction, molecular docking analysis provided the selection of better siRNAs. Based on the experimental evaluation only 3rd siRNA was found to be more effective than others and showed better antiviral efficiency. These siRNAs should also be evaluated in other cell lines either separately or in combination against SARS-CoV-2 to determine their antiviral efficiency.
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DNA damage response (DDR) is a highly conserved genome surveillance mechanism that preserves cell viability in the presence of chemotherapeutic drugs. Hence, small molecules that inhibit DDR are expected to enhance the anti-cancer effect of chemotherapy. Through a recent chemical library screen, we identified shikonin as an inhibitor that strongly suppressed DDR activated by various chemotherapeutic drugs in cancer cell lines derived from different origins. Mechanistically, shikonin inhibited the activation of ataxia telangiectasia mutated (ATM), and to a lesser degree ATM and RAD3-related (ATR), two master upstream regulators of the DDR signal, through inducing degradation of ATM and ATR-interacting protein (ATRIP), an obligate associating protein of ATR, respectively. As a result of DDR inhibition, shikonin enhanced the anti-cancer effect of chemotherapeutic drugs in both cell cultures and in mouse models. While degradation of ATRIP is proteasome dependent, that of ATM depends on caspase- and lysosome-, but not proteasome. Overexpression of ATM significantly mitigated DDR inhibition and cell death induced by shikonin and chemotherapeutic drugs. These novel findings reveal shikonin as a pan DDR inhibitor and identify ATM as a primary factor in determining the chemo sensitizing effect of shikonin. Our data may facilitate the development of shikonin and its derivatives as potential chemotherapy sensitizers through inducing ATM degradation.
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Objectives: The COVID-19 was identified for the first time from the sea food market, Wuhan city, China in 2019 and the pathogenic organism was identified as SARS-CoV-2. Currently, this virus has spread to 223 countries and territories and known as a serious issue for the global human community. Many vaccines have been developed and used for immunization. Methods: We have reported the insilico prediction, designing, secondary structure prediction, molecular docking analysis, and in vitro assessment of siRNAs against SARS-CoV-2. The online bioinformatic approach was used for siRNAs selection and designing. The selected siRNAs were evaluated for antiviral efficacy by using Lipofectamine 2000 as delivery agent to HEK-293 cells. The MTT assay was used for cytotoxicity determination. The antiviral efficacy of potential siRNAs was determined based on the Ct value of q-RT-PCR and the data analysis was done by Prism-GraphPad software. Results: The analyzed data resulted in the selection of only three siRNAs out of twenty-six siRNAs generated by online software. The secondary structure prediction and molecular docking analysis of siRNAs revealed the efficient binding to the target. There was no cellular toxicity observed in the HEK-293 cells at any tested concentrations of siRNAs. The purification of RNA was completed from inoculated cells and subjected to q-RT-PCR. The highest Ct value was observed in siRNA 3 than the others. The results offered valuable evidence and invigorated us to assess the potency of siRNAs by using alone or in combination in other human cells. Conclusion: The data generated from this study indicates the significance of in silico prediction and narrow down the potential siRNA' against SARS-CoV-2, and molecular docking investigation offered the effective siRNAs binding with the target. Finally, it is concluded that the online bioinformatics approach provided the prediction and selection of siRNAs with better antiviral efficacy. The siRNA-3 was observed to be the best for reduction of viral RNA in cells.
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Alzheimer's disease (AD), the most prominent form of dementia in the elderly, has no cure. Strategies focused on the reduction of amyloid beta or hyperphosphorylated Tau protein have largely failed in clinical trials. Novel therapeutic targets and strategies are urgently needed. Emerging data suggest that in response to environmental stress, mitochondria initiate an integrated stress response (ISR) shown to be beneficial for healthy aging and neuroprotection. Here, we review data that implicate mitochondrial electron transport complexes involved in oxidative phosphorylation as a hub for small molecule-targeted therapeutics that could induce beneficial mitochondrial ISR. Specifically, partial inhibition of mitochondrial complex I has been exploited as a novel strategy for multiple human conditions, including AD, with several small molecules being tested in clinical trials. We discuss current understanding of the molecular mechanisms involved in this counterintuitive approach. Since this strategy has also been shown to enhance health and life span, the development of safe and efficacious complex I inhibitors could promote healthy aging, delaying the onset of age-related neurodegenerative diseases.
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The outbreak of the COVID-19 pandemic has cost five million lives to date, and was caused by a positive-sense RNA virus named SARS-CoV2. The lack of drugs specific to SARS-CoV2, leads us to search for an effective and specific therapeutic approach. Small interfering RNA (siRNA) is able to activate the RNA interference (RNAi) pathway to silence the specific targeted gene and inhibit the viral replication, and it has not yet attracted enough attention as a SARS-CoV2 antiviral agent. It could be a potential weapon to combat this pandemic until the completion of full scale, effective mass vaccination. For this study, specific siRNAs were designed using a web-based bioinformatics tool (siDirect2.0) against 14 target sequences. These might have a high probability of silencing the essential proteins of SARS-CoV2. such as: 3CLpro/Mpro/nsp5, nsp7, Rd-Rp/nsp12, ZD, NTPase/HEL or nsp13, PLpro/nsp3, envelope protein (E), spike glycoprotein (S), nucleocapsid phosphoprotein (N), membrane glycoprotein (M), ORF8, ORF3a, nsp2, and its respective 5' and 3'-UTR. Among these potential drug targets, the majority of them contain highly conserved sequences; the rest are chosen on the basis of their role in viral replication and survival. The traditional vaccine development technology using SARS-CoV2 protein takes 6-8 months; meanwhile the virus undergoes several mutations in the candidate protein chosen for vaccine development. By the time the protein-based vaccine reaches the market, the virus would have undergone several mutations, such that the antibodies against the viral sequence may not be effective in restricting the newly mutated viruses. However, siRNA technology can make sequences based on real time viral mutation status. This has the potential for suppressing SARS-CoV2 viral replication, through RNAi technology.
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The use of small interfering RNAs (siRNAs) has been under investigation for the treatment of several unmet medical needs, including acute lung injury/acute respiratory distress syndrome (ALI/ARDS) wherein siRNA may be implemented to modify the expression of pro-inflammatory cytokines and chemokines at the mRNA level. The properties such as clear anatomy, accessibility, and relatively low enzyme activity make the lung a good target for local siRNA therapy. However, the translation of siRNA is restricted by the inefficient delivery of siRNA therapeutics to the target cells due to the properties of naked siRNA. Thus, this review will focus on the various delivery systems that can be used and the different barriers that need to be surmounted for the development of stable inhalable siRNA formulations for human use before siRNA therapeutics for ALI/ARDS become available in the clinic.
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The invasive plant Mikania micrantha Kunth (M. micrantha) from South America poses a significant threat to the stability and biodiversity of ecosystems. However, an effective and economical method to control M. micrantha is still lacking. RNA interference (RNAi) has been widely studied and applied in agriculture for trait improvement. Spray-induced gene silencing (SIGS) can produce RNAi silencing effects without introducing heritable modifications to the plant genome and is becoming a novel nontransformation strategy for plant protection. In this study, the genes encoding chlorophyll a/b-binding proteins were selected as targets of RNAi, based on high-throughput sequencing of M. micrantha transcriptome and bioinformatic analyses of sequence specificity. Three types of RNAi molecules, double-stranded RNA, RNAi nanomicrosphere, and short hairpin RNA (shRNA), with their corresponding short interfering RNA sequences were designed and synthesized for SIGS vector construction, from which each RNAi molecule was transcribed and extracted to be sprayed on M. micrantha leaves. Whereas water-treated control leaves remained green, leaves treated with RNAi molecules turned yellow and eventually wilted. Quantitative real-time PCR showed that the expression levels of target genes were significantly reduced in the RNAi-treated groups compared with those of the control, suggesting that all three types of RNAi herbicides effectively silenced the endogenous target genes, which are essential for the growth of M. micrantha. We also found that shRNA showed better silencing efficiency than the other two molecules. Taken together, our study successfully designed three types of RNAi-based herbicides that specifically silenced endogenous target genes and controlled the growth of M. micrantha. Moreover, we identified a gene family encoding chlorophyll a/b-binding proteins that is important for the growth and development of M. micrantha and could serve as potential targets for controlling the spread of M. micrantha.
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The salmon louse, Lepeophtheirus salmonis is an ectoparasite of salmonid fish in the Northern Hemisphere, causing large economical losses in the aquaculture industry and represent a threat to wild populations of salmonids. Like other oviparous animals, it is likely that female lice use lipoproteins for lipid transport to maturing oocytes and other organs of the body. As an important component of lipoproteins, apolipoproteins play a vital role in the transport of lipids through biosynthesis of lipoproteins. Apolipoproteins have been studied in detail in different organisms, but no studies have been done in salmon lice. Two apolipoprotein encoding genes (LsLp1 and LsLp2) were identified in the salmon lice genome. Transcriptional analysis revealed both genes to be expressed at all stages from larvae to adult with some variation, LsLp1 generally higher than LsLp2 and both at their highest levels in adult stages of the louse. In adult female louse, the LsLp1 and LsLp2 transcripts were found in the sub-epidermal tissue and the intestine. RNA interference-mediated knockdown of LsLp1 and LsLp2 in female lice resulted in reduced expression of both transcripts. LsLp1 knockdown female lice produced significantly less offspring than control lice, while knockdown of LsLp2 in female lice caused no reduction in the number of offspring. These results suggest that LsLp1 has an important role in reproduction in female salmon lice.
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The need for long-term treatments of chronic diseases has motivated the widespread development of long-acting parenteral formulations (LAPFs) with the aim of improving drug pharmacokinetics and therapeutic efficacy. LAPFs have been proven to extend the half-life of therapeutics, as well as to improve patient adherence; consequently, this enhances the outcome of therapy positively. Over past decades, considerable progress has been made in designing effective LAPFs in both preclinical and clinical settings. Here we review the latest advances of LAPFs in preclinical and clinical stages, focusing on the strategies and underlying mechanisms for achieving long acting. Existing strategies are classified into manipulation of in vivo clearance and manipulation of drug release from delivery systems, respectively. And the current challenges and prospects of each strategy are discussed. In addition, we also briefly discuss the design principles of LAPFs and provide future perspectives of the rational design of more effective LAPFs for their further clinical translation.
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Ginsenosides are a series of glycosylated triterpenoids which belong to protopanaxadiol (PPD)-, protopanaxatriol (PPT)-, ocotillol (OCT)- and oleanane (OA)-type saponins known as active compounds of Panax genus. They are accumulated in plant roots, stems, leaves, and flowers. The content and composition of ginsenosides are varied in different ginseng species, and in different parts of a certain plant. In this review, we summarized the representative saponins structures, their distributions and the contents in nearly 20 Panax species, and updated the biosynthetic pathways of ginsenosides focusing on enzymes responsible for structural diversified ginsenoside biosynthesis. We also emphasized the transcription factors in ginsenoside biosynthesis and non-coding RNAs in the growth of Panax genus plants, and highlighted the current three major biotechnological applications for ginsenosides production. This review covered advances in the past four decades, providing more clues for chemical discrimination and assessment on certain ginseng plants, new perspectives for rational evaluation and utilization of ginseng resource, and potential strategies for production of specific ginsenosides.
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The coronavirus disease 2019 (COVID-19) is an ongoing pandemic caused by an RNA virus termed as severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). SARS-CoV-2 possesses an almost 30kbp long genome. The genome contains open-reading frame 1ab (ORF1ab) gene, the largest one of SARS-CoV-2, encoding polyprotein PP1ab and PP1a responsible for viral transcription and replication. Several vaccines have already been approved by the respective authorities over the world to develop herd immunity among the population. In consonance with this effort, RNA interference (RNAi) technology holds the possibility to strengthen the fight against this virus. Here, we have implemented a computational approach to predict potential short interfering RNAs including small interfering RNAs (siRNAs) and microRNAs (miRNAs), which are presumed to be intrinsically active against SARS-CoV-2. In doing so, we have screened miRNA library and siRNA library targeting the ORF1ab gene. We predicted the potential miRNA and siRNA candidate molecules utilizing an array of bioinformatic tools. By extending the analysis, out of 24 potential pre-miRNA hairpins and 131 siRNAs, 12 human miRNA and 10 siRNA molecules were sorted as potential therapeutic agents against SARS-CoV-2 based on their GC content, melting temperature (Tm), heat capacity (Cp), hybridization and minimal free energy (MFE) of hybridization. This computational study is focused on lessening the extensive time and labor needed in conventional trial and error based wet lab methods and it has the potential to act as a decent base for future researchers to develop a successful RNAi therapeutic.
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Insects rely on an innate immune system to recognize and eliminate pathogens. Key components of this system are highly conserved across all invertebrates. To detect pathogens, insects use Pattern recognition receptors (PRRs) that bind to signature motifs on the surface of pathogens called Pathogen Associated Molecular Patterns (PAMPs). In general, insects use peptidoglycan recognition proteins (PGRPs) in the Immune Deficiency (IMD) pathway to detect Gram-negative bacteria, and other PGRPs and Gram-negative binding proteins (GNBPs) in the Toll pathway to detect Gram-positive bacteria and fungi, although there is crosstalk and cooperation between these and other pathways. Once pathogens are recognized, these pathways activate the production of potent antimicrobial peptides (AMPs). Most PRRs in insects have been reported from genome sequencing initiatives but few have been characterized functionally. The initial studies on insect PRRs were done using established dipteran model organisms such as Drosophila melanogaster, but there are differences in the numbers and functional role of PRRs in different insects. Here we describe the genomic repertoire of PGRPs in Rhodnius prolixus, a hemimetabolous hemipteran vector of the parasite Trypanosoma cruzi that causes Chagas disease in humans. Using a de novo transcriptome from the fat body of immune activated insects, we found 5 genes encoding PGRPs. Phylogenetic analysis groups R. prolixus PGRPs with D. melanogaster PGRP-LA, which is involved in the IMD pathway in the respiratory tract. A single R. prolixus PGRP gene encodes isoforms that contain an intracellular region or motif (cryptic RIP Homotypic Interaction Motif-cRHIM) that is involved in the IMD signaling pathway in D. melanogaster. We characterized and silenced this gene using RNAi and show that the PGRPs that contain cRHIMs are involved in the recognition of Gram-negative bacteria, and activation of the IMD pathway in the fat body of R. prolixus, similar to the PGRP-LC of D. melanogaster. This is the first functional characterization of a PGRP containing a cRHIM motif that serves to activate the IMD pathway in a hemimetabolous insect.
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In many ways, cancer cells are different from healthy cells. A lot of tactical nano-based drug delivery systems are based on the difference between cancer and healthy cells. Currently, nanotechnology-based delivery systems are the most promising tool to deliver DNA-based products to cancer cells. This review aims to highlight the latest development in the lipids and polymeric nanocarrier for siRNA delivery to the cancer cells. It also provides the necessary information about siRNA development and its mechanism of action. Overall, this review gives us a clear picture of lipid and polymer-based drug delivery systems, which in the future could form the base to translate the basic siRNA biology into siRNA-based cancer therapies.
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Gene therapy is rapidly emerging as a powerful therapeutic strategy for a wide range of neurodegenerative disorders, including Alzheimer's disease (AD), Parkinson's disease (PD) and Huntington's disease (HD). Some early clinical trials have failed to achieve satisfactory therapeutic effects. Efforts to enhance effectiveness are now concentrating on three major fields: identification of new vectors, novel therapeutic targets, and reliable of delivery routes for transgenes. These approaches are being assessed closely in preclinical and clinical trials, which may ultimately provide powerful treatments for patients. Here, we discuss advances and challenges of gene therapy for neurodegenerative disorders, highlighting promising technologies, targets, and future prospects.
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Defining genes that are essential for life has major implications for understanding critical biological processes and mechanisms. Although essential genes have been identified and characterised experimentally using functional genomic tools, it is challenging to predict with confidence such genes from molecular and phenomic data sets using computational methods. Using extensive data sets available for the model organism Caenorhabditis elegans, we constructed here a machine-learning (ML)-based workflow for the prediction of essential genes on a genome-wide scale. We identified strong predictors for such genes and showed that trained ML models consistently achieve highly-accurate classifications. Complementary analyses revealed an association between essential genes and chromosomal location. Our findings reveal that essential genes in C. elegans tend to be located in or near the centre of autosomal chromosomes; are positively correlated with low single nucleotide polymorphim (SNP) densities and epigenetic markers in promoter regions; are involved in protein and nucleotide processing; are transcribed in most cells; are enriched in reproductive tissues or are targets for small RNAs bound to the argonaut CSR-1. Based on these results, we hypothesise an interplay between epigenetic markers and small RNA pathways in the germline, with transcription-based memory; this hypothesis warrants testing. From a technical perspective, further work is needed to evaluate whether the present ML-based approach will be applicable to other metazoans (including Drosophila melanogaster) for which comprehensive data sets (i.e. genomic, transcriptomic, proteomic, variomic, epigenetic and phenomic) are available.