Silencing Of Target Genes Research Articles

94 (PB084) - Novel inhibitors of the luminal lineage transcription factor peroxisome proliferator-activated receptor gamma (PPARG) durably eradicate tumors in urothelial cancer (UC) animal models

Background: Small molecule targeting of cell lineage-defining transcription factors is a demonstrated and effective therapeutic strategy in oncology, exemplified by estrogen receptor (ER) agents in luminal breast cancer. Two-thirds of advanced UC is classified as luminal and overexpression of PPARG is characteristic of this molecular subtype. Currently, there is a poor understanding of how recurrent missense mutations in PPARG and its obligate heterodimer retinoid X receptor alpha (RXRA) impact PPARG function; previous tool compounds designed to inhibit PPARG have minimal phenotypic activity in UC cell lines. Material and Methods: The PPARG inhibitor FTX-6746 was evaluated in UC cell lines including UMUC9 (PPARG amplification) and HT1197 (RXRA-S427F hotspot mutation), which activate PPARG by different genetic mechanisms. In vitro analyses included clonogenic growth assays and assessment of target gene modulation after treatment. in vivo studies were performed in NCG or Balb/C nude mice with oral administration of FTX-6746 at doses indicated below. Results: Our biochemical studies indicate that patient-derived missense mutations in PPARG and RXRA bias an active conformation of PPARG, mimicking an agonist-bound state. Addressing the limitations of previous tool compounds, we discovered novel inhibitors that drive a powerful repressive conformation of PPARG with high specificity (>100X selective over PPARA/ PPARD). This results in robust PPARG target gene silencing in cells (IC50 = 5 nM), and in vitro growth inhibition preferentially observed in cell lines with activated PPARG signaling. Tumor growth inhibition or regression was observed in two xenograft models of UC at well-tolerated oral doses with no tumor regrowth upon cessation of treatment. Treatment of UMUC9 xenograft tumors with 3, 10, 30 and 60 mg/kg twice daily of FTX-6746 resulted in up to 80% target gene suppression in tumor tissue at day 2 and up to >100% tumor growth inhibition at day 21. Similar efficacy was observed in HT1197 xenograft studies treated with 30 and 60 mg/kg twice daily of FTX-6746, with >45% target gene suppression at day 2 and 85–112% tumor growth inhibition at day 42.Tabled 1ModelDose (mg/kg)Schedule%Tumor Growth Inhibition%Target Gene Suppression (D2)UMUC93BID*205916UMUC910BID*2070NDUMUC930BID*208373UMUC930BID*2110580UMUC960BID*21108NDHT119730BID*428546HT119760BID*4211259 Open table in a new tab Conclusions: These collective results suggest that PPARG is a lineage-defining transcription factor in UC and small molecule PPARG inhibition will be an effective therapy for patients with advanced urothelial cancer harboring the luminal subtype. Conflict of interest: Ownership: All authors are shareholders in Flare Therapeutics.

EFFECT OF PERIOSTIN ON AUTOPHAGY AND FACTORS RELATED TO BONE METABOLISM IN OSTEOBLASTS

The purpose of this study was to explore the effect of periostin on autophagy and factors related to bone metabolism in osteoblasts. According to experimental design, some osteoblast-like cells (MC3T3-E1) were transfected by lentiviral vector to silence periostin gene. Celigo[Formula: see text] Image Cytometer and Real-Time PCR were used to assess target gene silencing effects. Western blotting was used to evaluate autophagy-associated factors (LC3B,Beclin1) and factors related to bone metabolism (RANKL, OPG, NF-[Formula: see text]B[Formula: see text]P65). Another part of osteoblasts was added with recombinant periostin protein at concentration of 75[Formula: see text]ng/mL. Monodansylcadaverine (MDC) detection was conducted to trace formation of autophagy vesicles. Factors associated with autophagy and bone metabolism were reassessed using western blotting. When periostin gene was silenced, western blotting analysis showed that the expression of LC3B-I, Beclin1, RANKL and NF-[Formula: see text]B[Formula: see text]P65 in experimental group was significantly increased; however, OPG expression was decreased. After adding recombinant periostin protein, MDC detection revealed significantly fewer green fluorescent particles in the experimental group. Western blotting results showed that the expression of LC3B, Beclin1, RANKL and NF-[Formula: see text]B[Formula: see text]P65 was decreased; however, OPG expression was increased. This study examined the regulatory mechanism of periostin in bone metabolism, which will provide a future reference for the treatment of primary teeth abnormal replacement caused by inflammation.

RUNX1/CEBPA Mutation in Acute Myeloid Leukemia Promotes Hypermethylation and Indicates for Demethylation Therapy.

Acute myeloid leukemia (AML) is a rapidly progressing heterogeneous disease with a high mortality rate, which is characterized by hyperproliferation of atypical immature myeloid cells. The number of AML patients is expected to increase in the near future, due to the old-age-associated nature of AML and increased longevity in the human population. RUNX1 and CEBPA, key transcription factors (TFs) of hematopoiesis, are frequently and independently mutated in AML. RUNX1 and CEBPA can bind TET2 demethylase and attract it to their binding sites (TFBS) in cell lines, leading to DNA demethylation of the regions nearby. Since TET2 does not have a DNA-binding domain, TFs are crucial for its guidance to target genomic locations. In this paper, we show that RUNX1 and CEBPA mutations in AML patients affect the methylation of important regulatory sites that resulted in the silencing of several RUNX1 and CEBPA target genes, most likely in a TET2-dependent manner. We demonstrated that hypermethylation of TFBS in AML cells with RUNX1 mutations was associated with resistance to anticancer chemotherapy. Demethylation therapy restored expression of the RUNX1 target gene, BIK, and increased sensitivity of AML cells to chemotherapy. If our results are confirmed, mutations in RUNX1 could be an indication for prescribing the combination of cytotoxic and demethylation therapies.

Mechanisms of action of cytoplasmic microRNAs. Part 3. TNRC6-associated mechanism of miRNA-mediated mRNA degradation

The scientific review presents the mechanisms of action of cytoplasmic miRNAs, namely posttranscriptional silencing: the TNRC6-associated mechanism of miRNA-mediated mRNA degradation. To write the article, information was searched using databases Scopus, Web of Science, MedLine, PubMed, Google Scholar, EMBASE, Global Health, The Cochrane Library, CyberLeninka. It is known that in the cytoplasm of cells in cases of short region, miRNA complementarities cause posttranscriptional silencing, using the first of the main molecular mechanisms: the TNRC6-associated mechanism of miRNA-mediated mRNA degradation. Mammalian AGO proteins have been shown to contain the conserved m7G-cap-binding protein motif (known as the MID domain), which is required to induce microRNA-mediated translation repression. After binding of this AGO motif to microRNAs, TNRC6 proteins (GW182) are recruited that, in turn, recruits various proteins (PABPC1, PAN3 and NOT1) involved in the induction of the target gene silencing. The authors state that tryptophan residues, which are placed in the hydrophobic pockets of TNRC6 protein partners, cause a high degree of affinity and specificity of interactions. Scientists believe that the TNRC6 protein when interacting with AGO proteins can simultaneously use three GW/WG repeats (motif 1, motif 2 and hook motif), which are located in the Argonaute-binding domain. Therefore, the TNRC6 protein can bind to three AGO molecules simultaneously. TNRC6 proteins are known to be PABP-interacting proteins whose interaction with PABP is mediated by conservative PABP-binding motif 2. TNRC6 proteins have been shown to interact with the cytoplasmic PABPC1 protein during mRNA translation and stabilization. It is shown that the CCR4-NOT protein complex is a highly conserved multifunctional multiprotein formation having 3’-5’-exoribonuclease activity, due to which it controls mRNA metabolism. Thus, the TNRC6-associated mechanism of miRNA-mediated mRNA degradation in the cytoplasm of the cell causes posttranscriptional silencing. In this mechanism, there is an interaction of TNRC6 with PABPC1 protein, recruitment of deadenylating complexes PAN2-PAN3 and CCR4-NOT by the TNRC6 proteins.

Visible gland constantly traces virus-induced gene silencing in cotton.

A virus-induced gene silencing (VIGS) system was established to induce endogenous target gene silencing by post-transcriptional gene silencing (PTGS), which is a powerful tool for gene function analysis in plants. Compared with stable transgenic plant via Agrobacterium-mediated gene transformation, phenotypes after gene knockdown can be obtained rapidly, effectively, and high-throughput through VIGS system. This approach has been successfully applied to explore unknown gene functions involved in plant growth and development, physiological metabolism, and biotic and abiotic stresses in various plants. In this system, GhCLA1 was used as a general control, however, silencing of this gene leads to leaf albino, wilting, and plant death ultimately. As such, it cannot indicate the efficiency of target gene silencing throughout the whole plant growth period. To address this question, in this study, we developed a novel marker gene, Gossypium PIGMENT GLAND FORMATION GENE (GoPGF), as the control to trace the efficiency of gene silencing in the infected tissues. GoPGF has been proved a key gene in gland forming. Suppression of GoPGF does not affect the normal growth and development of cotton. The number of gland altered related to the expression level of GoPGF gene. So it is a good marker that be used to trace the whole growth stages of plant. Moreover, we further developed a method of friction inoculation to enhance and extend the efficiency of VIGS, which facilitates the analysis of gene function in both the vegetative stage and reproductive stage. This improved VIGS technology will be a powerful tool for the rapid functional identification of unknown genes in genomes.

Combination treatment of mannose and GalNAc conjugated small interfering RNA protects against lethal Marburg virus infection

Marburg virus (MARV) infection results in severe viral hemorrhagic fever with mortalities up to 90%, and there is a pressing need for effective therapies. Here, we established a small interfering RNA (siRNA) conjugate platform that enabled successful subcutaneous delivery of siRNAs targeting the MARV nucleoprotein. We identified a hexavalent mannose ligand with high affinity to macrophages and dendritic cells, which are key cellular targets of MARV infection. This ligand enabled successful siRNA conjugate delivery to macrophages both invitro and invivo. The delivered hexa-mannose-siRNA conjugates rendered substantial target gene silencing in macrophages when supported by a mannose functionalized endosome release polymer. This hexa-mannose-siRNA conjugate was further evaluated alongside our hepatocyte-targeting GalNAc-siRNA conjugate, to expand targeting of infected liver cells. In MARV-Angola-infected guinea pigs, these platforms offered limited survival benefit when used as individual agents. However, in combination, they achieved up to 100% protection when dosed 24h post infection. This novel approach, using two different ligands to simultaneously deliver siRNA to multiple cell types relevant to infection, provides a convenient subcutaneous route of administration for treating infection by these dangerous pathogens. The mannose conjugate platform has potential application to other diseases involving macrophages and dendritic cells.

Osteoblastic microRNAs in skeletal diseases: Biological functions and therapeutic implications

Skeletal diseases normally represents a grievous imbalance between osteoblasts for bone formation and osteoclasts for bone resorption. A lack of osteogenic function can make it difficult to repair pathological bone erosion. Therefore, substantial efforts have been made to remedy these issues, with the aid of bioactive molecules, herbs and materials. Following recent insights, the importance of epigenetic gene regulation is increasingly evident, especially microRNAs. MicroRNAs can silence target genes by inhibiting mRNA translation or degrading mRNA molecules by binding to their 3′-untranslated region. There is accumulating evidence indicating that the miRNAs significantly involved in osteogenic gene expression, signaling pathway intervention and programmed cell death. Besides, numerous new target drugs (microRNA inhibitors or agonists) have been proposed to exploit its value in skeletal physiology and pathology. In this review, we mainly discuss the role of microRNAs in the context of skeletal disease-associated osteoblast differentiation, the applications of microRNA polymorphisms as biomarkers for diagnostic and therapeutic targets, and the challenges to meet this goal. Our summary provides novel horizon for improving the therapeutic effect of microRNAs, which may be beneficial to the further clinical translation of microRNAs in the treatments of skeletal diseases.

Artificial primary-miRNAs as a platform for simultaneous delivery of siRNA and antisense oligonucleotide for multimodal gene regulation.

Self-assembled nucleic acid nanostructures have been widely explored for gene therapy applications due to their unique advantages. Their roles are not limited to offer intracellular delivery platforms but additionally provide a biological function to induce targeted gene regulation. Here, we report a self-assembled artificial primary-miRNA (pri-miRNA) for achieving simultaneous multimodal gene regulation. Artificial pri-miRNAs are designed to play a role as substrate RNAs to recruit and interact with Drosha/DGCR8 (Microprocessor). Incorporation of functional RNA motifs and site-specific chemical modification of the primary miRNA are utilized for the biogenesis of two individual gene-regulating oligonucleotides. Once they are cleaved by the endogenous Drosha/DGCR8 complex, basal strands and pre-miRNA can be generated inside of cells. In this study, we integrated basal strands with either SMN2 ASO or anti-miR21 to induce multimodal gene regulation. Microprocessing and subsequent gene regulation were first evaluated by measuring the activity of reporter pre-miRNA. Chemical modification on the primary miRNA was optimized through a series of in vitro Drosha cleavage tests and targeted gene silencing in cells. Primary miRNA with the basal ASO or anti-miR strands showed a successful in vitro activity and resulted in simultaneous multimodal gene regulation in cells. Artificial primary miRNA may offer synergistic therapeutic effects for treating various diseases, including spinal muscular atrophy and cancer.

Mastering noninvasive predictive biomarkers to follow up renal transplant patients.

Renal transplantation is the treatment of choice for end-stage renal disease (ESRD) patients. Several cellular processes are regulated via non-coding RNAs by silencing target gene expression. Previous investigations have established a linkage between a number of human microRNAs and kidney failure. This study aims to identify the expression of urinary miR-199a-3p and miR-155-5p as non-invasive biomarkers during post and pre-transplantation over a six-month follow-up period. In addition to the classic chronic renal disease markers (estimated glomerular filtration rate eGFR, Serum creatinine, serum electrolytes, and Antinuclear antibodies ANA test). Urinary miR-199a-3p and miR-155-5p expression levels in 72 adults with diabetic nephropathy and, 42 adults with lupus nephropathy renal transplant recipients. Both were compared with 32 healthy controls prior and post-transplantation. miRNAs were evaluated by quantitative reverse transcription-polymerase chain reaction. Urinary miR-199a-3p significantly (p<0.0001) downregulated in diabetic and lupus nephropathy prior to transplantation and significantly upregulated post-transplantation compared to the control. While urinary miR-155-5p quantities were significantly higher in prior renal transplant patients in comparison with the same patients' post-renal transplantation(P<0.0001). In conclusion, Urinary miR-199a-3p and miR-155-5p can be used as a non-invasive biomarker with high specificity and sensitivity to follow up the renal transplant patients before and post-transplantation instead of biopsy which is complicated by a non-negligible factor.

Localized efficacy of environmental RNAi in Tetranychus urticae

Environmental RNAi has been developed as a tool for reverse genetics studies and is an emerging pest control strategy. The ability of environmental RNAi to efficiently down-regulate the expression of endogenous gene targets assumes efficient uptake of dsRNA and its processing. In addition, its efficiency can be augmented by the systemic spread of RNAi signals. Environmental RNAi is now a well-established tool for the manipulation of gene expression in the chelicerate acari, including the two-spotted spider mite, Tetranychus urticae. Here, we focused on eight single and ubiquitously-expressed genes encoding proteins with essential cellular functions. Application of dsRNAs that specifically target these genes led to whole mite body phenotypes—dark or spotless. These phenotypes were associated with a significant reduction of target gene expression, ranging from 20 to 50%, when assessed at the whole mite level. Histological analysis of mites treated with orally-delivered dsRNAs was used to investigate the spatial range of the effectiveness of environmental RNAi. Although macroscopic changes led to two groups of body phenotypes, silencing of target genes was associated with the distinct cellular phenotypes. We show that regardless of the target gene tested, cells that displayed histological changes were those that are in direct contact with the dsRNA-containing gut lumen, suggesting that the greatest efficiency of the orally-delivered dsRNAs is localized to gut tissues in T. urticae.

A circular RNA vector for targeted plant gene silencing based on an asymptomatic viroid.

Gene silencing for functional studies in plants has been largely facilitated by manipulating viral genomes with inserts from host genes to trigger virus-induced gene silencing (VIGS) against the corresponding mRNAs. However, viral genomes encode multiple proteins and can disrupt plant homeostasis by interfering with endogenous cell mechanisms. To try to circumvent this functional limitation, we have developed a silencing method based on the minimal autonomously-infectious nucleic acids currently known: viroids, which lack proven coding capability. The genome of Eggplant latent viroid, an asymptomatic viroid, was manipulated with insertions ranging between 21 and 42 nucleotides. Our results show that, although larger insertions might be tolerated, the maintenance of the secondary structure appears to be critical for viroid genome stability. Remarkably, these modified ELVd molecules are able to induce systemic infection promoting the silencing of target genes in eggplant. Inspired by the design of artificial microRNAs, we have developed a simple and standardized procedure to generate stable insertions into the ELVd genome capable of silencing a specific target gene. Analogously to VIGS, we have termed our approach viroid-induced gene silencing, and demonstrate that it is a promising tool for dissecting gene functions in eggplant.

Small RNA-based plant protection against diseases.

Plant diseases cause significant decreases in yield and quality of crops and consequently pose a very substantial threat to food security. In the continuous search for environmentally friendly crop protection, exploitation of RNA interferance machinery is showing promising results. It is well established that small RNAs (sRNAs) including microRNA (miRNA) and small interfering RNA (siRNA) are involved in the regulation of gene expression via both transcriptional and post-transcriptional RNA silencing. sRNAs from host plants can enter into pathogen cells during invasion and silence pathogen genes. This process has been exploited through Host-Induced Gene Silencing (HIGS), in which plant transgenes that produce sRNAs are engineered to silence pest and pathogen genes. Similarly, exogenously applied sRNAs can enter pest and pathogen cells, either directly or via the hosts, and silence target genes. This process has been exploited in Spray-Induced Gene Silencing (SIGS). Here, we focus on the role of sRNAs and review how they have recently been used against various plant pathogens through HIGS or SIGS-based methods and discuss advantages and drawbacks of these approaches.

A 2′-modified uridine analog, 2′-O-(methylthiomethoxy)methyl uridine, for siRNA applications

The medicinal applications of siRNAs have been intensively examined but are still hindered by their low molecular stability under biological conditions and off-target effects, etc. The introduction of chemical modifications to the nucleoside is a promising strategy for solving these limitations. Herein, we describe the development of a new uridine analog, U*, that has a (methylthiomethoxy)methoxy group at the 2′ position. The phosphoramidite reagent corresponding to U* was easily synthesized and the RNA oligonucleotides containing U* were stably prepared using a standard protocol for oligonucleotide synthesis. The introduction of U* into the siRNA resulted in positive or negative effects on the targeted gene silencing in a position-dependent manner, and the positive effects were attributed to the improved stability under biological conditions. The thermodynamic analysis of the U*-modified RNAs revealed a slight destabilization of the dsRNA, based depending on which U was strategically utilized to restrain the off-target effects of the siRNA. This study describes a rare example of nucleoside analogs with a large substitution at the 2′-position in the context of an siRNA application and is informative for the development of other analogs to further improve the molecular properties of siRNAs for medicinal applications.

Nanoparticles-Based Strategies to Improve the Delivery of Therapeutic Small Interfering RNA in Precision Oncology.

Small interfering RNA (siRNA) can selectively suppress the expression of disease-causing genes, holding great promise in the treatment of human diseases, including malignant cancers. In recent years, with the development of chemical modification and delivery technology, several siRNA-based therapeutic drugs have been approved for the treatment of non-cancerous liver diseases. Nevertheless, the clinical development of siRNA-based cancer therapeutics remains a major translational challenge. The main obstacles of siRNA therapeutics in oncology include both extracellular and intracellular barriers, such as instability under physiological conditions, insufficient tumor targeting and permeability (particularly for extrahepatic tumors), off-target effects, poor cellular uptake, and inefficient endosomal escape. The development of clinically suitable and effective siRNA delivery systems is expected to overcome these challenges. Herein, we mainly discuss recent strategies to improve the delivery and efficacy of therapeutic siRNA in cancer, including the application of non-viral nanoparticle-based carriers, the selection of target genes for therapeutic silencing, and the combination with other therapeutic modalities. In addition, we also provide an outlook on the ongoing challenges and possible future developments of siRNA-based cancer therapeutics during clinical translation.

Targeted delivery of self-assembled nanocomplex between fusion peptides and siRNAs for breast cancer treatment

Breast cancer is one of the serious diseases and has the second-highest mortality in women worldwide. RNA interference has been developed as a promising way of specific cancer treatment by silencing oncogenes efficiently. However, small RNAs exhibit difficulties in specific cellular uptake and instability. Therefore, we developed novel self-assembled nanocomplexes between newly designed fusion peptides and small RNAs for an efficient, stable, targeted delivery to breast cancer cells. Designed R11-SP82 (RS) and R11-TT1 (RT) peptides could form self-assembled nanocomplexes via electrostatic attraction. As a result, RS nanocomplexes exhibited prolonged stability, enhanced cellular uptake, and target gene silencing by siRNAs to MDA-MB-231 breast cancer cells. Moreover, RS nanocomplexes successfully inhibited breast cancer cell growth via specific and efficient siRNA delivery. Furthermore, in vitro and in vivo safety tests showed negligible cytotoxicity and neither tissue damage nor significant inflammatory cytokine release. Therefore, the RS nanocomplex strategy could be expected to become a promising siRNA delivery platform for treating breast cancer or other cancers.

Extracellular vesicles isolated from dsRNA-sprayed barley plants exhibit no growth inhibition or gene silencing in Fusarium graminearum

Numerous reports have shown that incorporating a double-stranded RNA (dsRNA)-expressing transgene into plants or applying dsRNA by spraying it onto their leaves successfully protects them against invading pathogens exploiting the mechanism of RNA interference (RNAi). How dsRNAs or siRNAs are transferred between donor host cells and recipient fungal cells is largely unknown. It is speculated that plant extracellular vesicles (EVs) function as RNA shuttles between plants and their pathogens. Recently, we found that EVs isolated from host-induced gene silencing (HIGS) or spray-induced gene silencing (SIGS) plants contained dsRNA-derived siRNAs. In this study, we evaluated whether isolated EVs from dsRNA-sprayed barley (Hordeum vulgare) plants affected the growth of the phytopathogenic ascomycete Fusarium graminearum. Encouraged by our previous finding that dropping barley-derived EVs on F. graminearum cultures caused fungal stress phenotypes, we conducted an in vitro growth experiment in microtiter plates where we co-cultivated F. graminearum with plant EVs isolated from dsRNA-sprayed barley leaves. We observed that co-cultivation of F. graminearum macroconidia with barley EVs did not affect fungal growth. Furthermore, plant EVs containing SIGS-derived siRNA appeared not to affect F. graminearum growth and showed no gene silencing activity on F. graminearum CYP51 genes. Based on our findings, we concluded that either the amount of SIGS-derived siRNA was insufficient to induce target gene silencing in F. graminearum, indicating that the role of EVs in SIGS is minor, or that F. graminearum uptake of plant EVs from liquid cultures was inefficient or impossible.

Expression of miR-221 and miR-18a in patients with hepatocellular carcinoma and its clinical significance.

Recently, microRNA (miRNA) has been evaluated to provide a new diagnostic and therapeutic modality hepatocellular carcinoma (HCC) and other tumors. They are small non-coding RNA molecules that function as transcriptional and post-transcriptional regulators of gene expression by silencing target genes. The aim of this study was to evaluate the clinical significance of microRNA-18a, 221 (miR-18a, miR-221) expression in HCC formalin-fixed paraffin-embedded (FFPE) tissue. miR-18a and miR-221 expressions were assessed by reverse transcription and real-time quantitative reverse transcription polymerase chain reaction in 50 pairs of FFPE HCC and the adjacent noncancerous liver tissues. And we evaluated the expression level in HCC tissues as compared with their adjacent noncancerous counterparts. And the relationship between miR-18a, miR-221 level and clinicopathological data and survival rates were analyzed. miR-221 and miR-18a were overexpressed in HCC tissue as compared with their adjacent noncancerous liver tissue (P<0.001). miR-221 expression was found to be correlated with larger tumor size (P=0.048). miR-18a expression was correlated with modified Union for International Cancer Control stage (P=0.05). The overall survival (P=0.02) of HCC patients with high miR-221 expression was significantly poorer compared to those patients with low expression. Multivariate analyses demonstrated that miR-221 may be a poor prognostic factor of HCC patients. High expression of miR-221 in FFPE tissues could provide significance for prognosis of HCC patients. Although, miR-18a expression was significantly upregulated in HCC tissues, they are not correlated with prognosis. Further large prospective studies are needed to determine their clinical significance.

Recent Developments and Strategies for the Application of Agrobacterium-Mediated Transformation of Apple Malus × domestica Borkh.

Genetic transformation has become an important tool in plant genome research over the last three decades. This applies not only to model plants such as Arabidopsis thaliana but also increasingly to cultivated plants, where the establishment of transformation methods could still pose many problems. One of such plants is the apple (Malus spp.), the most important fruit of the temperate climate zone. Although the genetic transformation of apple using Agrobacterium tumefaciens has been possible since 1989, only a few research groups worldwide have successfully applied this technology, and efficiency remains poor. Nevertheless, there have been some developments, especially in recent years, which allowed for the expansion of the toolbox of breeders and breeding researchers. This review article attempts to summarize recent developments in the Agrobacterium-mediated transformation strategies of apple. In addition to the use of different tissues and media for transformation, agroinfiltration, as well as pre-transformation with a Baby boom transcription factor are notable successes that have improved transformation efficiency in apple. Further, we highlight targeted gene silencing applications. Besides the classical strategies of RNAi-based silencing by stable transformation with hairpin gene constructs, optimized protocols for virus-induced gene silencing (VIGS) and artificial micro RNAs (amiRNAs) have emerged as powerful technologies for silencing genes of interest. Success has also been achieved in establishing methods for targeted genome editing (GE). For example, it was recently possible for the first time to generate a homohistont GE line into which a biallelic mutation was specifically inserted in a target gene. In addition to these methods, which are primarily aimed at increasing transformation efficiency, improving the precision of genetic modification and reducing the time required, methods are also discussed in which genetically modified plants are used for breeding purposes. In particular, the current state of the rapid crop cycle breeding system and its applications will be presented.

Silencing of multiple target genes via ingestion of dsRNA and PMRi affects development and survival in Helicoverpa armigera

RNA interference (RNAi) triggered by exogenous double-stranded RNA (dsRNA) is a powerful tool to knockdown genetic targets crucial for the growth and development of agriculturally important insect pests. Helicoverpa armigera is a pest feeding on more than 30 economically important crops worldwide and a major threat. Resistance to insecticides and Bt toxins has been gradually increasing in the field. RNAi-mediated knockdown of H. armigera genes by producing dsRNAs homologous to genetic targets in bacteria and plants has a high potential for insect management to decrease agricultural loss. The acetylcholinesterase (AChE), ecdysone receptor (EcR) and v-ATPase-A (vAA) genes were selected as genetic targets. Fragments comprising a coding sequence of < 500 bp were cloned into the L4440 vector for dsRNA production in bacteria and in a TRV-VIGS vector in antisense orientation for transient expression of dsRNA in Solanum tuberosum leaves. After ingesting bacterial-expressed dsRNA, the mRNA levels of the target genes were significantly reduced, leading to mortality and abnormal development in larva of H. armigera. Furthermore, the S. tuberosum plants transformed with TRV-VIGS expressing AChE exhibited higher mortality > 68% than the control plants 17%, recorded ten days post-feeding and significant resistance in transgenic (transient) plants was observed. Moreover, larval lethality and molting defects were observed in larva fed on potato plants expressing dsRNA specific to EcR. Analysis of transcript levels by quantitative RT–PCR revealed that larval mortality was attributable to the knockdown of genetic targets by RNAi. The results demonstrated that down-regulation of H. armigera genes involved in ATP hydrolysis, transcriptional stimulation of development genes and neural conduction has aptitude as a bioinsecticide to control H. armigera population sizes and therefore decreases crop loss.

The Impact of Inflammatory Stimuli on Xylosyltransferase-I Regulation in Primary Human Dermal Fibroblasts.

Inflammation plays a vital role in regulating fibrotic processes. Beside their classical role in extracellular matrix synthesis and remodeling, fibroblasts act as immune sentinel cells participating in regulating immune responses. The human xylosyltransferase-I (XT-I) catalyzes the initial step in proteoglycan biosynthesis and was shown to be upregulated in normal human dermal fibroblasts (NHDF) under fibrotic conditions. Regarding inflammation, the regulation of XT-I remains elusive. This study aims to investigate the effect of lipopolysaccharide (LPS), a prototypical pathogen-associated molecular pattern, and the damage-associated molecular pattern adenosine triphosphate (ATP) on the expression of XYLT1 and XT-I activity of NHDF. We used an in vitro cell culture model and mimicked the inflammatory tissue environment by exogenous LPS and ATP supplementation. Combining gene expression analyses, enzyme activity assays, and targeted gene silencing, we found a hitherto unknown mechanism involving the inflammasome pathway components cathepsin B (CTSB) and caspase-1 in XT-I regulation. The suppressive role of CTSB on the expression of XYLT1 was further validated by the quantification of CTSB expression in fibroblasts from patients with the inflammation-associated disease Pseudoxanthoma elasticum. Altogether, this study further improves the mechanistic understanding of inflammatory XT-I regulation and provides evidence for fibroblast-targeted therapies in inflammatory diseases.