Effects Of Gene Silencing Research Articles

Spray-induced gene silencing to control plant pathogenic fungi: A step-by-step guide.

RNA interference (RNAi)-based control technologies are gaining popularity as potential alternatives to synthetic fungicides in the ongoing effort to manage plant pathogenic fungi. Among these methods, spray-induced gene silencing (SIGS) emerges as particularly promising due to its convenience and feasibility for development. This approach is a new technology for plant disease management, in which double-stranded RNAs (dsRNAs) targeting essential or virulence genes are applied to plants or plant products and subsequently absorbed by plant pathogens, triggering a gene silencing effect and the inhibition of the infection process. Spray-induced gene silencing has demonstrated efficacy in laboratory settings against various fungal pathogens. However, as research progressed from the laboratory to the greenhouse and field environments, novel challenges arose, such as ensuring the stability of dsRNAs and their effective delivery to fungal targets. Here, we provide a practical guide to SIGS for the control of plant pathogenic fungi. This guide outlines the essential steps and considerations needed for designing and assessing dsRNA molecules. It also addresses key challenges inherent to SIGS, including delivery and stability of dsRNA molecules, and how nanoencapsulation of dsRNAs can aid in overcoming these obstacles. Additionally, the guide underscores existing knowledge gaps that warrant further research and aims to provide assistance to researchers, especially those new to the field, encouraging the advancement of SIGS for the control of a broad range of fungal pathogens.

PbMADS49 Regulates Lignification During Stone Cell Development in 'Dangshansuli' (Pyrus bretschneideri) Fruit.

Lignified stone cell content is one of the critical factors affecting 'Dangshansuli' fruit quality. The function of MADS-box transcription factors in regulating lignin biosynthesis in pear fruit is still less. In this study, PbMADS49 gene silencing inhibited the lignin biosynthesis and stone cell secondary wall development of pear fruit mainly through reducing the expression levels of lignin monomer polymerisation key enzymes (PbPRX33 and PbPRX45). PbMADS49 was a transcriptional repressor inhibiting its transcription by binding to the CArG element in the target gene promoter. Combined with the co-expression network and promoter cis-acting element analysis, we hypothesised that PbMADS49 positively regulates the transcription of PbPRX33 through PbWRKY63. The gene silencing effect of homologous genes PbPRX33-1 and PbPRX33-2 was consistent with PbMADS49, and PbPRX33-2 was more significant than PbPRX33-1. This study shows that PbMADS49 is a positive regulator of stone cell lignification, providing new insights into the development mechanism of pear stone cells.

HcCnAα regulates NF-κB signaling in Hyriopsis cumingii by interacting with HcIKK and facilitating IκB phosphorylation.

Calcineurin (CN), a serine/threonine protein phosphatase regulated by Ca2+ and calmodulin, plays a crucial role in the immune response of bivalves. In this study, we examined the effects of gene silencing of the CN subunit (HcCnAα) in Hyriopsis cumingii on the expression of genes associated with the NF-κB signaling pathway, as well as the phosphorylation of the inhibitor of NF-κB (IκB), through RNA interference (RNAi), quantitative PCR (qPCR), and Western blot (WB) analyses. The IκB kinase (HcIKK) and B-cell CLL/lymphoma 10 (HcBcl10) genes of H. cumingii were cloned using rapid amplification of cDNA ends, and protein interactions with HcCnAα were investigated through yeast two-hybrid assays. The results demonstrated that RNAi-mediated knockdown of HcCnAα significantly reduced the expression of IKK, p65, and downstream immune-related genes in the NF-κB signaling pathway-Lys, The, Def, α2-M, TNF-α, and IL-17-all showing significant decreases (P<0.05). Additionally, phosphorylation of IκB was inhibited. These findings suggest that HcCnAα plays a regulatory role in the NF-κB signaling pathway, influencing the expression of downstream immune response-related genes and defense proteins. Furthermore, yeast two-hybrid assay results indicated a direct protein-protein interaction between HcIKK and HcCnAα, while no interaction was observed between HcBcl10 and HcCnAα. This study elucidates the specific molecular mechanisms by which HcCnAα regulates the immune response in H. cumingii, providing a foundational basis for further exploration of immune regulation mechanisms and the development of disease prevention strategies in bivalves.

Integrating cotyledon-based virus-induced gene silencing with visual marker promises a rapid, highly effective validation of gene functions in Nepeta cataria.

Nepeta spp. generate volatile nepetalactone iridoids that have cat-attractant and insect-repellent activities. They differ from typical mint family (Lamiaceae) iridoids, which are non-volatile glucosides, and also vary from other species in the Nepetoideae sub-family, which do not generate iridoids. The chemistry and evolution of Nepeta make it suitable for further investigation. However, the lack of transgenic technology hampers the molecular and genetic investigations in Nepeta. Virus-induced gene silencing (VIGS) is a powerful tool to detect gene functions in vivo. Here, we constructed a modified VIGS method in Nepeta cataria, using cotyledon infiltration, with the gene silencing effect spreading to the first two pairs of true leaves. The VIGS efficiency reached as high as 84.4%, and the procedure takes only 3 weeks. We employed this method to validate the role of geraniol 8-hydroxylase in nepetalactone biosynthesis with ChlH as a visual marker in N. cataria. The method is also applicable to Nepeta mussinii. Thus, we developed an easy and effective VIGS approach, which will be advantageous for endogenous gene studies in two Nepeta species and holds the potential for application in other plants.

A dual-locked cyclopeptide-siRNA conjugate for tumor-specific gene silencing.

Strategies allowing tumor-selective siRNA delivery while minimizing off-tumor gene silencing effects are highly demanded to advance cancer gene therapy, which however still remain challenging. We herein report a dual-locking bioconjugation approach to address this challenge. A dual-locked cyclopeptide-siRNA conjugate (DPRC) was designed to simultaneously endow siRNA with tumor-targeting properties and tumor-biomarker/visible-light dually controllable action. The DPRC consisted of a programmed death-ligand 1 (PD-L1)-targeting cyclopeptide as a tumor-homing ligand and B-cell lymphoma-2 (Bcl-2)-targeting siRNA as a payload. They were conjugated via a tandem-responsive cleavable linker containing a photocleavable coumarin moiety quenched by naphthylamide through a disulfide linkage. Owing to the interaction between cell-membrane PD-L1 and the cyclopeptide, the DPRC was efficiently taken up by PD-L1-positive cancer cells. Notably, the internalized DPRC could only release and restore the gene silencing activity of siBcl-2 upon GSH-mediated disulfide bond breakage followed by visible light irradiation on the coumarin moiety to induce photo-cleavage. The released siBcl-2 further silenced the expression of anti-apoptotic Bcl-2 to suppress cancer cell growth. We demonstrated the tumor-targeting and dual-locked action of siRNA by the DPRC in both two-dimensional and three-dimensional cancer cell cultures. This study thus presents a novel strategy for precise tumor-specific gene silencing by siRNA.

Practical laboratory class to assess gene silencing using CRISPR interference (CRISPRi) technology in the archaeon Haloferax volcanii.

Perturbation of gene expression using RNA interference (RNAi) or CRISPR interference (CRISPRi) is a useful strategy to explore the function of essential genes. In the archaeon Haloferax volcanii, the CRISPR-Cas system has been adapted as a CRISPRi tool to silence the expression of specific genes. We developed a laboratory class (LC) to conceptualize gene silencing through inactivation of the H. volcanii LonB protease gene, a negative regulator of carotenoid pigments biosynthesis, using CRISPRi. This LC has been successfully applied in the Biology and Biochemistry of Microorganisms course for undergraduate students of Biology in 2022 and 2023. The following objectives were proposed: (a) generate H. volcanii mutant strains with reduced expression of the lonB gene using CRISPRi; (b) examine the effect of lonB gene silencing on cell pigmentation and growth rate; (c) assess lonB gene repression by Western blotting (WB). This LC allows students to obtain and screen CRISPRi silenced-mutants by means of simple procedures using a non-pathogenic organism as well as handle basic microbiology, biochemistry and molecular biology protocols. Additionally, the LC fosters social actions through collaborative work (experimental work), the interpretation and discussion of data and the ability to communicate outcomes orally and in a written format (scientific report).

Investigating the Expression and Function of HIF-1α in Neocaridina davidi During Embryo Cleavage Stage

Neocaridina davidi, a member of Decapoda within Crustacea, stands out due to its petite stature, robust reproductive capabilities and short molting cycle. Consequently, it has emerged as a valuable experimental model for investigations spanning ecology, development, physiology, and toxicology. Despite the pivotal role of Hypoxia Inducible Factor-1α (HIF-1α) in diverse physiological processes like biological hypoxia stress, cellular growth, and stress resistance, its functionality in crustaceans remains underexplored. Moreover, the role and function of HIF-1α in crustaceans, especially in embryonic stages, have been insufficiently addressed. This study delves into the function of HIF-1α during embryonic development. Initially, the complete sequence of the HIF-1α gene was acquired. Notably, the expression of HIF-1α during the cleavage stage surpassed that of subsequent phases. Subsequently, dsRNAHIF-1α was introduced into sexually mature shrimp, revealing that the inhibitory impact of dsRNA on gene expression in the parental generation could be inherited by the offspring, exerting a specific gene silencing effect in the embryo. The consequences of silencing HIF-1α in embryos manifested as varying degrees of premature division termination, besides, the glycolysis in the embryos was also affected by HIF-1α suppression. These results provide data support for understanding the early embryonic development of crustaceans and HIF-1α functions within it.

Shielding siRNA by peptide-based nanofibers: An efficient approach for turning off EGFR gene in breast cancer.

Peptide-based self-assembled nanosystems show great promise as non-viral gene and siRNA delivery vectors. In the current study, we designed and functionalized nanofibers for the delivery of siRNA, targeting and silencing EGFR gene overexpressed in triple-negative breast cancer. The nanofiber-mediated siRNA delivery was characterized in terms of zeta potential, morphology, and structural stability by circular dichroism spectroscopy. In cytotoxicity studies, nanofibers presented high biocompatibility showing a negligible effect on cell viability both on healthy and cancer cell lines. The binding between nanofibers and EGFR-siRNA was investigated and ascertained by performing different biophysical studies. The complex siRNA:NF was stable over time, under fetal bovine serum, temperature and ionic strength effects. Moreover, nanofibers effectiveness in stabilizing and delivering an ad hoc selected siRNA for EGFR gene expression silencing was verified in a preclinical model of triple-negative breast cancer. Specifically, a significant gene knockdown was obtained with the complex siRNA:NF, that is comparable with the effect obtained by lipofectamine/siRNA transfection. This effective gene silencing derived from the successful internalization of nanofibers by cancer cells as observed by confocal microscopy. These results suggested that this peptide-based nanofiber could be an effective and safe systemic siRNA delivery system for application in biomedical areas.

REST-dependent glioma progression occurs independently of the repression of the long non-coding RNA HAR1A.

The long non-coding RNA (lncRNA), HAR1A is emerging as a putative tumour suppressor. In non-neoplastic brain cells, REST suppresses HAR1A expression. In gliomas REST acts as an oncogene and is a potential therapeutic target. It is therefore conceivable that REST promotes glioma progression by down-regulating HAR1A. To test this hypothesis, glioma clinical databases were analysed to study: (I) HAR1A/REST correlation; (II) HAR1A and REST prognostic role; (III) molecular pathways associated with these genes. HAR1A expression and subcellular localization were studied in glioblastoma and paediatric glioma cells. REST function was also studied in these cells, by observing the effects of gene silencing on: (I) HAR1A expression; (II) cancer cell proliferation, apoptosis, migration; (III) expression of neural differentiation genes. The same phenotypes (and cell morphology) were studied in HAR1A overexpressing cells. Our results show that REST and HAR1A are negatively correlated in gliomas. Higher REST expression predicts worse prognosis in low-grade gliomas (the opposite is true for HAR1A). REST-silencing induces HAR1A upregulation. HAR1A is primarily detected in the nucleus. REST-silencing dramatically reduces cell proliferation and induces apoptosis, but HAR1A overexpression has no major effect on investigated cell phenotypes. We also show that REST regulates the expression of neural differentiation genes and that its oncogenic function is primarily HAR1A-independent.

Rabies virus-mimicking liposomes for targeted gene therapy in Alzheimer’s disease

RNA interference (RNAi) harbors significant potential for treating neurological disorders; nevertheless, limited efficacy has been discerned. The presence of barriers within the central nervous system, coupled with the inherent instability of nucleic acids within biological conditions, poses formidable challenges in advancing effective gene delivery strategies. In this study, we designed and prepared a virus-mimic non-viral gene vector, rabies virus glycoprotein (RVG29)-decorated liposome (f(Lipo)-RVG29), to deliver small interfering RNAs to the brain. Alzheimer’s disease (AD) was chosen as a model of neurodegenerative disease in this context, and b-site APP cleaving enzyme silencing siRNA (siBACE1) was used. The developed liposomal delivery system has a particle size of under 80 nm with a spherical shape, positive zeta potential, and the ability to protect siRNA against nucleases. In vitro studies demonstrate that functionalizing the cationic liposome by the RVG29 targeting ligand significantly enhances the effectiveness of gene delivery and silencing. Examination through ex vivo imaging illustrates an increased deposition of fluorescent-labeled f(Lipo)-RVG29 within brain tissue after 12 h post application. Additionally, the in vivo delivery of f(Lipo)-RVG29 carrying siRNA has substantially suppressed BACE1 expression at both mRNA and protein levels within the brain tissue. Our results suggest that the developed non-viral vector could be a promising gene carrier system combining the synergistic effect of virus-mimic RVG29 ligand with bioinspired liposome that imitates the natural lipid bilayers of cell membranes for brain-targeted RNAi therapeutics.

MMP-2 Responsive Gold Nanorods Loaded with HSP-70 siRNA for Enhanced Photothermal Tumor Therapy.

Gold nanorods (Au NRs) are a valuable photothermal nanomaterial for tumor therapy. However, when treated with Au NRs for photothermal therapy, the expression of heat shock proteins in tumors will increase, which will induce heat resistance in tumor cells and reduce the photothermal therapeutic effect of Au NRs. By RNA interference, the expression of heat shock proteins would be effectively inhibited to improve the efficasy of tumor photothermal therapy. However, deep and noninvasive tissue penetration remains a great obstacle to applying siRNA successfully. Thus, the nanoplatform AGC/HSP-70 siRNA was designed for enhanced photothermal tumor therapy by RNA interference. In the AGC/HSP-70 siRNA complex, the Au-S bond modified the matrix metalloproteinase-2 (MMP-2)-sensitive peptide GPLGLAG on the surface of gold nanorods. Moreover, the natural basic polysaccharide (chitosan) was reacted with the peptide by an amide bond for delivering heat shock protein 70 silencing siRNA (HSP-70 siRNA). Modifying the MMP-2-sensitive linker could cause more Au NRs to accumulate in tumors to exert a photothermal effect and promote the penetration of HSP-70 siRNA and chitosan complexes into deep tumor tissues. In vitro experiments indicated that the enzymolysis of the MMP-2-sensitive linker for AGC/HSP-70 siRNA could promote the cellular uptake and perinuclear distribution of HSP-70 siRNA in tumor cells, which may be due to the smaller size and positive electricity of the complexes. All of these results ensured the efficient gene silencing effect of HSP-70 siRNA to enhance the photothermal therapeutic effect of Au NRs in tumor tissues, as demonstrated by the gene silencing and cellular apoptotic experiments. In vivo experiments further proved that the AGC/HSP-70 siRNA nanoplatform efficiently improved the photothermal effect of Au NRs. In summary, this work proved that AGC/HSP-70 siRNA is a promising drug delivery strategy for enhancing the photothermal therapy of tumors by regulating the photothermal sensitivity of deep tumor cells as well as retaining more Au NRs in tumor tissues, and also provides a novel strategy for tumor photothermal therapy.

Pdk3's role in RANKL-induced osteoclast differentiation: insights from a bone marrow macrophage model.

Osteoporosis (OP) is a chronic disease characterized by decreased bone mass, loss of skeletal structural integrity and increased susceptibility to fracture. Available studies have shown that the pyruvate dehydrogenase kinase (PDK) family is associated with osteoclastogenesis and bone loss, but the specific role of Pdk3 in bone pathology has not been systematically investigated. A cell OP model was established in receptor activator for nuclear factor-κB Ligand (RANKL)-induced bone marrow macrophages (BMMs). Hereafter, the expression levels of Pdk3 and osteoclastogenesis feature genes including nuclear factor of activated T cells 1 (Nfatc1), Cathepsin K (Ctsk), osteoclast associated Ig-like receptor (Oscar) in BMMs-derived osteoclasts were examined based on real-time quantitative PCR and western blotting methods. Further, the phosphorylation of ERK, P65 and JAK/STAT and their correlation was Pdk3 was gauged. In particular, changes in the activity of these signaling pathways were observed by silencing experiments of the Pdk3 gene (using small interfering RNA). Finally, the effects of Pdk3 gene silencing on signaling pathway activity, osteoclastogenesis, and related inflammatory and apoptotic indicators were observed by transfection with PDK3-specific siRNA. Following RANKL exposure, the levels of Pdk3 and osteoclastogenesis feature genes were all elevated, and a positive correlation between Pdk3 and osteoclastogenesis feature genes was seen. Meanwhile, ERK, P65 and JAK/STAT phosphorylation was increased by RANKL, and Pdk3 was confirmed to be positively correlated with the phosphorylation of ERK, P65 and JAK/STAT. Additionally, in RANKL-exposed osteoclasts, Pdk3 knockdown diminished the phosphorylation of ERK, P65 and JAK/STAT, reduced the expressions of osteoclastogenesis feature genes. Importantly, knockdown of Pdk3 also reduced the expression of inflammatory cytokines and resulted in elevated levels of Bax and Casp3 expression, as well as downregulation of Bcl2 expression. This study reveals for the first time the role of Pdk3 in RANKL-induced osteoclastogenesis and OP. These findings provide a foundation for future studies on the role of Pdk3 in other bone diseases and provide new ideas for the development of OP therapeutics targeting Pdk3.

Recent Advancements in Cancer RNA-interference Therapy with Nanotechnology Strategies

Cancer is a global challenge, and genetics play a major role in onsets and in designing the next generation of cancer therapies. One of the key approaches is the downregulation of genes through RNA interference (RNAi) which has been proposed as a promising tool for controlling cancer-causing genes by employing a complementary base-pairing mechanism. Crucial tools in RNAi; small interfering RNA (siRNA), and microRNA (miRNA) have been extensively utilized in cancer therapy. Despite their discovery nearly two decades ago, only a handful of RNAi-based products have recently gained approval from regulatory authorities like the FDA, principally due to inherent limitations. The efficacy of RNAi therapies is significantly hindered by barriers related to absorption, distribution, metabolism, and excretion, leading to rapid elimination from the systemic circulation before reaching the cytosol of targeted cells. Nevertheless, RNAi therapeutics hold immense promise in various diseases, especially in various types of cancer. Overcoming these challenges demands the design of suitable drug delivery systems and the implementation of strategies aimed at enhancing pharmacokinetic parameters associated with RNAi therapies. Nanotechnology-based vehicles such as polymer-based nanoparticles, lipid nanoparticles, liposomes, dendrimers, and inorganic nanoparticles may serve as efficient carriers for targeting RNAi therapies to their desired sites. This review discusses the recent advances in nanotechnology strategies for RNAi delivery, with the overarching objective of facilitating effective targeting and gene silencing for the advancement of RNAi in cancer therapy.

6455 The Effect of Gene Silencing on Inhibiting Stress Hormones in Wild Birds Raised Under Stressful Conditions

Abstract Disclosure: S.M. Abdulateef: None. T. Mohammed: None. W. Al-Jugif: None. M.Q. Abed: None. Wild birds are notable for their ease of rearing, tolerance to extreme environmental conditions such as heat and cold, and their resistance to diseases. They are also recognized for their ability to withstand stress. However, their primary challenge is low egg production, stemming from their aggressive nature. This aggression elevates the secretion of stress and fear hormones, significantly impacting reproductive hormones. This study aims to explore the effects of gene silencing on the physiological traits of wild birds, particularly its influence on physiological responses and the reduction of stress hormone levels. Three treatments were employed: T1 (control), T2 (welfare treatment, wherein birds were raised with enhanced well-being), and T3 (using siRNA technology to silence the gene responsible for stress hormone corticosterone production). The silencing of CYP11B1 and CYP11B2 involved obtaining the cDNA of each gene from the NCBI, associated with corticosterone production. The coding sequence from the 5' end at the cap direction was reversed to complement mRNA. Two siRNAs were procured from Macrogene Company (South Korea) in lyophilized form at high concentration, with sequences: CYP11B2 5'-acacctctgcctttgccctgagtgccat-3' and CYP11B1 5'-ctaaagtcaaactcagccccacattcat-3'. These were dissolved in molecular-grade distilled water for a final concentration of 500 pmol/µl. Each hen was injected in the wing in the jugular artery with 125 µl of the siRNA solution at 14 and 18 weeks of age, before the commencement of the egg-laying cycle. Corticosterone levels were measured using the Sunlong Biotech CO., LTD kit. The results showed a significant decrease (p&amp;lt;0.01) in stress hormone levels in the third treatment group (gene silencing treatment), leading to notable physiological responses in the birds compared to the other treatments. This study highlights the impact of gene silencing on the physiological traits of wild birds and its effects on their physiological responses. The findings of this study are significant in the context of avian physiology and poultry farming. By elucidating the impact of stress hormones on egg production and demonstrating the efficacy of gene silencing as a method to enhance reproductive efficiency, this research opens new avenues for improving the productivity and well-being of wild birds in controlled environments. It also underscores the potential of genetic interventions in addressing physiological challenges in wildlife. Presentation: 6/1/2024

Iron and siRNA co-encapsulated ferritin nanocages induce ferroptosis synergistically for cancer therapy

Ferroptosis has received great attention as an iron-dependent programmed cell death for efficient cancer therapy. However, with the accumulation of iron in tumor cells, the antioxidant system is activated by reducing glutathione (GSH) with glutathione peroxidase 4 (GPX4), which critically limits the ferroptosis therapeutic effect. Herein, an iron and GPX4 silencing siRNA (siGPX4) co-encapsulated ferritin nanocage (HFn@Fe/siGPX4) was developed to enhance ferroptosis by disruption of redox homeostasis and inhibition of antioxidant enzyme synergistically. The siGPX4 were loaded into the nanocages by pre-incubated with iron, which could significantly improve the loading efficiency of the gene drugs when compared with the reported gene drug loading strategy by ferritin nanocages. And more iron was overloaded into the ferritin through the diffusion method. When HFn@Fe/siGPX4 was taken up by human breast cancer cell MCF-7 in a TfR1-mediated pathway, the excess iron ions in the drug delivery system could for one thing induce ferroptosis by the production of reactive oxygen species (ROS), for another promote siGPX4 escaping from the lysosome to exert gene silencing effect more effectively. Both the in vitro and in vivo results demonstrated that HFn@Fe/siGPX4 could significantly inhibit tumor growth by synergistical ferroptosis. Thus, the developed HFn@Fe/siGPX4 afforded a combined ferroptosis strategy for ferroptosis-based antitumor as well as a novel and efficient gene drug delivery system.

Direct cytosolic delivery of siRNA via cell membrane fusion using cholesterol-enriched exosomes.

Efficient cytosolic delivery is a significant hurdle when using short interfering RNA (siRNA) in therapeutic applications. Here we show that cholesterol-rich exosomes are prone to entering cancer cells through membrane fusion, achieving direct cytosolic delivery of siRNA. Molecular dynamics simulations suggest that deformation and increased contact with the target cell membrane facilitate membrane fusion. In vitro we show that cholesterol-enriched milk-derived exosomes (MEs) achieve a significantly higher gene silencing effect of siRNA, inducing superior cancer cell apoptosis compared with the native and cholesterol-depleted MEs, as well as conventional transfection agents. When administered orally or intravenously to mice bearing orthotopic or subcutaneous tumours, the cholesterol-enriched MEs/siRNA exhibit antitumour activity superior to that of lipid nanoparticles. Collectively, by modulating the cholesterol content of exosome membranes to facilitate cell entry via membrane fusion, we provide a promising approach for siRNA-based gene therapy, paving the way for effective, safe and simple gene therapy strategies.

BAG2, MAD2L1, and MDK are cancer-driver genes and candidate targets for novel therapies in malignant pleural mesothelioma

Malignant pleural mesothelioma (MPM) is an aggressive cancer with a poor prognosis and the identification of novel druggable targets is urgently needed. In previous work, we identified 15 deregulated genes highly expressed in MPM tissues and correlated with a poor prognosis. Here, we validated these findings on an independent dataset of 211 MPM patients (EGA, EGAD00001001915) and on a panel of MPM cell lines. Furthermore, we carried out in vitro gene silencing followed by proliferation, cytotoxicity, caspase, and migration assays to define whether these targets could be cancer-driver genes. We ended up with three novel candidates (i.e., BAG2, MAD2L1, and MDK), whose encoded proteins could be exploited as druggable targets. Moreover, of novelty, immunohistochemistry analysis on tissues revealed that the overexpression of BAG2 and MAD2L1 could differentiate MPM from RMP patients. Furthermore, when we tested Neratinib (an inhibitor of MAD2L1) and iMDK (an inhibitor of MDK) we found that they are effective on MPM cells, in part phenocopying the effects of MAD2L1 and MDK gene silencing. In summary, in the present work, we report that BAG2, MAD2L1, and MDK are bona fide cancer-driver genes for MPM worth of further studies.

How carbon sources drive cellulose synthesis in two Komagataeibacter xylinus strains

Bacterial cellulose synthesis from defined media and waste products has attracted increasing interest in the circular economy context for sustainable productions. In this study, a glucose dehydrogenase-deficient Δgdh K2G30 strain of Komagataeibacter xylinus was obtained from the parental wild type through homologous recombination. Both strains were grown in defined substrates and cheese whey as an agri-food waste to assess the effect of gene silencing on bacterial cellulose synthesis and carbon source metabolism. Wild type K2G30 boasted higher bacterial cellulose yields when grown in ethanol-based medium and cheese whey, although showing an overall higher d-gluconic acid synthesis. Conversely, the mutant Δgdh strain preferred d-fructose, d-mannitol, and glycerol to boost bacterial cellulose production, while displaying higher substrate consumption rates and a lower d-gluconic acid synthesis. This study provides an in-depth investigation of two K. xylinus strains, unravelling their suitability for scale-up BC production.

LncRNA PVT1 silencing inhibits gastric cancer cells’ progression via enhancing chemosensitivity to paclitaxel

Gastric cancer (GC) is one of the leading causes of cancer-related deaths worldwide because of its high morbidity and the absence of effective therapies. Even though paclitaxel is a powerful anticancer chemotherapy drug, recent studies have indicated its ineffectiveness against GC cells. Long non-coding RNA (lncRNA) PVT1 has a high expression in GC cells and increases the progression of tumors via inducing drug resistance. In the present study, the effects of the siRNA-mediated lncRNA PVT1 gene silencing along with paclitaxel treatment on the rate of apoptosis, growth, and migration of AGS GC cells were investigated. AGS cells were cultured and then transfected with siRNA PVT1 using electroporation. The MTT test was used to examine the effect of treatments on the viability of cultured cells. Furthermore, the flow cytometry method was used to evaluate the impact of treatments on the cell cycle process and apoptosis induction in GC cells. Finally, the mRNA expression of target genes was assessed using the qRT-PCR method. The results showed that lncRNA PVT1 gene suppression, along with paclitaxel treatment, reduces the viability of cancer cells and significantly increases the apoptosis rate of cancer cells and the number of cells arrested in the G2/M phase compared to the control group. Based on the results of qRT-PCR, combined treatment significantly decreased the expression of MMP3, MMP9, MDR1, MRP1, Bcl-2, k-Ras, and c-Myc genes and increased the expression of the Bax gene compared to the control group. The results of our study showed that lncRNA PVT1 gene targeting, together with paclitaxel treatment, induces apoptosis, inhibits growth, alleviates drug resistance, and reduces the migratory capability of GC cells. Therefore, there is a need for further investigations to evaluate the feasibility and effectiveness of this approach in vivo in animal models.

Intracellular Delivery of Antisense Oligonucleotides by Tri-Branched Cyclic Disulfide Units.

Therapeutic oligonucleotides, such as antisense DNA, show promise in treating previously untreatable diseases. However, their applications are still hindered by the poor membrane permeability of naked oligonucleotides. Therefore, it is necessary to develop efficient methods for intracellular oligonucleotide delivery. Previously, our group successfully developed disulfide-based Membrane Permeable Oligonucleotides (MPON), which achieved enhanced cellular uptake and gene silencing effects through an endocytosis-free uptake mechanism. Herein, we report a new molecular design for the next generation of MPON, called trimer MPON. The trimer MPON consists of a tri-branched backbone, three α-lipoic acid units, and a spacer linker between the oligonucleotides and tri-branched cyclic disulfide unit. We describe the design, synthesis, and functional evaluation of the trimer MPON, offering new insights into the molecular design for efficient oligonucleotide delivery.