Ultrasound-Mediated Lysozyme Microbubbles Targeting NOX4 Knockdown Alleviate Cisplatin-Exposed Cochlear Hair Cell Ototoxicity.

The nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 (NOX4) protein plays an essential role in the cisplatin (CDDP)-induced generation of reactive oxygen species (ROS). In this study, we evaluated the suitability of ultrasound-mediated lysozyme microbubble (USMB) cavitation to enhance NOX4 siRNA transfection in vitro and ex vivo. Lysozyme-shelled microbubbles (LyzMBs) were constructed and designed for siNOX4 loading as siNOX4/LyzMBs. We investigated different siNOX4-based cell transfection approaches, including naked siNOX4, LyzMB-mixed siNOX4, and siNOX4-loaded LyzMBs, and compared their silencing effects in CDDP-treated HEI-OC1 cells and mouse organ of Corti explants. Transfection efficiencies were evaluated by quantifying the cellular uptake of cyanine 3 (Cy3) fluorescein-labeled siRNA. In vitro experiments showed that the high transfection efficacy (48.18%) of siNOX4 to HEI-OC1 cells mediated by US and siNOX4-loaded LyzMBs significantly inhibited CDDP-induced ROS generation to almost the basal level. The ex vivo CDDP-treated organ of Corti explants of mice showed an even more robust silencing effect of the NOX4 gene in the siNOX4/LyzMB groups treated with US sonication than without US sonication, with a marked abolition of CDDP-induced ROS generation and cytotoxicity. Loading of siNOX4 on LyzMBs can stabilize siNOX4 and prevent its degradation, thereby enhancing the transfection and silencing effects when combined with US sonication. This USMB-derived therapy modality for alleviating CDDP-induced ototoxicity may be suitable for future clinical applications.

Post-GWAS functional analyses of CNTNAP5 suggests its role in glaucomatous neurodegeneration.

Primary angle closure glaucoma (PACG) affects more than 20 million people worldwide, with an increased prevalence in south-east Asia. In a prior haplotype-based GWAS, we identified a novel CNTNAP5 genic region, significantly associated with PACG. In the current study, we have extended our perception of CNTNAP5 involvement in glaucomatous neurodegeneration in a zebrafish model, through investigating phenotypic consequences pertinent to retinal degeneration upon knockdown of cntnap5 by translation-blocking morpholinos. While cntnap5 knockdown was successfully validated using an antibody, immunofluorescence followed by western blot analyses in cntnap5-morphant (MO) zebrafish revealed increased expression of acetylated tubulin indicative of perturbed cytoarchitecture of retinal layers. Moreover, significant loss of Nissl substance is observed in the neuro-retinal layers of cntnap5-MO zebrafish eye, indicating neurodegeneration. Additionally, in spontaneous movement behavioural analysis, cntnap5-MO zebrafish have a significantly lower average distance traversed in light phase compared to mismatch-controls, whereas no significant difference was observed in the dark phase, corroborating with vision loss in the cntnap5-MO zebrafish. This study provides the first direct functional evidence of a putative role of CNTNAP5 in visual neurodegeneration.

RNAi-mediated pest control targeting the Troponin I (wupA) gene in sweet potato weevil, Cylas formicarius.

The sweet potato weevil (Cylas formicarius) is a critical pest producing enormous global losses in sweet potato crops. Traditional pest management approaches for sweet potato weevil, primarily using chemical pesticides, causes pollution, food safety issues, and harming natural enemies. While RNA interference (RNAi) is a promising environmentally friendly approach to pest control, its efficacy in controlling the sweet potato weevil has not been extensively studied. In this study, we selected a potential target for controlling C. formicarius, the Troponin I gene (wupA), which is essential for musculature composition and crucial for fundamental life activities. We determined that wupA is abundantly expressed throughout all developmental stages of the sweet potato weevil. We evaluated the efficiency of double-stranded RNAs in silencing the wupA gene via microinjection and oral feeding of sweet potato weevil larvae at different ages. Our findings demonstrate that both approaches significantly reduced the expression of wupA and produced high mortality. Moreover, the 1st instar larvae administered dswupA exhibited significant growth inhibition. We assessed the toxicity of dswupA on the no-target insect silkworm and assessed its safety. Our study indicates that wupA knockdown can inhibit the growth and development of C. formicarius and offer a potential target gene for environmentally friendly control.

Spatially Precise Genetic Engineering at the Electrode-Tissue Interface.

The interface between electrodes and neural tissues plays a pivotal role in determining the efficacy and fidelity of neural activity recording and modulation. While considerable efforts have been made to improve the electrode-tissue interface, the majority of studies have primarily concentrated on the development of biocompatible neural electrodes through abiotic materials and structural engineering. In this study, an approach is presented that seamlessly integrates abiotic and biotic engineering principles into the electrode-tissue interface. Specifically, ultraflexible neural electrodes with short hairpin RNAs (shRNAs) designed to silence the expression of endogenous genes within neural tissues are combined. The system facilitates shRNA-mediated knockdown of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and polypyrimidine tract-binding protein 1 (PTBP1), two essential genes associated in neural survival/growth and neurogenesis, within specific cell populations located at the electrode-tissue interface. Additionally, it is demonstrated that the downregulation of PTEN in neurons can result in an enlargement of neuronal cell bodies at the electrode-tissue interface. Furthermore, the system enables long-term monitoring of neuronal activities following PTEN knockdown in a mouse model of Parkinson's disease and traumatic brain injury. The system provides a versatile approach for genetically engineering the electrode-tissue interface with unparalleled precision, paving the way for the development of regenerative electronics and next-generation brain-machine interfaces.

RNA therapeutics to control fibrinolysis: review on applications in biology and medicine.

Regulation of fibrinolysis, the process that degrades blood clots, is pivotal in maintaining hemostasis. Dysregulation leads to thrombosis or excessive bleeding. Proteins in the fibrinolysis system include fibrinogen, coagulation factor XIII, plasminogen, tissue plasminogen activator, urokinase plasminogen activator, α2-antiplasmin, thrombin-activatable fibrinolysis inhibitor, plasminogen activator inhibitor-1, α2-macroglobulin, and others. While each of these is a potential therapeutic target for diseases, they lack effective or long-acting inhibitors. Rapid advances in RNA-based technologies are creating powerful tools to control the expression of proteins. RNA agents can be long-acting and tailored to either decrease or increase production of a specific protein. Advances in nucleic acid delivery, such as by lipid nanoparticles, have enabled the delivery of RNA to the liver, where most proteins of coagulation and fibrinolysis are produced. This review will summarize the classes of RNA that induce 1) inhibition of protein synthesis, including small interfering RNA and antisense oligonucleotides; 2) protein expression, including messenger RNA and self-amplifying RNA; and 3) gene editing for gene knockdown and precise editing. It will review specific examples of RNA therapies targeting proteins in the coagulation and fibrinolysis systems and comment on the wide range of opportunities for controlling fibrinolysis for biological applications and future therapeutics using state-of-the-art RNA therapies.

Lipid Nanoparticle-Based Inhibitors for SARS-CoV-2 Host Cell Infection.

Purpose: The global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the lingering threat to public health has fueled the search for effective therapeutics to treat SARS-CoV-2. This study aimed to develop lipid nanoparticle (LNP) inhibitors of SARS-CoV-2 entry to reduce viral infection in the nose and upper airway. Methods: Two types of LNP formulations were prepared following a microfluidic mixing method. The LNP-Trap consisted of DOPC, DSPC, cholesterol, and DSPE-PEG-COOH modified with various spike protein binding ligands, including ACE2 peptide, recombinant human ACE2 (rhACE2) or monoclonal antibody to spike protein (mAb). The LNP-Trim consisted of ionizing cationic DLin-MC3-DMA, DSPC, cholesterol, and DMG-PEG lipids encapsulating siACE2 or siTMPRSS2. Both formulations were assayed for biocompatibility and cell uptake in airway epithelial cells (Calu-3). Functional assessment of activity was performed using SARS-CoV-2 spike protein binding assays (LNP-Trap), host receptor knockdown (LNP-Trim), and SARS-CoV-2 pseudovirus neutralization assay (LNP-Trap and LNP-Trim). Localization and tissue distribution of fluorescently labeled LNP formulations were assessed in mice following intranasal administration. Results: Both LNP formulations were biocompatible based on cell impedance and MTT cytotoxicity studies in Calu-3 cells at concentrations as high as 1 mg/mL. LNP-Trap formulations were able to bind spike protein and inhibit pseudovirus infection by 90% in Calu-3 cells. LNP-Trim formulations reduced ACE2 and TMPRSS2 at the mRNA (70% reduction) and protein level (50% reduction). The suppression of host targets in Calu-3 cells treated with LNP-Trim resulted in over 90% inhibition of pseudovirus infection. In vivo studies demonstrated substantial retention of LNP-Trap and LNP-Trim in the nasal cavity following nasal administration with minimal systemic exposure. Conclusion: Both LNP-Trap and LNP-Trim formulations were able to safely and effectively inhibit SARS-CoV-2 pseudoviral infection in airway epithelial cells. These studies provide proof-of-principle for a localized treatment approach for SARS-CoV-2 in the upper airway.

Spray-drying of PEI-/PPI-based nanoparticles for DNA or siRNA delivery.

Spray-drying of nucleic acid-based drugs designed for gene therapy or gene knockdown is associated with many advantages including storage stability and handling as well as the possibility of pulmonary application. The encapsulation of nucleic acids in nanoparticles prior to spray-drying is one strategy for obtaining efficient formulations. This, however, strongly relies on the definition of optimal nanoparticles, excipients and spray-drying conditions. Among polymeric nanoparticles, polyethylenimine (PEI)-based complexes with or without chemical modifications have been described previously as very efficient for gene or oligonucleotide delivery. The tyrosine-modification of linear or branched low molecular weight PEIs, or of polypropylenimine (PPI) dendrimers, has led to high complex stability, improved cell uptake and transfection efficacy as well as high biocompatibility. In this study, we identify optimal spray-drying conditions for PEI-based nanoparticles containing large plasmid DNA or small siRNAs, and further explore the spray-drying of nanoparticles containing chemically modified polymers. Poly(vinyl alcohol) (PVA), but not trehalose or lactose, is particularly well-suited as excipient, retaining or even enhancing transfection efficacies compared to fresh complexes. A big mesh size is critically important as well, while the variation of the spray-drying temperature plays a minor role. Upon spray-drying, microparticles in a âˆ¼ 3.3 - 8.5 µm size range (laser granulometry) are obtained, dependent on the polymers. Upon their release from the spray-dried material, the nanoparticles show increased sizes and markedly altered zeta potentials as compared to their fresh counterparts. This may contribute to their high efficacy that is seen also after prolonged storage of the spray-dried material. We conclude that these spray-dried systems offer a great potential for the preparation of nucleic acid drug storage forms with facile reconstitution, as well as for their direct pulmonary application as dry powder.

Analysis of the Expression of PRDX6 in Patients with Hepatocellular Carcinoma and its Effect on the Phenotype of Hepatocellular Carcinoma Cells.

Objectives: This research aimed to study the expression of PRDX6 mRNA in hepatocellular carcinoma (HCC) and its effect on the prognosis of HCC. Moreover, the effect of PRDX6 gene knockdown on the proliferation, migration, and invasion of HepG2 cells mediated by lentivirus was also examined. This study offers a theoretical and experimental basis for further research on the mechanism of PRDX6 in liver cancer and new methods for clinical diagnosis and treatment. Methods: RNA sequence data of 369 HCC patients were screened through the TCGA database, and the expression and clinical characteristics of PRDX6 mRNA were analyzed based on high-throughput RNA sequencing data. HepG2 cells were divided into WT, sh-NC and sh-PRDX6 groups. Real-time PCR and Western blot were used to detect the expression levels of the PRDX6 gene and protein, respectively. CCK8 method was used to detect the proliferation activity of HepG2 cells, scratch healing test was used to detect the migration ability, Transwell chamber was used to detect the invasion ability, and Western blot was used to detect the expression levels of PI3K/Akt/mTOR signaling pathway and Notch signaling pathway-related proteins. Results: The expression of PRDX6 was significantly correlated with the gender, race, clinical stage, histological grade, and survival time of HCC patients (P < 0.05). Compared with that in WT and sh-NC groups, the expression level of PRDX6 protein in HCC patients was significantly lower (P < 0.01), the proliferation activity of HCC cells was significantly decreased (P < 0.05), and the migration and invasion ability was significantly decreased (P < 0.05) in the sh-PRDX6 group. The expression levels of PI3K, p-Akt, p-mTOR, Notch1, and Hes1 proteins in the sh-PRDX6 group were significantly lower than those in WT and sh-NC groups (P < 0.05). Conclusion: The expression of PRDX6 may be closely related to the prognosis of HCC. Lentivirus-mediated PRDX6 knockdown can inhibit the proliferation, migration and invasion of HCC cells, which may be related to its regulating the PI3K/Akt/mTOR and Notch1 signaling pathways. PRDX6 is expected to be a new target for the diagnosis and treatment of liver cancer.

Generation and utilization of endostatin-sensitive cell lines for assessing the biological activity of endostatin.

Recombinant proteins are gaining increasing popularity for treating human diseases. The clinical effectiveness of recombinant proteins is directly related to their biological activity, which is an important indicator in drug development and quality control. However, certain recombinant proteins have unclear or complex signal pathways, making detecting their activity in vitro difficult. For instance, recombinant human endostatin (endostatin), a new antitumor drug developed in China, lacks a sensitive and stable assay for its biological activity since being market approval. To address this issue, we performed a genome-wide screening of immortalized human umbilical vein endothelial cells (HUVECs) using a CRISPR/Cas9 knockout library containing 20,000 targeted genes. We identified two potential endostatin-resistant genes, NEPSPP and UTS2, and successfully constructed a highly sensitive cell line, HUVEC-UTS2-3#, by knocking down the UTS2 gene. Based on the optimized parameters of HUVEC-UTS2-3# cells, we established a new method for detecting the biological activity of endostatin. The method was validated, and it produced results consistent with primary HUVEC cells but with higher sensitivity and more stable data. The use of gene-editing technology provides a novel solution for detecting the biological activity of recombinant proteins that other methods cannot detect.

Direct Activation of Nucleobases with Small Molecules for the Conditional Control of Antisense Function.

Conditionally controlled antisense oligonucleotides provide precise interrogation of gene function at different developmental stages in animal models. Only one example of small molecule-induced activation of antisense function exist. This has been restricted to cyclic caged morpholinos that, based on sequence, can have significant background activity in the absence of the trigger. Here, we provide a new approach using azido-caged nucleobases that are site-specifically introduced into antisense morpholinos. The caging group design is a simple azidomethylene (Azm) group that, despite its very small size, efficiently blocks Watson-Crick base pairing in a programmable fashion. Furthermore, it undergoes facile decaging via Staudinger reduction when exposed to a small molecule phosphine, generating the native antisense oligonucleotide under conditions compatible with biological environments. We demonstrated small molecule-induced gene knockdown in mammalian cells, zebrafish embryos, and frog embryos. We validated the general applicability of this approach by targeting three different genes.

Effect of Ferroptosis Inducers and Inhibitors on Cell Proliferation in Acute Leukemia.

BACKGROUND/AIM: Ferroptosis refers to an iron-dependent mechanism of regulated cell death that is attributable to lipid peroxidation. Ferroptosis has been documented as a therapeutic target for various solid cancers; nonetheless, its implication in leukemia remains ambiguous. Therefore, this study aimed at investigating the impact of ferroptosis inducers and inhibitors on in vitro leukemia cell line proliferation. MATERIALS AND METHODS: Six leukemia cell lines, including acute myeloid leukemia (AML)-derived MV4-11, THP-1, HL-60, and U-937, and T-lymphoblastic leukemia (T-ALL)-derived Jurkat and KOPT-K1 with activating NOTCH1 mutations, were assessed. Erastin, which interrupts cystine uptake and depletes intracellular glutathione, and RAS-selective lethal 3 (RSL3), which suppresses glutathione peroxidase 4 (GPX4), were employed as ferroptosis inducers. Lipid peroxidation-arresting ferrostatin-1 and deferoxamine were used as ferroptosis inhibitors. Cells were cultured with these compounds and cell proliferation was assessed using a colorimetric assay. Additionally, signaling protein expression was monitored using immunoblotting, and the outcome of GPX4 knockdown was evaluated. RESULTS: Ferroptosis inducers suppressed proliferation in all cell lines except THP-1 for Erastin and THP-1 and Jurkat for RSL3. Although the ferroptosis inhibitors did not affect cell proliferation, they rescued inducer-mediated growth suppression. Ferroptosis inducers impeded MYC and cyclin D3 expression in certain cell lines and NOTCH1 signaling in T-ALL cells. GPX4 knockdown and RSL3 treatment interrupted MYC and cyclin D3 expression, respectively, in four cell lines. CONCLUSION: Ferroptosis inducers may serve as potential candidates for novel molecular therapy against AML and T-ALL.

Glut-3 Gene Knockdown as a Potential Strategy to Overcome Glioblastoma Radioresistance.

The hypoxic pattern of glioblastoma (GBM) is known to be a primary cause of radioresistance. Our study explored the possibility of using gene knockdown of key factors involved in the molecular response to hypoxia, to overcome GBM radioresistance. We used the U87 cell line subjected to chemical hypoxia generated by CoCl2 and exposed to 2 Gy of X-rays, as single or combined treatments, and evaluated gene expression changes of biomarkers involved in the Warburg effect, cell cycle control, and survival to identify the best molecular targets to be knocked-down, among those directly activated by the HIF-1α transcription factor. By this approach, glut-3 and pdk-1 genes were chosen, and the effects of their morpholino-induced gene silencing were evaluated by exploring the proliferative rates and the molecular modifications of the above-mentioned biomarkers. We found that, after combined treatments, glut-3 gene knockdown induced a greater decrease in cell proliferation, compared to pdk-1 gene knockdown and strong upregulation of glut-1 and ldha, as a sign of cell response to restore the anaerobic glycolysis pathway. Overall, glut-3 gene knockdown offered a better chance of controlling the anaerobic use of pyruvate and a better proliferation rate reduction, suggesting it is a suitable silencing target to overcome radioresistance.

A knock down strategy for rapid, generic, and versatile modelling of muscular dystrophies in 3D-tissue-engineered-skeletal muscle.

BACKGROUND: Human iPSC-derived 3D-tissue-engineered-skeletal muscles (3D-TESMs) offer advanced technology for disease modelling. However, due to the inherent genetic heterogeneity among human individuals, it is often difficult to distinguish disease-related readouts from random variability. The generation of genetically matched isogenic controls using gene editing can reduce variability, but the generation of isogenic hiPSC-derived 3D-TESMs can take up to 6 months, thereby reducing throughput. METHODS: Here, by combining 3D-TESM and shRNA technologies, we developed a disease modelling strategy to induce distinct genetic deficiencies in a single hiPSC-derived myogenic progenitor cell line within 1 week. RESULTS: As proof of principle, we recapitulated disease-associated pathology of Duchenne muscular dystrophy and limb-girdle muscular dystrophy type 2A caused by loss of function of DMD and CAPN3, respectively. shRNA-mediated knock down of DMD or CAPN3 induced a loss of contractile function, disruption of tissue architecture, and disease-specific proteomes. Pathology in DMD-deficient 3D-TESMs was partially rescued by a candidate gene therapy treatment using micro-dystrophin, with similar efficacy compared to animal models. CONCLUSIONS: These results show that isogenic shRNA-based humanized 3D-TESM models provide a fast, cheap, and efficient tool to model muscular dystrophies and are useful for the preclinical evaluation of novel therapies.

Diverse temporal and spatial mechanisms work, partially through Stanniocalcin-1, V-ATPase and senescence, to activate the extracellular ATP-mediated drug resistance in human cancer cells.

Introduction: Resistance to drug therapies is associated with a large majority of cancer-related deaths. ATP-binding cassette (ABC) transporter-mediated drug efflux, epithelial-mesenchymal transition (EMT), cancer stem cells (CSCs), glutathione (GSH), senescence, and vacuole-type ATPase (V-ATPase) all contribute to the resistance. We recently showed that extracellular ATP (eATP) induces and regulates EMT, CSC formation, and ABC transporters in human cancer cells and tumors. eATP also consistently upregulates Stanniocalcin-1 (STC1), a gene that significantly contributes to EMT, CSC formation, and tumor growth. We also found that eATP enhances drug resistance in cancer cells through eATP internalization mediated by macropinocytosis, leading to an elevation of intracellular ATP (iATP) levels, induction of EMT, and CSC formation. However, these factors have never been systematically investigated in the context of eATP-induced drug resistance. Methods: In this study, we hypothesized that eATP increases drug resistance via inducing ABC efflux, EMT, CSCs, STC1, and their accompanied processes such as GSH reducing activity, senescence, and V-ATPase. RNA sequencing, metabolomics, gene knockdown and knockout, and functional assays were performed to investigate these pathways and processes. Results and discussion: Our study results showed that, in multiple human cancer lines, eATP induced genes involved in drug resistance, elevated ABC transporters' efflux activity of anticancer drugs; generated transcriptomic and metabolic profiles representing a drug resistant state; upregulated activities of GSH, senescence, and V-ATPase to promote drug resistance. Collectively, these newly found players shed light on the mechanisms of eATP-induced as well as STC1- and V-ATPase-mediated drug resistance and offer potential novel targets for combating drug resistance in cancers.

Self-Assembled Oligopeptoplex-Loaded Dissolving Microneedles for Adipocyte-Targeted Anti-Obesity Gene Therapy.

Advancements in gene delivery systems are pivotal for gene-based therapeutics in oncological, inflammatory, and infectious diseases. This study delineates the design of a self-assembled oligopeptoplex (SA-OP) optimized for shRNA delivery to adipocytes, targeting obesity and associated metabolic syndromes. Conventional systems face challenges, including instability due to electrostatic interactions between genetic materials and cationic oligopeptides. Additionally, repeated injections induce discomfort and compromise patient well-being. To circumvent these issues, a dissolvable hyaluronic acid-based, self-locking microneedle (LMN) patch is developed, with improved micro-dose efficiency, for precise SA-OP delivery. This platform offers pain-free administration and improved SA-OP storage stability. In vitro studies in 3T3-L1 cells demonstrated improvements in SA-OP preservation and gene silencing efficacy. In vivo evaluation in a mice model of diet-induced type 2 diabetes yielded significant gene silencing in adipose tissue and a 21.92 ± 2.51% reduction in body weight with minimum relapse risk at 6-weeks post-treatment, representing a superior therapeutic efficacy in a truncated timeframe relative to the GLP-1 analogues currently available on the market. Additionally, SA-OP (LMN) mitigated insulin resistance, inflammation, and hepatic steatosis. These findings establish SA-OP (LMN) as a robust, minimally invasive transdermal gene delivery platform with prolonged storage stability for treating obesity and its metabolic comorbidities.

Optimal dsRNA Concentration for RNA Interference in Asian Citrus Psyllid.

The Asian citrus psyllid (ACP) is a citrus pest and insect vector of "Candidatus Liberibacter asiaticus", the causal agent of citrus greening disease. Double-stranded RNA (dsRNA) biopesticides that trigger RNA interference (RNAi) offer an alternative to traditional insecticides. Standardized laboratory screening of dsRNA requires establishing the minimal effective concentration(s) that result in effective RNAi "penetrance" and trigger RNAi, resulting in one or more measurable phenotypes, herein, significant gene knockdown and the potential for mortality. In this study, knockdown was evaluated for a range of dsRNA concentrations of three ACP candidate genes, clathrin heavy chain (CHC), vacuolar ATPase subunit A (vATPase-A), and sucrose non-fermenting protein 7 (Snf7). Gene knockdown was quantified for ACP teneral adults and 3rd instar nymphs allowed a 48 h ingestion-access period (IAP) on 10, 50,100, 200, and 500 ng/µL dsRNA dissolved in 20% sucrose followed by a 5-day post-IAP on orange jasmine shoots. Significant gene knockdown (p < 0.05) in ACP third instar nymphs and adults ranged from 12-34% and 18-39%, 5 days post-IAP on dsRNA at 10-500 and 100-500 ng/µL, respectively. The threshold concentration beyond which no significant gene knockdown and adult mortality was observed post-48 h IAP and 10-day IAP, respectively, was determined as 200 ng/µL, a concentration indicative of optimal RNAi penetrance.

Halloween genes are expressed with a circadian rhythm during development in prothoracic glands of the insect RHODNIUS PROLIXUS.

We analyse the developmental and circadian profiles of expression of the genes responsible for ecdysteroidogenesis (Halloween genes) in the PGs of Rhodnius prolixus throughout larval-adult development. Extensive use of in vitro techniques enabled multiple different parameters to be measured in individual PGs. Expression of disembodied and spook closely paralleled the ecdysteroid synthesis of the same PGs, and the ecdysteroid titre in vivo, but with functionally significant exceptions. Various tissues other than PGs expressed one, both or neither genes. Both gonads express both genes in pharate adults (larvae close to ecdysis). Both genes were expressed at low, but significant, levels in UF Rhodnius, raising questions concerning how developmental arrest is maintained in UF animals. IHC confirmed the subcellular localisation of the coded proteins. Gene knockdown suppressed transcription of both genes and ecdysteroid synthesis, with spook apparently regulating the downstream gene disembodied. Transcription of both genes occurred with a daily rhythm (with peaks at night) that was confirmed to be under circadian control using aperiodic conditions. The complex behaviour of the rhythm in LL implied two anatomically distinct oscillators regulate this transcription rhythm. First, the circadian clock in the PGs and second, the circadian rhythm of of Rhodnius PTTH which is released rhythmically from the brain under control of the circadian clock therein, both of which were described previously. We conclude ecdysteroidogenesis in Rhodnius PGs employs a similar pathway as other insects, but its control is complex, involving mechanisms both within and outside the PGs.

Redox Protein Thioredoxin Mediates Neurite Outgrowth in Primary Cultured Mouse Cerebral Cortical Neurons.

Thioredoxin system plays an important role in maintaining the cellular redox balance. Recent evidence suggests that thioredoxin (Trx) system may promote cell survival and neuroprotection. In this study, we explored the role of thioredoxin system in neuronal differentiation using a primary mouse cortical neuronal cell culture. First, Trx and Trx reductase (TrxR) protein levels were analyzed in cultured neurons from 1 to 32 days in vitro (DIV). The result showed that Trx and TrxR protein levels time-dependently increased in the neuron cell culture from 1 to 18 DIV. To establish the role of Trx in neuronal differentiation, Trx gene expression was knockdown in cultured neurons using Trx sgRNA CRISPR/Cas9 technology. Treatment with CRISPR/Cas9/Trx sgRNA decreased Trx protein levels and caused a reduction in dendritic outgrowth and branching of cultured neurons. Then, primary cortical neurons were treated with the Trx inhibitor PX12 to block Trx reducing activity. Treatment with PX12 also reduced dendritic outgrowth and branching. Furthermore, PX12 treatment reduced the ratio of phosphorylated cyclic AMP response element-binding protein (CREB)/total CREB protein levels. To investigate whether CREB phosphorylation is redox regulated, SH-SY5Y cells were treated with H2O2, which reduced phosphorylated CREB protein levels and increased CREB thiol oxidation. However, treatment with CB3, a Trx-mimetic tripeptide, rescued H2O2-decreased CREB phosphorylation. Our results suggest that Trx regulates neuronal differentiation and maturation of primary mouse cortical neurons by targeting CREB neurotrophic pathway. Trx may regulate CREB activation by maintaining the cellular redox balance.

UV-LASER adjuvant-surfactant-facilitated delivery of mobile dsRNA to tomato plant vasculature and evidence of biological activity by gene knockdown in the potato psyllid.

BACKGROUND: Double-stranded RNA (dsRNA) biopesticides are of interest for the abatement of insect vectors of pathogenic bacteria such as 'Candidatus Liberibacter', which infects both its psyllid and plant hosts. Silencing of genes essential for psyllids, or for Liberibacter, is anticipated to lead to mortality or impeded bacterial multiplication. Foliar delivery is preferred for biopesticide application; however, the cuticle impedes dsRNA penetration into the vasculature. Here, conditions were established for wounding tomato leaves using ultraviolet light amplification by stimulated emissions of radiation (UV-LASER) to promote dsRNA penetration into leaves and vasculature. RESULTS: UV-LASER treatment with application of select adjuvants/surfactants resulted in vascular delivery of 100-, 300- and 600-bp dsRNAs that, in general, were correlated with size. The 100-bp dsRNA required no pretreatment, whereas 300- and 600-bp dsRNAs entered the vasculature after UV-LASER treatment only and UV-LASER adjuvant/surfactant treatment, respectively. Of six adjuvant/surfactants evaluated, plant-derived oil combined with an anionic organosilicon compound performed most optimally. Localization of dsRNAs in the tomato vasculature was documented using fluorometry and fluorescence confocal microscopy. The biological activity of in planta-delivered dsRNA (200-250 bp) was determined by feeding third-instar psyllids on tomato leaves post UV-LASER adjuvant/surfactant treatment, with or without psyllid cdc42- and gelsolin dsRNAs. Gene knockdown was quantified by quantitative, real-time polymerase chain reaction with reverse transcription (RT-qPCR) amplification. At 10 days post the ingestion-access period, knockdown of cdc42 and gelsolin expression was 61% and 56%, respectively, indicating that the dsRNAs delivered to the tomato vasculature were mobile and biologically active. CONCLUSION: Results indicated that UV-LASER adjuvant/surfactant treatments facilitated the delivery of mobile, biologically active dsRNA molecules to the plant vasculature. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Establishment of a high-content imaging assay for tau aggregation in hiPSC-derived neurons differentiated from two protocols to routinely evaluate compounds and genetic perturbations.

Aberrant protein aggregation is a pathological cellular hallmark of many neurodegenerative diseases, such as Alzheimer's disease (AD) and frontotemporal dementia (FTD), where the tau protein is aggregating, forming neurofibrillary tangles (NFTs), and propagating from neuron to neuron. These processes have been linked to disease progression and a decline in cognitive function. Various therapeutic approaches aim at the prevention or reduction of tau aggregates in neurons. Human induced pluripotent stem cells (hiPSCs) are a very valuable tool in neuroscience discovery, as they offer access to potentially unlimited amounts of cell types that are affected in disease, including cortical neurons, for in vitro studies. We have generated an in vitro model for tau aggregation that uses hiPSC - derived neurons expressing an aggregation prone, fluorescently tagged version of the human tau protein after lentiviral transduction. Upon addition of tau seeds in the form of recombinant sonicated paired helical filaments (sPHFs), the neurons show robust, disease-like aggregation of the tau protein. The model was developed as a plate-based high content screening assay coupled with an image analysis algorithm to evaluate the impact of small molecules or genetic perturbations on tau. We show that the assay can be used to evaluate small molecules or screen targeted compound libraries. Using siRNA-based gene knockdown, genes of interest can be evaluated, and we could show that a targeted gene library can be screened, by screening nearly 100 deubiquitinating enzymes (DUBs) in that assay. The assay uses an imaging-based readout, a relatively short timeline, quantifies the extent of tau aggregation, and also allows the assessment of cell viability. Furthermore, it can be easily adapted to different hiPSC lines or neuronal subtypes. Taken together, this complex and highly relevant approach can be routinely applied on a weekly basis in the screening funnels of several projects and generates data with a turnaround time of approximately five weeks.